register interest

Professor Peter Simmonds

Research Area: Microbiology
Technology Exchange: Bioinformatics, Computational biology and Vaccine production and evaluation
Scientific Themes: Immunology & Infectious Disease and Genetics & Genomics
Keywords: Viral pathogenesis, Virus evolution, Hepatitis C virus, Vaccines, RNA viruses and RNA secondary structure
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My main current research programme is in the investigation of the evolutionary and functional basis for the pervasive suppression of CpG and UpA dinucleotides in RNA viruses. Modelling evidence I obtained for selection against them in cytoplasmically-expressed RNA sequences demanded functional investigations. The discovery that viruses with high frequencies of CpG/UpA are recognised and suppressed by as yet uncharacterised intrinsic defence mechanisms suddenly opens exciting, new areas of enquiry into innate immunity in mammalian cells. Conversely, accelerated replication of viruses with suppressed frequencies confronts us with a seeming evolutionary paradox which we will investigate through experimental investigations of relationships between disease processes and transmission dynamics. Enhanced expression of genes with low CpG and UpA frequencies can be exploited as a generic technology in vaccine and vector design.

Other ongoing and recent research has been focussed on the characterisation of RNA secondary structure in viruses, and its effect on virus evolution. Discovery of large scale RNA structure in many families of positive-stranded RNA viruses followed the development of large-scale bioinformatic methods to quantify and characterise RNA secondary structure formation in viral genomes. The association of genome-scale organised RNA structure (GORS) with host persistence, and the finding that their genomes are structured in a fundamentally different way from those causing acute infections has been the subject of ongoing investigations of the nature of virus interactions with host cell defences modulated by double-stranded RNA, the physical structure of the predicted RNA structures and the influence of RNA structure as a sequence constraint on the evolution of persistent viruses. This area of research has also initiated a series of functional studies of the role of RNA secondary structure elements in the replication of HCV (Professor David Evans, University of Warwick), caliciviruses (Dr Ian Goodfellow, ImperialCollege, London) and hepatitis A virus (Professor S. Lemon, University of Texas Medical   Branch).

A second area of research focuses on the molecular epidemiology, evolution and emergence of a wide range of human pathogenic viruses. This includes aetiological investigations of the role of known and recently discovered viruses in respiratory, enteric and neurological disease. I am closely involved in an active ongoing programme of research in clinical virology in its broadest sense with investigations ranging from basic science molecular epidemiology, evolutionary and pathogenicity studies through to investigations of the impact and newly discovered and emerging viruses in human health, and the development of diagnostics for patient screening and monitoring.

Collectively, these investigations fit into a broader theme of research work in the nature of evolutionary process in RNA viruses, how it differs from that of their mammalian hosts, how this and recombination shapes their molecular epidemiology and clinical manifestations.

DPhil (PhD) projects for:

a) Exploring aspects of the cellular pathways that control virus replication of viruses with high CpG frequencies

b) Development of a novel infection model for the development of an effective vaccine for hepatitis C virus (HCV)

will be offered for the 2018 academic year. Please contact me ( for further details.

Name Department Institution Country
Professor Ellie (Eleanor) Barnes Experimental Medicine Division Oxford University, Peter Medawar Building United Kingdom
Professor Paul Klenerman Experimental Medicine Division Oxford University, Peter Medawar Building United Kingdom
Professor Oliver Pybus Zoology University of Oxford United Kingdom
Dr Katie Jeffery FRCPath Department of Microbiology and Infectious DIseases Oxford University Hospitals NHS Foundation Trust United Kingdom
Dr Rory Bowden Wellcome Trust Centre for Human Genetics Oxford University, Henry Wellcome Building of Genomic Medicine United Kingdom
Prof Jan Rehwinkel (RDM) Investigative Medicine Division Oxford University, Weatherall Institute of Molecular Medicine United Kingdom
Zell R, Delwart E, Gorbalenya AE, Hovi T, King AMQ, Knowles NJ, Lindberg AM, Pallansch MA, Palmenberg AC, Reuter G et al. 2017. ICTV Virus Taxonomy Profile: Picornaviridae. J Gen Virol, 98 (10), pp. 2421-2422. | Show Abstract | Read more

The family Picornaviridae comprises small non-enveloped viruses with RNA genomes of 6.7 to 10.1 kb, and contains >30 genera and >75 species. Most of the known picornaviruses infect mammals and birds, but some have also been detected in reptiles, amphibians and fish. Many picornaviruses are important human and veterinary pathogens and may cause diseases of the central nervous system, heart, liver, skin, gastrointestinal tract or upper respiratory tract. Most picornaviruses are transmitted by the faecal-oral or respiratory routes. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of the Picornaviridae, which is available at

Trivedi S, Murthy S, Sharma H, Hartlage AS, Kumar A, Gadi S, Simmonds P, Chauhan LV, Scheel TKH, Billerbeck E et al. 2017. Viral persistence, liver disease and host response in Hepatitis C-like virus rat model. Hepatology, | Show Abstract | Read more

The lack of a relevant, tractable, and immunocompetent animal model for hepatitis C virus (HCV) has severely impeded investigations of viral persistence, immunity and pathogenesis. In the absence of immunocompetent models with robust HCV infection, homolog hepaciviruses in their natural host could potentially provide useful surrogate models. We isolated a rodent hepacivirus (RHV) from wild rats (Rattus norvegicus), RHV-rn1, acquired the complete viral genome sequence and developed an infectious reverse genetics system. RHV-rn1 resembles HCV in genomic features including the pattern of polyprotein cleavage sites and secondary structures in the viral 5' and 3' UTRs. We used site-directed and random mutagenesis to determine that only the first of the two miR-122 seed sites in viral 5'UTR is required for viral replication and persistence in rats. Next, we used the clone derived virus progeny to infect several inbred and outbred rat strains. Our results determined that RHV-rn1 possesses several HCV-defining hallmarks: hepatotropism, propensity to persist, and the ability of induce gradual liver damage. Histological examination of liver samples revealed the presence of lymphoid aggregates, parenchymal inflammation and macro/micro vesicular steatosis in chronically infected rats. Gene expression analysis demonstrated that the intrahepatic response during RHV-rn1 infection in rats mirrors that of HCV infection, including persistent activation of interferon signaling pathways. Finally, we determined that the backbone drug of HCV direct acting antiviral (DAA) therapy, Sofosbuvir, effectively suppresses chronic RHV-rn1 infection in rats. Taken together, we developed RHV-rn1 infected rats as a fully immunocompetent and informative surrogate model to delineate the mechanisms of HCV-related viral persistence, immunity and pathogenesis. This article is protected by copyright. All rights reserved.

Smith DB, Meyers G, Bukh J, Gould EA, Monath T, Scott Muerhoff A, Pletnev A, Rico-Hesse R, Stapleton JT, Simmonds P, Becher P. 2017. Proposed revision to the taxonomy of the genus Pestivirus, family Flaviviridae. J Gen Virol, 98 (8), pp. 2106-2112. | Show Abstract | Read more

We propose the creation of seven new species in the genus Pestivirus (family Flaviviridae) in addition to the four existing species, and naming species in a host-independent manner using the format Pestivirus X. Only the virus species names would change; virus isolates would still be referred to by their original names. The original species would be re-designated as Pestivirus A (original designation Bovine viral diarrhea virus 1), Pestivirus B (Bovine viral diarrhea virus 2), Pestivirus C (Classical swine fever virus) and Pestivirus D (Border disease virus). The seven new species (and example isolates) would be Pestivirus E (pronghorn pestivirus), Pestivirus F (Bungowannah virus), Pestivirus G (giraffe pestivirus), Pestivirus H (Hobi-like pestivirus), Pestivirus I (Aydin-like pestivirus), Pestivirus J (rat pestivirus) and Pestivirus K (atypical porcine pestivirus). A bat-derived virus and pestiviruses identified from sheep and goat (Tunisian sheep pestiviruses), which lack complete coding region sequences, may represent two additional species.

Colmant AMG, Hobson-Peters J, Bielefeldt-Ohmann H, van den Hurk AF, Hall-Mendelin S, Chow WK, Johansen CA, Fros J, Simmonds P, Watterson D et al. 2017. A New Clade of Insect-Specific Flaviviruses from Australian Anopheles Mosquitoes Displays Species-Specific Host Restriction. mSphere, 2 (4), pp. e00262-17-e00262-17. | Show Abstract | Read more

Flaviviruses are arthropod-borne viruses found worldwide and are responsible for significant human and veterinary diseases, including dengue, Zika, and West Nile fever. Some flaviviruses are insect specific and replicate only in mosquitoes. We report a genetically divergent group of insect-specific flaviviruses from Anopheles mosquitoes that do not replicate in arthropod cell lines or heterologous Anopheles species, exhibiting unprecedented specialization for their host species. Determination of the complete sequences of the RNA genomes of three of these viruses, Karumba virus (KRBV), Haslams Creek virus, and Mac Peak virus (McPV), that are found in high prevalence in some Anopheles mosquito populations and detection of virus-specific proteins, replicative double-stranded RNA, and small interfering RNA responses in the host mosquito species provided strong evidence of a functional replicating virus in the mosquito midgut. Analysis of nucleotide composition in the KRBV and McPV sequences also revealed a pattern consistent with the virus evolving to replicate only in insects. These findings represent a significant advance in our knowledge of mosquito-borne flavivirus ecology, host restriction, and evolution. IMPORTANCE Flaviviruses like dengue, Zika, or West Nile virus infect millions of people each year and are transmitted to humans via infected-mosquito bites. A subset of flaviviruses can only replicate in the mosquito host, and recent studies have shown that some can interfere with pathogenic flaviviruses in mosquitoes and limit the replication and transmission of the latter. The insect-specific flaviviruses (ISFs) reported here form a new Anopheles mosquito-associated clade separate from the Aedes- and Culex-associated ISF clades. The identification of distinct clades for each mosquito genus provides new insights into the evolution and ecology of flaviviruses. One of these viruses was shown to replicate in the midgut of the mosquito host and exhibit the most specialized host restriction reported to date for ISFs. Understanding this unprecedented host restriction in ISFs could help identify the mechanisms involved in the evolution of flaviviruses and their emergence as mosquito-borne pathogens.

Adams MJ, Lefkowitz EJ, King AMQ, Harrach B, Harrison RL, Knowles NJ, Kropinski AM, Krupovic M, Kuhn JH, Mushegian AR et al. 2017. Changes to taxonomy and the International Code of Virus Classification and Nomenclature ratified by the International Committee on Taxonomy of Viruses (2017). Arch Virol, 162 (8), pp. 2505-2538. | Show Abstract | Read more

This article lists the changes to virus taxonomy approved and ratified by the International Committee on Taxonomy of Viruses (ICTV) in March 2017.

Berto A, Anh PH, Carrique-Mas JJ, Simmonds P, Van Cuong N, Tue NT, Van Dung N, Woolhouse ME, Smith I, Marsh GA et al. 2017. Detection of potentially novel paramyxovirus and coronavirus viral RNA in bats and rats in the Mekong Delta region of southern Viet Nam. Zoonoses Public Health, | Show Abstract | Read more

Bats and rodents are being increasingly recognized as reservoirs of emerging zoonotic viruses. Various studies have investigated bat viruses in tropical regions, but to date there are no data regarding viruses with zoonotic potential that circulate in bat and rat populations in Viet Nam. To address this paucity of data, we sampled three bat farms and three wet markets trading in rat meat in the Mekong Delta region of southern Viet Nam. Faecal and urine samples were screened for the presence of RNA from paramyxoviruses, coronaviruses and filoviruses. Paramyxovirus RNA was detected in 4 of 248 (1%) and 11 of 222 (4.9%) bat faecal and urine samples, respectively. Coronavirus RNA was detected in 55 of 248 (22%) of bat faecal samples; filovirus RNA was not detected in any of the bat samples. Further, coronavirus RNA was detected in 12 of 270 (4.4%) of rat faecal samples; all samples tested negative for paramyxovirus. Phylogenetic analysis revealed that the bat paramyxoviruses and bat and rat coronaviruses were related to viruses circulating in bat and rodent populations globally, but showed no cross-species mixing of viruses between bat and rat populations within Viet Nam. Our study shows that potentially novel variants of paramyxoviruses and coronaviruses commonly circulate in bat and rat populations in Viet Nam. Further characterization of the viruses and additional human and animal surveillance is required to evaluate the likelihood of viral spillover and to assess whether these viruses pose a risk to human health.

Ansari MA, Pedergnana V, L C Ip C, Magri A, Von Delft A, Bonsall D, Chaturvedi N, Bartha I, Smith D, Nicholson G et al. 2017. Genome-to-genome analysis highlights the effect of the human innate and adaptive immune systems on the hepatitis C virus. Nat Genet, 49 (5), pp. 666-673. | Show Abstract | Read more

Outcomes of hepatitis C virus (HCV) infection and treatment depend on viral and host genetic factors. Here we use human genome-wide genotyping arrays and new whole-genome HCV viral sequencing technologies to perform a systematic genome-to-genome study of 542 individuals who were chronically infected with HCV, predominantly genotype 3. We show that both alleles of genes encoding human leukocyte antigen molecules and genes encoding components of the interferon lambda innate immune system drive viral polymorphism. Additionally, we show that IFNL4 genotypes determine HCV viral load through a mechanism dependent on a specific amino acid residue in the HCV NS5A protein. These findings highlight the interplay between the innate immune system and the viral genome in HCV control.

Sharp CP, Gregory WF, Hattingh L, Malik A, Adland E, Daniels S, van Zyl A, Carlson JM, Wareing S, Ogwu A et al. 2017. PARV4 prevalence, phylogeny, immunology and coinfection with HIV, HBV and HCV in a multicentre African cohort. Wellcome Open Res, 2 pp. 26. | Show Abstract | Read more

Background: The seroprevalence of human parvovirus-4 (PARV4) varies considerably by region. In sub-Saharan Africa, seroprevalence is high in the general population, but little is known about the transmission routes or the prevalence of coinfection with blood-borne viruses, HBV, HCV and HIV.  Methods: To further explore the characteristics of PARV4 in this setting, with a particular focus on the prevalence and significance of coinfection, we screened a cohort of 695 individuals recruited from Durban and Kimberley (South Africa) and Gaborone (Botswana) for PARV4 IgG and DNA, as well as documenting HIV, HBV and HCV status.  Results: Within these cohorts, 69% of subjects were HIV-positive. We identified no cases of HCV by PCR, but 7.4% were positive for HBsAg. PARV4 IgG was positive in 42%; seroprevalence was higher in adults (69%) compared to children (21%) (p<0.0001) and in HIV-positive (52%) compared to HIV-negative individuals (24%) (p<0.0001), but there was no association with HBsAg status. We developed an on-line tool to allow visualization of coinfection data ( We identified five subjects who were PCR-positive for PARV4 genotype-3. Ex vivo CD8+ T cell responses spanned the entire PARV4 proteome and we propose a novel HLA-B*57:03-restricted epitope within the NS protein.  Conclusions: This characterisation of PARV4 infection provides enhanced insights into the epidemiology of infection and co-infection in African cohorts, and provides the foundations for planning further focused studies to elucidate transmission pathways, immune responses, and the clinical significance of this organism.

Adams MJ, Lefkowitz EJ, King AM, Harrach B, Harrison RL, Knowles NJ, Kropinski AM, Krupovic M, Kuhn JH, Mushegian AR et al. 2017. 50 years of the International Committee on Taxonomy of Viruses: progress and prospects. Arch Virol, 162 (5), pp. 1441-1446. | Show Abstract | Read more

We mark the 50th anniversary of the International Committee on Taxonomy of Viruses (ICTV) by presenting a brief history of the organization since its foundation, showing how it has adapted to advancements in our knowledge of virus diversity and the methods used to characterize it. We also outline recent developments, supported by a grant from the Wellcome Trust (UK), that are facilitating substantial changes in the operations of the ICTV and promoting dialogue with the virology community. These developments will generate improved online resources, including a freely available and regularly updated ICTV Virus Taxonomy Report. They also include a series of meetings between the ICTV and the broader community focused on some of the major challenges facing virus taxonomy, with the outcomes helping to inform the future policy and practice of the ICTV.

Simmonds P, Adams MJ, Benkő M, Breitbart M, Brister JR, Carstens EB, Davison AJ, Delwart E, Gorbalenya AE, Harrach B et al. 2017. Consensus statement: Virus taxonomy in the age of metagenomics. Nat Rev Microbiol, 15 (3), pp. 161-168. | Show Abstract | Read more

The number and diversity of viral sequences that are identified in metagenomic data far exceeds that of experimentally characterized virus isolates. In a recent workshop, a panel of experts discussed the proposal that, with appropriate quality control, viruses that are known only from metagenomic data can, and should be, incorporated into the official classification scheme of the International Committee on Taxonomy of Viruses (ICTV). Although a taxonomy that is based on metagenomic sequence data alone represents a substantial departure from the traditional reliance on phenotypic properties, the development of a robust framework for sequence-based virus taxonomy is indispensable for the comprehensive characterization of the global virome. In this Consensus Statement article, we consider the rationale for why metagenomic sequence data should, and how it can, be incorporated into the ICTV taxonomy, and present proposals that have been endorsed by the Executive Committee of the ICTV.

Simmonds P, Becher P, Bukh J, Gould EA, Meyers G, Monath T, Muerhoff S, Pletnev A, Rico-Hesse R, Smith DB et al. 2017. ICTV Virus Taxonomy Profile: Flaviviridae. J Gen Virol, 98 (1), pp. 2-3. | Show Abstract | Read more

The Flaviviridae is a family of small enveloped viruses with RNA genomes of 9000-13 000 bases. Most infect mammals and birds. Many flaviviruses are host-specific and pathogenic, such as hepatitis C virus in the genus Hepacivirus. The majority of known members in the genus Flavivirus are arthropod borne, and many are important human and veterinary pathogens (e.g. yellow fever virus, dengue virus). This is a summary of the current International Committee on Taxonomy of Viruses (ICTV) report on the taxonomy of the Flaviviridae, which is available at

Matthews PC, Sharp C, Simmonds P, Klenerman P. 2017. Human parvovirus 4 'PARV4' remains elusive despite a decade of study. F1000Res, 6 pp. 82. | Show Abstract | Read more

Human parvovirus 4 ('PARV4') is a small DNA tetraparvovirus, first reported in 2005. In some populations, PARV4 infection is uncommon, and evidence of exposure is found only in individuals with risk factors for parenteral infection who are infected with other blood-borne viruses. In other settings, seroprevalence studies suggest an endemic, age-associated transmission pattern, independent of any specific risk factors. The clinical impact of PARV4 infection remains uncertain, but reported disease associations include an influenza-like syndrome, encephalitis, acceleration of HIV disease, and foetal hydrops. In this review, we set out to report progress updates from the recent literature, focusing on the investigation of cohorts in different geographical settings, now including insights from Asia, the Middle East, and South America, and discussing whether attributes of viral or host populations underpin the striking differences in epidemiology. We review progress in understanding viral phylogeny and biology, approaches to diagnostics, and insights that might be gained from studies of closely related animal pathogens. Crucial questions about pathogenicity remain unanswered, but we highlight new evidence supporting a possible link between PARV4 and an encephalitis syndrome. The unequivocal evidence that PARV4 is endemic in certain populations should drive ongoing research efforts to understand risk factors and routes of transmission and to gain new insights into the impact of this virus on human health.

Thomson E, Ip CL, Badhan A, Christiansen MT, Adamson W, Ansari MA, Bibby D, Breuer J, Brown A, Bowden R et al. 2016. Comparison of Next-Generation Sequencing Technologies for Comprehensive Assessment of Full-Length Hepatitis C Viral Genomes. J Clin Microbiol, 54 (10), pp. 2470-2484. | Show Abstract | Read more

Affordable next-generation sequencing (NGS) technologies for hepatitis C virus (HCV) may potentially identify both viral genotype and resistance genetic motifs in the era of directly acting antiviral (DAA) therapies. This study compared the ability of high-throughput NGS methods to generate full-length, deep, HCV sequence data sets and evaluated their utility for diagnostics and clinical assessment. NGS methods using (i) unselected HCV RNA (metagenomics), (ii) preenrichment of HCV RNA by probe capture, and (iii) HCV preamplification by PCR implemented in four United Kingdom centers were compared. Metrics of sequence coverage and depth, quasispecies diversity, and detection of DAA resistance-associated variants (RAVs), mixed HCV genotypes, and other coinfections were compared using a panel of samples with different viral loads, genotypes, and mixed HCV genotypes/subtypes [geno(sub)types]. Each NGS method generated near-complete genome sequences from more than 90% of samples. Enrichment methods and PCR preamplification generated greater sequence depth and were more effective for samples with low viral loads. All NGS methodologies accurately identified mixed HCV genotype infections. Consensus sequences generated by different NGS methods were generally concordant, and majority RAVs were consistently detected. However, methods differed in their ability to detect minor populations of RAVs. Metagenomic methods identified human pegivirus coinfections. NGS provided a rapid, inexpensive method for generating whole HCV genomes to define infecting genotypes, RAVs, comprehensive viral strain analysis, and quasispecies diversity. Enrichment methods are particularly suited for high-throughput analysis while providing the genotype and information on potential DAA resistance.

Adams MJ, Lefkowitz EJ, King AM, Harrach B, Harrison RL, Knowles NJ, Kropinski AM, Krupovic M, Kuhn JH, Mushegian AR et al. 2016. Ratification vote on taxonomic proposals to the International Committee on Taxonomy of Viruses (2016). Arch Virol, 161 (10), pp. 2921-2949. | Show Abstract | Read more

This article lists the changes to virus taxonomy approved and ratified by the International Committee on Taxonomy of Viruses (ICTV) in April 2016.Changes to virus taxonomy (the Universal Scheme of Virus Classification of the International Committee on Taxonomy of Viruses [ICTV]) now take place annually and are the result of a multi-stage process. In accordance with the ICTV Statutes ( ), proposals submitted to the ICTV Executive Committee (EC) undergo a review process that involves input from the ICTV Study Groups (SGs) and Subcommittees (SCs), other interested virologists, and the EC. After final approval by the EC, proposals are then presented for ratification to the full ICTV membership by publication on an ICTV web site ( ) followed by an electronic vote. The latest set of proposals approved by the EC was made available on the ICTV website by January 2016 ( ). A list of these proposals was then emailed on 28 March 2016 to the 148 members of ICTV, namely the EC Members, Life Members, ICTV Subcommittee Members (including the SG chairs) and ICTV National Representatives. Members were then requested to vote on whether to ratify the taxonomic proposals (voting closed on 29 April 2016).

Puenpa J, Vongpunsawad S, Österback R, Waris M, Eriksson E, Albert J, Midgley S, Fischer TK, Eis-Hübinger AM, Cabrerizo M et al. 2016. Molecular epidemiology and the evolution of human coxsackievirus A6. J Gen Virol, 97 (12), pp. 3225-3231. | Show Abstract | Read more

Coxsackievirus A6 (CV-A6) is a major aetiologic agent for hand, foot and mouth disease (HFMD) in recent years. HFMD outbreaks associated with CV-A6 resulted from the evolutionary dynamics of CV-A6 and the appearance of novel recombinant forms (RFs). To examine this, 151 variants collected in 2013 and 2014 from Germany, Spain, Sweden, Denmark and Thailand were genotyped for the VP1 capsid and 3Dpol genes. Analysis of the VP1 gene showed an increasing correspondence between CV-A6 genome recombination and sequence divergence (estimated substitution rate of 8.1×10-3 substitutions site-1 year-1 and RF half-life of 3.1 years). Bayesian phylogenetic analysis showed that recent recombination groups (RF-E, -F, -H, -J and -K) shared a common ancestor (RF-A). Thirty-nine full-length genomes of different RFs revealed recombination breakpoints between the 2A-2C and the 5' UTRs. The emergence of new CV-A6 recombination groups has become widespread in Europe and Asia within the last 8 years.

Smith DB, Becher P, Bukh J, Gould EA, Meyers G, Monath T, Muerhoff AS, Pletnev A, Rico-Hesse R, Stapleton JT, Simmonds P. 2016. Proposed update to the taxonomy of the genera Hepacivirus and Pegivirus within the Flaviviridae family. J Gen Virol, 97 (11), pp. 2894-2907. | Show Abstract | Read more

Proposals are described for the assignment of recently reported viruses, infecting rodents, bats and other mammalian species, to new species within the Hepacivirus and Pegivirus genera (family Flaviviridae). Assignments into 14 Hepacivirus species (Hepacivirus A-N) and 11 Pegivirus species (Pegivirus A-K) are based on phylogenetic relationships and sequence distances between conserved regions extracted from complete coding sequences for members of each proposed taxon. We propose that the species Hepatitis C virus is renamed Hepacivirus C in order to acknowledge its unique historical position and so as to minimize confusion. Despite the newly documented genetic diversity of hepaciviruses and pegiviruses, members of these genera remain phylogenetically distinct, and differ in hepatotropism and the possession of a basic core protein; pegiviruses in general lack these features. However, other characteristics that were originally used to support their division into separate genera are no longer definitive; there is overlap between the two genera in the type of internal ribosomal entry site and the presence of miR-122 sites in the 5' UTR, the predicted number of N-linked glycosylation sites in the envelope E1 and E2 proteins, the presence of poly U tracts in the 3' UTR and the propensity of viruses to establish a persistent infection. While all classified hepaciviruses and pegiviruses have mammalian hosts, the recent description of a hepaci-/pegi-like virus from a shark and the likely existence of further homologues in other non-mammalian species indicate that further species or genera remain to be defined in the future.

Brown A, Halliday JS, Swadling L, Madden RG, Bendall R, Hunter JG, Maggs J, Simmonds P, Smith DB, Vine L et al. 2016. Characterization of the Specificity, Functionality, and Durability of Host T-Cell Responses Against the Full-Length Hepatitis E Virus. Hepatology, 64 (6), pp. 1934-1950. | Show Abstract | Read more

The interplay between host antiviral immunity and immunopathology during hepatitis E virus (HEV) infection determines important clinical outcomes. We characterized the specificity, functionality, and durability of host T-cell responses against the full-length HEV virus and assessed a novel "Quantiferon" assay for the rapid diagnosis of HEV infection. Eighty-nine volunteers were recruited from Oxford, Truro (UK), and Toulouse (France), including 44 immune-competent patients with acute HEV infection, 18 HEV-exposed immunosuppressed organ-transplant recipients (8 with chronic HEV), and 27 healthy volunteers. A genotype 3a peptide library (616 overlapping peptides spanning open reading frames [ORFs] 1-3) was used in interferon-gamma (IFN-γ) T-cell ELISpot assays. CD4(+) /CD8(+) T-cell subsets and polyfunctionality were defined using ICCS and SPICE analysis. Quantification of IFN-γ used whole-blood stimulation with recombinant HEV-capsid protein in the QuantiFERON kit. HEV-specific T-cell responses were detected in 41/44 immune-competent HEV exposed volunteers (median magnitude: 397 spot-forming units/10(6) peripheral blood mononuclear cells), most frequently targeting ORF2. High-magnitude, polyfunctional CD4 and CD8(+) T cells were detected during acute disease and maintained to 12 years, but these declined over time, with CD8(+) responses becoming more monofunctional. Low-level responses were detectable in immunosuppressed patients. Twenty-three novel HEV CD4(+) and CD8(+) T-cell targets were mapped predominantly to conserved genomic regions. QuantiFERON testing demonstrated an inverse correlation between IFN-γ production and the time from clinical presentation, providing 100% specificity, and 71% sensitivity (area under the receiver operator characteristic curve of 0.86) for HEV exposure at 0.3 IU/mL. CONCLUSION: Robust HEV-specific T-cell responses generated during acute disease predominantly target ORF2, but decline in magnitude and polyfunctionality over time. Defining HEV T-cell targets will be important for the investigation of HEV-associated autoimmune disease. (Hepatology 2016;64:1934-1950).

Harvala H, Simmonds P. 2016. Viral meningitis: epidemiology and diagnosis. Lancet Infect Dis, 16 (11), pp. 1211-1212. | Read more

Salmona M, Caporossi A, Simmonds P, Thélu MA, Fusillier K, Mercier-Delarue S, De Castro N, LeGoff J, Chaix ML, François O et al. 2016. First next-generation sequencing full-genome characterization of a hepatitis C virus genotype 7 divergent subtype. Clin Microbiol Infect, 22 (11), pp. 947.e1-947.e8. | Show Abstract | Read more

We report the near-full-length genome sequence of a hepatitis C virus (HCV) isolate from a man originating from Democratic Republic of Congo, the genotype of which could not be determined by the routinely used sequencing technique. The near-complete genome sequence of this variant BAK1 was obtained by the association of two next-generation sequencing technologies. Evolutionary analysis indicates that this isolate, BAK1, could be the first reported strain belonging to a new HCV-7b subtype. This new subtype has been incorrectly identified as genotype 2 by the Versant HCV Genotype 2.0 assay (LiPA). The requirement of three independent isolates has been filled, and a new subtype can be assigned. More examples of HCV-7 are required to better understand its origin, its pathogenicity and its relationship with genotype 2.

Magri A, Ozerov AA, Tunitskaya VL, Valuev-Elliston VT, Wahid A, Pirisi M, Simmonds P, Ivanov AV, Novikov MS, Patel AH. 2016. Exploration of acetanilide derivatives of 1-(ω-phenoxyalkyl)uracils as novel inhibitors of Hepatitis C Virus replication. Sci Rep, 6 (1), pp. 29487. | Show Abstract | Read more

Hepatitis C Virus (HCV) is a major public health problem worldwide. While highly efficacious directly-acting antiviral agents have been developed in recent years, their high costs and relative inaccessibility make their use limited. Here, we describe new 1-(ω-phenoxyalkyl)uracils bearing acetanilide fragment in 3 position of pyrimidine ring as potential antiviral drugs against HCV. Using a combination of various biochemical assays and in vitro virus infection and replication models, we show that our compounds are able to significantly reduce viral genomic replication, independently of virus genotype, with their IC50 values in the nanomolar range. We also demonstrate that our compounds can block de novo RNA synthesis and that effect is dependent on a chemical structure of the compounds. A detailed structure-activity relationship revealed that the most active compounds were the N(3)-substituted uracil derivatives containing 6-(4-bromophenoxy)hexyl or 8-(4-bromophenoxy)octyl fragment at N(1) position.

Smith DB, Paddy JO, Simmonds P. 2016. The use of human sewage screening for community surveillance of hepatitis E virus in the UK. J Med Virol, 88 (5), pp. 915-918. | Show Abstract | Read more

Hepatitis E virus sequences were detected by RT-PCR in 14/15 (93%) of untreated sewage samples from Edinburgh, Scotland, UK. Phylogenetic analysis of amplicons at limiting dilution revealed the co-circulation of multiple variants of HEV-3, with a pattern of diversity matching that observed in a local cohort of HEV-infected hepatitis patients.

Witteveldt J, Martin-Gans M, Simmonds P. 2016. Enhancement of the Replication of Hepatitis C Virus Replicons of Genotypes 1 to 4 by Manipulation of CpG and UpA Dinucleotide Frequencies and Use of Cell Lines Expressing SECL14L2 for Antiviral Resistance Testing. Antimicrob Agents Chemother, 60 (5), pp. 2981-2992. | Show Abstract | Read more

Treatment for hepatitis C virus (HCV) has improved greatly through the use of direct-acting antivirals (DAAs). However, their effectiveness and potential for drug resistance development in non-genotype 1 variants of HCV remain relatively unexplored, as in vitro assays to assess drug susceptibility are poorly developed and unsuited for a transient-transfection format. In the current study, we have evaluated the effects of dinucleotide frequency changes in the replicon and the use of a SEC14L2-expressing cell line on the replication of HCVs of different genotypes and evaluated the resulting assay formats for measurements of susceptibility to the DAA sofosbuvir. Removal of CpG and UpA dinucleotides from the luciferase gene used in HCV replicons of genotype 1b (Con1) and genotype 2a (JFH-1) achieved between 10- and 100-fold enhancement of replication over that of the wild type posttransfection. Removal of CpG and UpA dinucleotides in the neomycin gene or deletion of the whole gene in replicons of genotype 3a (S52) and genotype 4a (ED43) enhanced replication, but phenotypic effects on altering luciferase gene composition were minimal. A further 10-fold replication enhancement of replicons from all four genotypes was achieved by using a transgenic Huh7.5 cell line expressing SECL14L2, whose expression showed a dose-dependent effect on HCV replication that was reversible by small interfering RNA (siRNA) knockdown of gene expression. By combining these strategies, the 100- to 1,000-fold enhancement of replication allowed the susceptibility of all four genotypes to the RNA polymerase inhibitor sofosbuvir to be robustly determined in a transient-transfection assay format. These methods of replication enhancement provide new tools for monitoring the susceptibility and resistance of a wide range of HCV genotypes to DAAs.

Bonsall D, Gregory WF, Ip CL, Donfield S, Iles J, Ansari MA, Piazza P, Trebes A, Brown A, Frater J et al. 2016. Evaluation of Viremia Frequencies of a Novel Human Pegivirus by Using Bioinformatic Screening and PCR. Emerg Infect Dis, 22 (4), pp. 671-678. | Show Abstract | Read more

Next-generation sequencing has critical applications in virus discovery, diagnostics, and environmental surveillance. We used metagenomic sequence libraries for retrospective screening of plasma samples for the recently discovered human hepegivirus 1 (HHpgV-1). From a cohort of 150 hepatitis C virus (HCV)-positive case-patients, we identified 2 persons with HHpgV-1 viremia and a high frequency of human pegivirus (HPgV) viremia (14%). Detection of HHpgV-1 and HPgV was concordant with parallel PCR-based screening using conserved primers matching groups 1 (HPgV) and 2 (HHPgV-1) nonstructural 3 region sequences. PCR identified 1 HHPgV-1-positive person with viremia from a group of 195 persons with hemophilia who had been exposed to nonvirally inactivated factor VII/IX; 18 (9%) were HPgV-positive. Relative to HCV and HPgV, active infections with HHpgV-1 were infrequently detected in blood, even in groups that had substantial parenteral exposure. Our findings are consistent with lower transmissibility or higher rates of virus clearance for HHpgV-1 than for other bloodborne human flaviviruses.

Kokki I, Smith D, Simmonds P, Ramalingam S, Wellington L, Willocks L, Johannessen I, Harvala H. 2016. Hepatitis E virus is the leading cause of acute viral hepatitis in Lothian, Scotland. New Microbes New Infect, 10 pp. 6-12. | Show Abstract | Read more

Acute viral hepatitis affects all ages worldwide. Hepatitis E virus (HEV) is increasingly recognized as a major cause of acute hepatitis in Europe. Because knowledge of its characteristics is limited, we conducted a retrospective study to outline demographic and clinical features of acute HEV in comparison to hepatitis A, B and C in Lothian over 28 months (January 2012 to April 2014). A total of 3204 blood samples from patients with suspected acute hepatitis were screened for hepatitis A, B and C virus; 913 of these samples were also screened for HEV. Demographic and clinical information on patients with positive samples was gathered from electronic patient records. Confirmed HEV samples were genotyped. Of 82 patients with confirmed viral hepatitis, 48 (59%) had acute HEV. These patients were older than those infected by hepatitis A, B or C viruses, were more often male and typically presented with jaundice, nausea, vomiting and/or malaise. Most HEV cases (70%) had eaten pork or game meat in the few months before infection, and 14 HEV patients (29%) had a recent history of foreign travel. The majority of samples were HEV genotype 3 (27/30, 90%); three were genotype 1. Acute HEV infection is currently the predominant cause of acute viral hepatitis in Lothian and presents clinically in older men. Most of these infections are autochthonous, and further studies confirming the sources of infection (i.e. food or blood transfusion) are required.

Smith DB, Simmonds P, Izopet J, Oliveira-Filho EF, Ulrich RG, Johne R, Koenig M, Jameel S, Harrison TJ, Meng XJ et al. 2016. Proposed reference sequences for hepatitis E virus subtypes. J Gen Virol, 97 (3), pp. 537-542. | Show Abstract | Read more

The nomenclature of hepatitis E virus (HEV) subtypes is inconsistent and makes comparison of different studies problematic. We have provided a table of proposed complete genome reference sequences for each subtype. The criteria for subtype assignment vary between different genotypes and methodologies, and so a conservative pragmatic approach has been favoured. Updates to this table will be posted on the International Committee on Taxonomy of Viruses website ( The use of common reference sequences will facilitate communication between researchers and help clarify the epidemiology of this important human pathogen. This subtyping procedure might be adopted for other taxa of the genus Orthohepevirus.

Gaunt E, Wise HM, Zhang H, Lee LN, Atkinson NJ, Nicol MQ, Highton AJ, Klenerman P, Beard PM, Dutia BM et al. 2016. Elevation of CpG frequencies in influenza A genome attenuates pathogenicity but enhances host response to infection. Elife, 5 (FEBRUARY2016), pp. e12735. | Show Abstract | Read more

Previously, we demonstrated that frequencies of CpG and UpA dinucleotides profoundly influence the replication ability of echovirus 7 (Tulloch et al., 2014). Here, we show that that influenza A virus (IAV) with maximised frequencies of these dinucleotides in segment 5 showed comparable attenuation in cell culture compared to unmodified virus and a permuted control (CDLR). Attenuation was also manifested in vivo, with 10-100 fold reduced viral loads in lungs of mice infected with 200PFU of CpG-high and UpA-high mutants. However, both induced powerful inflammatory cytokine and adaptive (T cell and neutralising antibody) responses disproportionate to their replication. CpG-high infected mice also showed markedly reduced clinical severity, minimal weight loss and reduced immmunopathology in lung, yet sterilising immunity to lethal dose WT challenge was achieved after low dose (20PFU) pre-immunisation with this mutant. Increasing CpG dinucleotide frequencies represents a generic and potentially highly effective method for generating safe, highly immunoreactive vaccines.

Smith DB, Gaunt ER, Digard P, Templeton K, Simmonds P. 2016. Detection of influenza C virus but not influenza D virus in Scottish respiratory samples. J Clin Virol, 74 pp. 50-53. | Show Abstract | Read more

BACKGROUND: A newly proposed genus of influenza virus (influenza D) is associated with respiratory disease in pigs and cattle. The novel virus is most closely related to human influenza C virus and can infect ferrets but infection has not been reported in humans. OBJECTIVES: To ascertain if influenza D virus can be detected retrospectively in patient respiratory samples. STUDY DESIGN: 3300 human respiratory samples from Edinburgh, Scotland, covering the period 2006-2008, were screened in pools of 10 by RT-PCR using primers capable of detecting both influenza C and D viruses. RESULTS: Influenza D was not detected in any sample. Influenza C was present in 6 samples (0.2%), compared with frequencies of 3.3% and 0.9% for influenza A and B viruses from RT-PCR testing of respiratory samples over the same period. Influenza C virus was detected in samples from individuals <2 years or >45 years old, with cases occurring throughout the year. Phylogenetic analysis of nearly complete sequences of all seven segments revealed the presence of multiple, reassortant lineages. CONCLUSION: We were unable to detect viruses related to influenza D virus in human respiratory samples. Influenza C virus was less prevalent than influenza A and B viruses, was associated with mild disease in the young (<2 years) and old (>45 years) and comprised multiple, reassortant lineages. Inclusion of influenza C virus as part of a diagnostic testing panel for respiratory infections would be of limited additional value.

Lu L, Van Dung N, Bryant JE, Carrique-Mas J, Van Cuong N, Anh PH, Rabaa MA, Baker S, Simmonds P, Woolhouse ME. 2016. Evolution and phylogeographic dissemination of endemic porcine picornaviruses in Vietnam. Virus Evol, 2 (1), pp. vew001. | Show Abstract | Read more

Members of the Picornaviridae are important and often zoonotic viruses responsible for a variety of human and animal diseases. However, the evolution and spatial dissemination of different picornaviruses circulating in domestic animals are not well studied. We examined the rate of evolution and time of origin of porcine enterovirus G (EV-G) and porcine kobuvirus species C lineages (PKV-C) circulating in pig farms in Vietnam and from other countries. We further explored the spatiotemporal spread of EV-G and PKV-C in Southwest Vietnam using phylogeographic models. Multiple types of EV-G are co-circulating in Vietnam. The two dominant EV-G types among isolates from Vietnam (G1 and G6) showed strong phylogenetic clustering. Three clades of PKV-C (PKV-C1-3) represent more recent introductions into Vietnam; PKV-C2 is closely related to PKV-C from Southwest China, indicating possible cross-border dissemination. In addition, high virus lineage migration rates were estimated within four districts in Dong Thap province in Vietnam for both EV-G types (G1, G6) and all PKV-C (C1-3) clades. We found that Chau Thanh district is a primary source of both EV-G and PKV-C clades, consistent with extensive pig trading in and out of the district. Understanding the evolution and spatial dissemination of endemic picornaviruses in pigs may inform future strategies for the surveillance and control of picornaviruses.

van Wilgenburg B, Scherwitzl I, Hutchinson EC, Leng T, Kurioka A, Kulicke C, de Lara C, Cole S, Vasanawathana S, Limpitikul W et al. 2016. MAIT cells are activated during human viral infections. Nat Commun, 7 pp. 11653. | Show Abstract | Read more

Mucosal-associated invariant T (MAIT) cells are abundant in humans and recognize bacterial ligands. Here, we demonstrate that MAIT cells are also activated during human viral infections in vivo. MAIT cells activation was observed during infection with dengue virus, hepatitis C virus and influenza virus. This activation-driving cytokine release and Granzyme B upregulation-is TCR-independent but dependent on IL-18 in synergy with IL-12, IL-15 and/or interferon-α/β. IL-18 levels and MAIT cell activation correlate with disease severity in acute dengue infection. Furthermore, HCV treatment with interferon-α leads to specific MAIT cell activation in vivo in parallel with an enhanced therapeutic response. Moreover, TCR-independent activation of MAIT cells leads to a reduction of HCV replication in vitro mediated by IFN-γ. Together these data demonstrate MAIT cells are activated following viral infections, and suggest a potential role in both host defence and immunopathology.

Smith DB, Simmonds P. 2015. Hepatitis E virus and fulminant hepatitis--a virus or host-specific pathology? Liver Int, 35 (4), pp. 1334-1340. | Show Abstract | Read more

BACKGROUND & AIMS: Fulminant hepatitis is a rare outcome of infection with hepatitis E virus. Several recent reports suggest that virus variation is an important determinant of disease progression. To critically examine the evidence that virus-specific factors underlie the development of fulminant hepatitis following hepatitis E virus infection. METHODS: Published sequence information of hepatitis E virus isolates from patients with and without fulminant hepatitis was collected and analysed using statistical tests to identify associations between virus polymorphisms and disease outcome. RESULTS: Fulminant hepatitis has been reported following infection with all four hepatitis E virus genotypes that infect humans comprising multiple phylogenetic lineages within genotypes 1, 3 and 4. Analysis of virus sequences from individuals infected by a common source did not detect any common substitutions associated with progression to fulminant hepatitis. Re-analysis of previously reported associations between virus substitutions and fulminant hepatitis suggests that these were probably the result of sampling biases. CONCLUSIONS: Host-specific factors rather than virus genotype, variants or specific substitutions appear to be responsible for the development of fulminant hepatitis.

Oude Munnink BB, Phan MVT, Kellam P, Cotten M. 2016. Complete Genome Characterization of Two Wild-Type Measles Viruses from Vietnamese Infants during the 2014 Outbreak Genome Announcements, 4 (2), pp. e00250-16-e00250-16. | Show Abstract | Read more

© 2016 Oude Munnink et al. A large measles virus outbreak occurred across Vietnam in 2014. We identified and obtained complete measles virus genomes in stool samples collected from two diarrheal pediatric patients in Dong Thap Province. These are the first complete genome sequences of circulating measles viruses in Vietnam during the 2014 measles outbreak.

Oude Munnink BB, Phan MVT, van der Hoek L, Kellam P, Cotten M. 2016. Genome Sequences of a Novel Vietnamese Bat Bunyavirus Genome Announcements, 4 (6), pp. e01366-16-e01366-16. | Show Abstract | Read more

© 2016 Oude Munnink et al. To document the viral zoonotic risks in Vietnam, fecal samples were systematically collected from a number of mammals in southern Vietnam and subjected to agnostic deep sequencing. We describe here novel Vietnamese bunyavirus sequences detected in bat feces. The complete L and S segments from 14 viruses were determined.

Van Dung N, Anh PH, Van Cuong N, Hoa NT, Carrique-Mas J, Hien VB, Sharp C, Rabaa M, Berto A, Campbell J et al. 2016. Large-scale screening and characterization of enteroviruses and kobuviruses infecting pigs in Vietnam. J Gen Virol, 97 (2), pp. 378-388. | Show Abstract | Read more

A recent survey of pigs in Dong Thap province, Vietnam identified a high frequency of enterovirus species G (EV-G) infection (144/198; 72.7%). Amongst these was a plethora of EV-G types (EV-G1, EV-G6 and four new types EV-G8-EV-G11). To better characterize the genetic diversity of EV-G and investigate the possible existence of further circulating types, we performed a larger-scale study on 484 pig and 45 farm-bred boar faecal samples collected in 2012 and 2014, respectively. All samples from the previous and current studies were also screened for kobuviruses. The overall EV infection frequency remained extremely high (395/484; 81.6%), but with comparable detection rates and viral loads between healthy and diarrhoeic pigs; this contrasted with less frequent detection of EV-G in boars (4/45; 8.9%). EV was most frequently detected in pigs ≤ 14 weeks old (∼ 95%) and declined in older pigs. Infections with EV-G1 and EV-G6 were most frequent, whilst less commonly detected types included EV-G3, EV-G4 and EV-G8-EV-G11, and five new types (EV-G12-EV-G16). In contrast, kobuvirus infection frequency was significantly higher in diarrhoeic pigs (40.9 versus 27.6%; P = 0.01). Kobuviruses also showed contrasting epizootiologies and age associations; a higher prevalence was found in boars (42%) compared with domestic pigs (29%), with the highest infection frequency amongst pigs >52 weeks old. Although genetically diverse, all kobuviruses identified belonged to the species Aichivirus C. In summary, this study confirms infection with EV-G was endemic in Vietnamese domestic pigs and exhibits high genetic diversity and extensive inter-type recombination.

Toppinen M, Perdomo MF, Palo JU, Simmonds P, Lycett SJ, Söderlund-Venermo M, Sajantila A, Hedman K. 2015. Bones hold the key to DNA virus history and epidemiology. Sci Rep, 5 (1), pp. 17226. | Show Abstract | Read more

DNA in human skeletal remains represents an important historical source of host genomic information and potentially of infecting viruses. However, little is known about viral persistence in bone. We searched ca. 70-year-old long bones of putative Finnish casualties from World War II for parvovirus B19 (B19V) DNA, and found a remarkable prevalence of 45%. The viral sequences were exclusively of genotypes 2 (n = 41), which disappeared from circulation in 1970´s, or genotype 3 (n = 2), which has never been reported in Northern Europe. Based on mitochondrial and Y-chromosome profiling, the two individuals carrying B19V genotype 3 were likely from the Soviet Red Army. The most recent common ancestor for all genotypes was estimated at early 1800s. This work demonstrates the forms of B19V that circulated in the first half of the 20(th) century and provides the first evidence of the suitability of bone for exploration of DNA viruses.

Rabaa MA, Tue NT, Phuc TM, Carrique-Mas J, Saylors K, Cotten M, Bryant JE, Nghia HD, Cuong NV, Pham HA et al. 2015. The Vietnam Initiative on Zoonotic Infections (VIZIONS): A Strategic Approach to Studying Emerging Zoonotic Infectious Diseases. Ecohealth, 12 (4), pp. 726-735. | Show Abstract | Read more

The effect of newly emerging or re-emerging infectious diseases of zoonotic origin in human populations can be potentially catastrophic, and large-scale investigations of such diseases are highly challenging. The monitoring of emergence events is subject to ascertainment bias, whether at the level of species discovery, emerging disease events, or disease outbreaks in human populations. Disease surveillance is generally performed post hoc, driven by a response to recent events and by the availability of detection and identification technologies. Additionally, the inventory of pathogens that exist in mammalian and other reservoirs is incomplete, and identifying those with the potential to cause disease in humans is rarely possible in advance. A major step in understanding the burden and diversity of zoonotic infections, the local behavioral and demographic risks of infection, and the risk of emergence of these pathogens in human populations is to establish surveillance networks in populations that maintain regular contact with diverse animal populations, and to simultaneously characterize pathogen diversity in human and animal populations. Vietnam has been an epicenter of disease emergence over the last decade, and practices at the human/animal interface may facilitate the likelihood of spillover of zoonotic pathogens into humans. To tackle the scientific issues surrounding the origins and emergence of zoonotic infections in Vietnam, we have established The Vietnam Initiative on Zoonotic Infections (VIZIONS). This countrywide project, in which several international institutions collaborate with Vietnamese organizations, is combining clinical data, epidemiology, high-throughput sequencing, and social sciences to address relevant one-health questions. Here, we describe the primary aims of the project, the infrastructure established to address our scientific questions, and the current status of the project. Our principal objective is to develop an integrated approach to the surveillance of pathogens circulating in both human and animal populations and assess how frequently they are exchanged. This infrastructure will facilitate systematic investigations of pathogen ecology and evolution, enhance understanding of viral cross-species transmission events, and identify relevant risk factors and drivers of zoonotic disease emergence.

Hoehn KB, Gall A, Bashford-Rogers R, Fidler SJ, Kaye S, Weber JN, McClure MO, SPARTAC Trial Investigators, Kellam P, Pybus OG. 2015. Dynamics of immunoglobulin sequence diversity in HIV-1 infected individuals. Philos Trans R Soc Lond B Biol Sci, 370 (1676), pp. 20140241-20140241. | Show Abstract | Read more

Advances in immunoglobulin (Ig) sequencing technology are leading to new perspectives on immune system dynamics. Much research in this nascent field has focused on resolving immune responses to viral infection. However, the dynamics of B-cell diversity in early HIV infection, and in response to anti-retroviral therapy, are still poorly understood. Here, we investigate these dynamics through bulk Ig sequencing of samples collected over 2 years from a group of eight HIV-1 infected patients, five of whom received anti-retroviral therapy during the first half of the study period. We applied previously published methods for visualizing and quantifying B-cell sequence diversity, including the Gini index, and compared their efficacy to alternative measures. While we found significantly greater clonal structure in HIV-infected patients versus healthy controls, within HIV patients, we observed no significant relationships between statistics of B-cell clonal expansion and clinical variables such as viral load and CD4(+) count. Although there are many potential explanations for this, we suggest that important factors include poor sampling resolution and complex B-cell dynamics that are difficult to summarize using simple summary statistics. Importantly, we find a significant association between observed Gini indices and sequencing read depth, and we conclude that more robust analytical methods and a closer integration of experimental and theoretical work is needed to further our understanding of B-cell repertoire diversity during viral infection.

Toppinen M, Perdomo MF, Palo J, Simmonds P, Lycett S, Soderlund-Venermo M, Sajantila A, Hedman K. 2015. Bones hold the key to virus history and epidemiology JOURNAL OF CLINICAL VIROLOGY, 70 pp. S81-S81. | Read more

Smith D, Harvala H, Bradley-Stewart A, Gunson R, Simmonds P. 2015. Pathogenicity of hepatitis E virus genotype 3 variaints JOURNAL OF CLINICAL VIROLOGY, 70 pp. S123-S123. | Read more

Smith D, Templeton K, Simmonds P. 2015. The Edinburgh microbiology archive JOURNAL OF CLINICAL VIROLOGY, 70 pp. S14-S14. | Read more

Smith D, Simmonds P. 2015. Development of a real time PCR for poliovirus in sewage JOURNAL OF CLINICAL VIROLOGY, 70 pp. S71-S71. | Read more

Simmonds P, Tulloch F, Evans DJ, Ryan MD. 2015. Attenuation of dengue (and other RNA viruses) with codon pair recoding can be explained by increased CpG/UpA dinucleotide frequencies. Proc Natl Acad Sci U S A, 112 (28), pp. E3633-E3634. | Read more

Adams MJ, Lefkowitz EJ, King AM, Bamford DH, Breitbart M, Davison AJ, Ghabrial SA, Gorbalenya AE, Knowles NJ, Krell P et al. 2015. Ratification vote on taxonomic proposals to the International Committee on Taxonomy of Viruses (2015). Arch Virol, 160 (7), pp. 1837-1850. | Show Abstract | Read more

Changes to virus taxonomy approved and ratified by the International Committee on Taxonomy of Viruses in February 2015 are listed.

Simmonds P. 2015. Methods for virus classification and the challenge of incorporating metagenomic sequence data. J Gen Virol, 96 (Pt 6), pp. 1193-1206. | Show Abstract | Read more

The division of viruses into orders, families, genera and species provides a classification framework that seeks to organize and make sense of the diversity of viruses infecting animals, plants and bacteria. Classifications are based on similarities in genome structure and organization, the presence of homologous genes and sequence motifs and at lower levels such as species, host range, nucleotide and antigenic relatedness and epidemiology. Classification below the level of family must also be consistent with phylogeny and virus evolutionary histories. Recently developed methods such as PASC, DEMaRC and NVR offer alternative strategies for genus and species assignments that are based purely on degrees of divergence between genome sequences. They offer the possibility of automating classification of the vast number of novel virus sequences being generated by next-generation metagenomic sequencing. However, distance-based methods struggle to deal with the complex evolutionary history of virus genomes that are shuffled by recombination and reassortment, and where taxonomic lineages evolve at different rates. In biological terms, classifications based on sequence distances alone are also arbitrary whereas the current system of virus taxonomy is of utility precisely because it is primarily based upon phenotypic characteristics. However, a separate system is clearly needed by which virus variants that lack biological information might be incorporated into the ICTV classification even if based solely on sequence relationships to existing taxa. For these, simplified taxonomic proposals and naming conventions represent a practical way to expand the existing virus classification and catalogue our rapidly increasing knowledge of virus diversity.

Boros Á, Pankovics P, Simmonds P, Pollák E, Mátics R, Phan TG, Delwart E, Reuter G. 2015. Genome analysis of a novel, highly divergent picornavirus from common kestrel (Falco tinnunculus): the first non-enteroviral picornavirus with type-I-like IRES. Infect Genet Evol, 32 pp. 425-431. | Show Abstract | Read more

Although the number of identified avian-borne picornaviruses (family Picornaviridae) is continuously increasing there remains several species-rich avian host groups, such as the order Falconiformes (with 290 bird species) from which picornaviruses have not been identified. This study reports the first complete genome of a novel, highly divergent picornavirus, named as Falcovirus A1 (KP230449), from the carnivorous bird, the common kestrel (Falco tinnunculus, order Falconiformes). Falcovirus A1 has the longest 3D(RdRp) genome region and distant phylogenetic relationship to the Hepatitis A virus 1 (Hepatovirus) and Avian encephalomyelitis virus 1 (Tremovirus). It has a type-I (enterovirus-like) IRES in the 5'UTR - identified for the first time among avian-borne picornaviruses suggesting that type-I IRES is not restricted only to enteroviruses and providing further evidence of mosaicism of this region among different picornavirus genera.

Scheel TK, Simmonds P, Kapoor A. 2015. Surveying the global virome: identification and characterization of HCV-related animal hepaciviruses. Antiviral Res, 115 pp. 83-93. | Show Abstract | Read more

Recent advances in sequencing technologies have greatly enhanced our abilities to identify novel microbial sequences. Thus, our understanding of the global virome and the virome of specific host species in particular is rapidly expanding. Identification of animal viruses is important for understanding animal disease, the origin and evolution of human viruses, as well as zoonotic reservoirs for emerging infections. Although the human hepacivirus, hepatitis C virus (HCV), was identified 25years ago, its origin has remained elusive. In 2011, the first HCV homolog was reported in dogs but subsequent studies showed the virus to be widely distributed in horses. This indicated a wider hepacivirus host range and paved the way for identification of rodent, bat and non-human primate hepaciviruses. The equine non-primate hepacivirus (NPHV) remains the closest relative of HCV and is so far the best characterized. Identification and characterization of novel hepaciviruses may in addition lead to development of tractable animal models to study HCV persistence, immune responses and pathogenesis. This could be particular important, given the current shortage of immunocompetent models for robust HCV infection. Much remains to be learned on the novel hepaciviruses, including their association with disease, and thereby how relevant they will become as HCV model systems and for studies of animal disease. This review discusses how virome analysis led to identification of novel hepaci- and pegiviruses, their genetic relationship and characterization and the potential use of animal hepaciviruses as models to study hepaciviral infection, immunity and pathogenesis. This article forms part of a symposium in Antiviral Research on "Hepatitis C: Next steps toward global eradication."

Gaunt E, Harvala H, Österback R, Sreenu VB, Thomson E, Waris M, Simmonds P. 2015. Genetic characterization of human coxsackievirus A6 variants associated with atypical hand, foot and mouth disease: a potential role of recombination in emergence and pathogenicity. J Gen Virol, 96 (Pt 5), pp. 1067-1079. | Show Abstract | Read more

Human coxsackievirus A6 (CVA6) is an enterically transmitted enterovirus. Until recently, CVA6 infections were considered as being of minor clinical significance, and only rarely aetiologically linked with hand, foot and mouth disease (HFMD) associated with other species A enteroviruses (particularly EV71 and CVA16). From 2008 onwards, however, CVA6 infections have been associated with several outbreaks worldwide of atypical HFMD (aHFMD) accompanied by a varicelliform rash. We recently reported CVA6-associated eczema herpeticum occurring predominantly in children and young adults in Edinburgh in January and February 2014. To investigate genetic determinants of novel clinical phenotypes of CVA6, we genetically characterized and analysed CVA6 variants associated with eczema herpeticum in Edinburgh in 2014 and those with aHFMD in CAV isolates collected from 2008. A total of eight recombinant forms (RFs) have circulated worldwide over the past 10 years, with the particularly recent appearance of RF-H associated with eczema herpeticum cases in Edinburgh in 2014. Comparison of phylogenies and divergence of complete genome sequences of CVA6 identified recombination breakpoints in 2A-2C, within VP3, and between 5' untranslated region and VP1. A Bayesian temporal reconstruction of CVA6 evolution since 2004 provided estimates of dates and the actual recombination events that generated more recently appearing recombination groups (RF-E, -F, -G and -H). Associations were observed between recombination groups and clinical presentations of herpangina, aHFMD and eczema herpeticum, but not with VP1 or other structural genes. These observations provided evidence that NS gene regions may potentially contribute to clinical phenotypes and outcomes of CVA6 infection.

Shah A, Connelly M, Whitaker P, McIntyre C, Etherington C, Denton M, Hale A, Harvala H, Simmonds P, Peckham DG. 2015. Pathogenicity of individual rhinovirus species during exacerbations of cystic fibrosis. Eur Respir J, 45 (6), pp. 1748-1751. | Read more

Simmonds P. 2015. Methods for virus classification and the challenge of incorporating metagenomic sequence data JOURNAL OF GENERAL VIROLOGY, 96 pp. 1193-1206. | Show Abstract | Read more

© 2015 The Author. The division of viruses into orders, families, genera and species provides a classification framework that seeks to organize and make sense of the diversity of viruses infecting animals, plants and bacteria. Classifications are based on similarities in genome structure and organization, the presence of homologous genes and sequence motifs and at lower levels such as species, host range, nucleotide and antigenic relatedness and epidemiology. Classification below the level of family must also be consistent with phylogeny and virus evolutionary histories. Recently developed methods such as PASC, DEMaRC and NVR offer alternative strategies for genus and species assignments that are based purely on degrees of divergence between genome sequences. They offer the possibility of automating classification of the vast number of novel virus sequences being generated by next-generation metagenomic sequencing. However, distance-based methods struggle to deal with the complex evolutionary history of virus genomes that are shuffled by recombination and reassortment, and where taxonomic lineages evolve at different rates. In biological terms, classifications based on sequence distances alone are also arbitrary whereas the current system of virus taxonomy is of utility precisely because it is primarily based upon phenotypic characteristics. However, a separate system is clearly needed by which virus variants that lack biological information might be incorporated into the ICTV classification even if based solely on sequence relationships to existing taxa. For these, simplified taxonomic proposals and naming conventions represent a practical way to expand the existing virus classification and catalogue our rapidly increasing knowledge of virus diversity.

Kapoor A, Kumar A, Simmonds P, Bhuva N, Singh Chauhan L, Lee B, Sall AA, Jin Z, Morse SS, Shaz B et al. 2015. Virome Analysis of Transfusion Recipients Reveals a Novel Human Virus That Shares Genomic Features with Hepaciviruses and Pegiviruses. MBio, 6 (5), pp. e01466-e01415. | Show Abstract | Read more

UNLABELLED: To investigate the transmission of novel infectious agents by blood transfusion, we studied changes in the virome composition of blood transfusion recipients pre- and posttransfusion. Using this approach, we detected and genetically characterized a novel human virus, human hepegivirus 1 (HHpgV-1), that shares features with hepatitis C virus (HCV) and human pegivirus (HPgV; formerly called GB virus C or hepatitis G virus). HCV and HPgV belong to the genera Hepacivirus and Pegivirus of the family Flaviviridae. HHpgV-1 was found in serum samples from two blood transfusion recipients and two hemophilia patients who had received plasma-derived clotting factor concentrates. In the former, the virus was detected only in the posttransfusion samples, indicating blood-borne transmission. Both hemophiliacs were persistently viremic over periods of at least 201 and 1,981 days. The 5' untranslated region (UTR) of HHpgV-1 contained a type IV internal ribosome entry site (IRES), structurally similar to although highly divergent in sequence from that of HCV and other hepaciviruses. However, phylogenetic analysis of nonstructural genes (NS3 and NS5B) showed that HHpgV-1 forms a branch within the pegivirus clade distinct from HPgV and homologs infecting other mammalian species. In common with some pegivirus variants infecting rodents and bats, the HHpgV-1 genome encodes a short, highly basic protein upstream of E1, potentially possessing a core-like function in packaging RNA during assembly. Identification of this new human virus, HHpgV-1, expands our knowledge of the range of genome configurations of these viruses and may lead to a reevaluation of the original criteria by which the genera Hepacivirus and Pegivirus are defined. IMPORTANCE: More than 30 million blood components are transfused annually in the United States alone. Surveillance for infectious agents in the blood supply is key to ensuring the safety of this critical resource for medicine and public health. Here, we report the identification of a new and highly diverse HCV/GB virus (GBV)-like virus from human serum samples. This new virus, human hepegivirus 1 (HHpgV-1), was found in serum samples from blood transfusion recipients, indicating its potential for transmission via transfusion products. We also found persistent long-term HHpgV-1 viremia in two hemophilia patients. HHpgV-1 is unique because it shares genetic similarity with both highly pathogenic HCV and the apparently nonpathogenic HPgV (GBV-C). Our results add to the list of human viruses and provide data to develop reagents to study virus transmission and disease association and for interrupting virus transmission and new human infections.

Smith DB, Ijaz S, Tedder RS, Hogema B, Zaaijer HL, Izopet J, Bradley-Stewart A, Gunson R, Harvala H, Kokki I, Simmonds P. 2015. Variability and pathogenicity of hepatitis E virus genotype 3 variants. J Gen Virol, 96 (11), pp. 3255-3264. | Show Abstract | Read more

Infection with hepatitis E virus (HEV) can be clinically inapparent or produce symptoms and signs of hepatitis of varying severity and occasional fatality. This variability in clinical outcomes may reflect differences in host susceptibility or the presence of virally encoded determinants of pathogenicity. Analysis of complete genome sequences supports the division of HEV genotype 3 (HEV-3) variants into three major clades: 3ra comprising HEV isolates from rabbits, and 3efg and 3abchij comprising the corresponding named subtypes derived from humans and pigs. Using this framework, we investigated associations between viral genetic variability of HEV-3 in symptomatic and asymptomatic infections by comparing HEV-3 subgenomic sequences previously obtained from blood donors with those from patients presenting with hepatitis in the UK (54 blood donors, 148 hepatitis patients), the Netherlands (38 blood donors, 119 hepatitis patients), France (24 blood donors, 55 hepatitis patients) and Germany (14 blood donors, 36 hepatitis patients). In none of these countries was evidence found for a significant association between virus variants and patient group (P>0.05 Fisher's exact test). Furthermore, within a group of 123 patients in Scotland with clinically apparent HEV infections, we found no evidence for an association between variants of HEV-3 and disease severity or alanine aminotransferase level. The lack of detectable virally encoded determinants of disease outcomes in HEV-3 infection implies a more important role for host factors in its clinical phenotype.

Matthews PC, Sharp CP, Malik A, Gregory WF, Adland E, Jooste P, Goulder PJ, Simmonds P, Klenerman P. 2015. Human parvovirus 4 infection among mothers and children in South Africa. Emerg Infect Dis, 21 (4), pp. 713-715. | Read more

Firth C, Bhat M, Firth MA, Williams SH, Frye MJ, Simmonds P, Conte JM, Ng J, Garcia J, Bhuva NP et al. 2014. Detection of zoonotic pathogens and characterization of novel viruses carried by commensal Rattus norvegicus in New York City. MBio, 5 (5), pp. e01933-e01914. | Show Abstract | Read more

Norway rats (Rattus norvegicus) are globally distributed and concentrate in urban environments, where they live and feed in closer proximity to human populations than most other mammals. Despite the potential role of rats as reservoirs of zoonotic diseases, the microbial diversity present in urban rat populations remains unexplored. In this study, we used targeted molecular assays to detect known bacterial, viral, and protozoan human pathogens and unbiased high-throughput sequencing to identify novel viruses related to agents of human disease in commensal Norway rats in New York City. We found that these rats are infected with bacterial pathogens known to cause acute or mild gastroenteritis in people, including atypical enteropathogenic Escherichia coli, Clostridium difficile, and Salmonella enterica, as well as infectious agents that have been associated with undifferentiated febrile illnesses, including Bartonella spp., Streptobacillus moniliformis, Leptospira interrogans, and Seoul hantavirus. We also identified a wide range of known and novel viruses from groups that contain important human pathogens, including sapoviruses, cardioviruses, kobuviruses, parechoviruses, rotaviruses, and hepaciviruses. The two novel hepaciviruses discovered in this study replicate in the liver of Norway rats and may have utility in establishing a small animal model of human hepatitis C virus infection. The results of this study demonstrate the diversity of microbes carried by commensal rodent species and highlight the need for improved pathogen surveillance and disease monitoring in urban environments. Importance: The observation that most emerging infectious diseases of humans originate in animal reservoirs has led to wide-scale microbial surveillance and discovery programs in wildlife, particularly in the developing world. Strikingly, less attention has been focused on commensal animals like rats, despite their abundance in urban centers and close proximity to human populations. To begin to explore the zoonotic disease risk posed by urban rat populations, we trapped and surveyed Norway rats collected in New York City over a 1-year period. This analysis revealed a striking diversity of known pathogens and novel viruses in our study population, including multiple agents associated with acute gastroenteritis or febrile illnesses in people. Our findings indicate that urban rats are reservoirs for a vast diversity of microbes that may affect human health and indicate a need for increased surveillance and awareness of the disease risks associated with urban rodent infestation.

Iles JC, Raghwani J, Harrison GL, Pepin J, Djoko CF, Tamoufe U, LeBreton M, Schneider BS, Fair JN, Tshala FM et al. 2014. Phylogeography and epidemic history of hepatitis C virus genotype 4 in Africa. Virology, 464-465 (1), pp. 233-243. | Show Abstract | Read more

HCV genotype 4 is prevalent in many African countries, yet little is known about the genotype׳s epidemic history on the continent. We present a comprehensive study of the molecular epidemiology of genotype 4. To address the deficit of data from the Democratic Republic of the Congo (DRC) we PCR amplified 60 new HCV isolates from the DRC, resulting in 33 core- and 48 NS5B-region sequences. Our data, together with genotype 4 database sequences, were analysed using Bayesian phylogenetic approaches. We find three well-supported intra-genotypic lineages and estimate that the genotype 4 common ancestor existed around 1733 (1650-1805). We show that genotype 4 originated in central Africa and that multiple lineages have been exported to north Africa since ~1850, including subtype 4a which dominates the epidemic in Egypt. We speculate on the causes of the historical intra-continental spread of genotype 4, including population movements during World War 2.

Van Nguyen D, Harvala H, Ngole EM, Delaporte E, Woolhouse ME, Peeters M, Simmonds P. 2014. High rates of infection with novel enterovirus variants in wild populations of mandrills and other old world monkey species. J Virol, 88 (11), pp. 5967-5976. | Show Abstract | Read more

UNLABELLED: Enteroviruses (EVs) are a genetically and antigenically diverse group of viruses infecting humans. A mostly distinct set of EV variants have additionally been documented to infect wild apes and several, primarily captive, Old World monkey (OWM) species. To investigate the prevalence and genetic characteristics of EVs infecting OWMs in the wild, fecal samples from mandrills (Mandrillus sphinx) and other species collected in remote regions of southern Cameroon were screened for EV RNA. Remarkably high rates of EV positivity were detected in M. sphinx (100 of 102 screened), Cercocebus torquatus (7/7), and Cercopithecus cephus (2/4), with high viral loads indicative of active infection. Genetic characterization in VP4/VP2 and VP1 regions allowed EV variants to be assigned to simian species H (EV-H) and EV-J (including one or more new types), while seven matched simian EV-B variants, SA5 and EV110 (chimpanzee). Sequences from the remaining 70 formed a new genetic group distinct in VP4/2 and VP1 region from all currently recognized human or simian EV species. Complete genome sequences were obtained from three to determine their species assignment. In common with EV-J and the EV-A A13 isolate, new group sequences were chimeric, being most closely related to EV-A in capsid genes and to EV-B in the nonstructural gene region. Further recombination events created different groupings in 5' and 3' untranslated regions. While clearly a distinct EV group, the hybrid nature of new variants prevented their unambiguous classification as either members of a new species or as divergent members of EV-A using current International Committee on Taxonomy of Viruses (ICTV) assignment criteria. IMPORTANCE: This study is the first large-scale investigation of the frequency of infection and diversity of enteroviruses (EVs) infecting monkeys (primarily mandrills) in the wild. Our findings demonstrate extremely high frequencies of active infection (95%) among mandrills and other Old World monkey species inhabiting remote regions of Cameroon without human contact. EV variants detected were distinct from those infecting human populations, comprising members of enterovirus species B, J, and H and a large novel group of viruses most closely related to species A in the P1 region. The viral sequences obtained contribute substantially to our growing understanding of the genetic diversity of EVs and the existence of interspecies chimerism that characterizes the novel variants in the current study, as well as in previously characterized species A and J viruses infecting monkeys. The latter findings will contribute to future development of consensus criteria for species assignments in enteroviruses and other picornavirus genera.

Matthews PC, Malik A, Simmons R, Sharp C, Simmonds P, Klenerman P. 2014. PARV4: an emerging tetraparvovirus. PLoS Pathog, 10 (5), pp. e1004036. | Read more

Atkinson NJ, Witteveldt J, Evans DJ, Simmonds P. 2014. The influence of CpG and UpA dinucleotide frequencies on RNA virus replication and characterization of the innate cellular pathways underlying virus attenuation and enhanced replication. Nucleic Acids Res, 42 (7), pp. 4527-4545. | Show Abstract | Read more

Most RNA viruses infecting mammals and other vertebrates show profound suppression of CpG and UpA dinucleotide frequencies. To investigate this functionally, mutants of the picornavirus, echovirus 7 (E7), were constructed with altered CpG and UpA compositions in two 1.1-1.3 Kbase regions. Those with increased frequencies of CpG and UpA showed impaired replication kinetics and higher RNA/infectivity ratios compared with wild-type virus. Remarkably, mutants with CpGs and UpAs removed showed enhanced replication, larger plaques and rapidly outcompeted wild-type virus on co-infections. Luciferase-expressing E7 sub-genomic replicons with CpGs and UpAs removed from the reporter gene showed 100-fold greater luminescence. E7 and mutants were equivalently sensitive to exogenously added interferon-β, showed no evidence for differential recognition by ADAR1 or pattern recognition receptors RIG-I, MDA5 or PKR. However, kinase inhibitors roscovitine and C16 partially or entirely reversed the attenuated phenotype of high CpG and UpA mutants, potentially through inhibition of currently uncharacterized pattern recognition receptors that respond to RNA composition. Generating viruses with enhanced replication kinetics has applications in vaccine production and reporter gene construction. More fundamentally, the findings introduce a new evolutionary paradigm where dinucleotide composition of viral genomes is subjected to selection pressures independently of coding capacity and profoundly influences host-pathogen interactions.




Sinclair C, Gaunt E, Simmonds P, Broomfield D, Nwafor N, Wellington L, Templeton K, Willocks L, Schofield O, Harvala H. 2014. Atypical hand, foot, and mouth disease associated with coxsackievirus A6 infection, Edinburgh, United Kingdom, January to February 2014 EUROSURVEILLANCE, 19 (12), pp. 6-10.

Witteveldt J, Blundell R, Maarleveld JJ, McFadden N, Evans DJ, Simmonds P. 2014. The influence of viral RNA secondary structure on interactions with innate host cell defences. Nucleic Acids Res, 42 (5), pp. 3314-3329. | Show Abstract | Read more

RNA viruses infecting vertebrates differ fundamentally in their ability to establish persistent infections with markedly different patterns of transmission, disease mechanisms and evolutionary relationships with their hosts. Although interactions with host innate and adaptive responses are complex and persistence mechanisms likely multi-factorial, we previously observed associations between bioinformatically predicted RNA secondary formation in genomes of positive-stranded RNA viruses with their in vivo fitness and persistence. To analyse this interactions functionally, we transfected fibroblasts with non-replicating, non-translated RNA transcripts from RNA viral genomes with differing degrees of genome-scale ordered RNA structure (GORS). Single-stranded RNA transcripts induced interferon-β mediated though RIG-I and PKR activation, the latter associated with rapid induction of antiviral stress granules. A striking inverse correlation was observed between induction of both cellular responses with transcript RNA structure formation that was independent of both nucleotide composition and sequence length. The consistent inability of cells to recognize RNA transcripts possessing GORS extended to downstream differences from unstructured transcripts in expression of TNF-α, other interferon-stimulated genes and induction of apoptosis. This functional association provides novel insights into interactions between virus and host early after infection and provides evidence for a novel mechanism for evading intrinsic and innate immune responses.

Harvala H, Wong V, Simmonds P, Johannessen I, Ramalingam S. 2014. Acute viral hepatitis - should the current screening strategy be modified? J Clin Virol, 59 (3), pp. 184-187. | Show Abstract | Read more

BACKGROUND: The epidemiology of viral hepatitis has changed. Since the introduction of safe and effective vaccines for hepatitis A and B in 1980s, the incidence of acute infections caused by these viruses has been declining in the UK. At the same time, hepatitis E virus (HEV) has been recognised as an increasingly important cause of acute hepatitis, but testing is not widely available. OBJECTIVES: The aim of this study was to establish the viral causes of acute hepatitis, and use that data to modify the current diagnostic algorithm. STUDY DESIGN: A Cognos search was performed to collate subjects tested for HAV, HBV, HCV, HEV, EBV and CMV between June 2010 and December 2012. Information included virological result and their ALT level if done within 5 days from virological testing. RESULTS: From 3462 subjects with suspected acute viral hepatitis, only 25% had biochemical evidence of acute hepatitis (n=854; ALT>100IU/l). The frequency of detection of acute HEV infection (25/409) was over 31-times higher than that of HAV (6/3462), and 7-times higher than that of HBV (24/3462). Most cases of acute HAV, HEV, EBV and CMV infections presented with abnormal ALT levels. Most EBV infections were associated with lymphadenopathy (23/34); in comparison most of CMV infections were not associated with lymphadenopathy (18/22). CONCLUSIONS: HEV screening should be included in the initial testing panel for acute hepatitis and screening at least for HAV and HEV might be limited to those with abnormal ALT levels.

Harvala H, Van Nguyen D, McIntyre C, Ahuka-Mundeke S, Ngole EM, Delaporte E, Peeters M, Simmonds P. 2014. Co-circulation of enteroviruses between apes and humans. J Gen Virol, 95 (Pt 2), pp. 403-407. | Show Abstract | Read more

A total of 139 stool samples from wild chimpanzees, gorillas and bonobos in Cameroon and Democratic Republic of Congo (DRC) were screened for enteroviruses (EVs) by reverse transcription PCR. Enterovirus RNA was detected in 10 % of samples, comprising eight from 58 sampled chimpanzees (13.8 %), one from 40 bonobos (2.5 %) and five from 40 gorillas (12.2 %). Three viruses isolated from chimpanzees grouped with human isolate EV-A89 and four (four chimpanzees, one gorilla) represented a newly identified type, EV-A119. These species A virus types overlapped with those circulating in human populations in the same area. The remaining six strains comprised a new species D type, EV-D120, infecting one chimpanzee and four gorillas, and a single EV variant infecting a bonobo that was remarkably divergent from other EVs and potentially constitutes a new enterovirus species. The study demonstrates both the circulation of genetically divergent EV variants in apes and monkeys as well as those shared with local human populations.

Smith DB, Simmonds P, Bell JE. 2014. Brain viral burden, neuroinflammation and neurodegeneration in HAART-treated HIV positive injecting drug users. J Neurovirol, 20 (1), pp. 28-38. | Show Abstract | Read more

The long-term impact of chronic human immunodeficiency virus (HIV) infection on brain status in injecting drug users (IDU) treated with highly active antiretroviral therapy (HAART) is unknown. Viral persistence in the brain with ongoing neuroinflammation may predispose to Alzheimer-like neurodegeneration. In this study, we investigated the brains of ten HAART-treated individuals (six IDU and four non-DU), compared with ten HIV negative controls (six IDU and four non-DU). HIV DNA levels in brain tissue were correlated with plasma and lymphoid tissue viral loads, cognitive status, microglial activation and Tau protein and amyloid deposition. Brain HIV proviral DNA levels were low in most cases but higher in HIV encephalitis (n = 2) and correlated significantly with levels in lymphoid tissue (p = 0.0075), but not with those in plasma. HIV positive subjects expressed more Tau protein and amyloid than HIV negative controls (highest in a 58 year old), as did IDU, but brain viral loads showed no relation to Tau and amyloid. Microglial activation linked significantly to HIV positivity (p = 0.001) and opiate abuse accentuated these microglial changes (p = 0.05). This study confirms that HIV DNA persists in brains despite HAART and that opiate abuse adds to the risk of brain damage in HIV positive subjects. Novel findings in this study show that (1) plasma levels are not a good surrogate indicator of brain status, (2) viral burden in brain and lymphoid tissues is related, and (3) while Tau and amyloid deposition is increased in HIV positive IDU, this is not specifically related to increased HIV burden within the brain.

Smith DB, Bukh J, Kuiken C, Muerhoff AS, Rice CM, Stapleton JT, Simmonds P. 2014. Expanded classification of hepatitis C virus into 7 genotypes and 67 subtypes: updated criteria and genotype assignment web resource. Hepatology, 59 (1), pp. 318-327. | Show Abstract | Read more

UNLABELLED: The 2005 consensus proposal for the classification of hepatitis C virus (HCV) presented an agreed and uniform nomenclature for HCV variants and the criteria for their assignment into genotypes and subtypes. Since its publication, the available dataset of HCV sequences has vastly expanded through advancement in nucleotide sequencing technologies and an increasing focus on the role of HCV genetic variation in disease and treatment outcomes. The current study represents a major update to the previous consensus HCV classification, incorporating additional sequence information derived from over 1,300 (near-)complete genome sequences of HCV available on public databases in May 2013. Analysis resolved several nomenclature conflicts between genotype designations and using consensus criteria created a classification of HCV into seven confirmed genotypes and 67 subtypes. There are 21 additional complete coding region sequences of unassigned subtype. The study additionally describes the development of a Web resource hosted by the International Committee for Taxonomy of Viruses (ICTV) that maintains and regularly updates tables of reference isolates, accession numbers, and annotated alignments ( The Flaviviridae Study Group urges those who need to check or propose new genotypes or subtypes of HCV to contact the Study Group in advance of publication to avoid nomenclature conflicts appearing in the literature. While the criteria for assigning genotypes and subtypes remain unchanged from previous consensus proposals, changes are proposed in the assignment of provisional subtypes, subtype numbering beyond "w," and the nomenclature of intergenotypic recombinant. CONCLUSION: This study represents an important reference point for the consensus classification of HCV variants that will be of value to researchers working in clinical and basic science fields.

Sinclair C, Gaunt E, Simmonds P, Broomfield D, Nwafor N, Wellington L, Templeton K, Willocks L, Schofield O, Harvala H. 2014. Atypical hand, foot, and mouth disease associated with coxsackievirus A6 infection, Edinburgh, United Kingdom, January to February 2014. Euro Surveill, 19 (12), pp. 20745. | Show Abstract

In January to February 2014, 16 hand, foot and mouth disease (HFMD) cases were identified in Edinburgh, United Kingdom. All presented with atypical features, with most (n=13) resembling eczema herpeticum or chickenpox. Coxsackievirus A6 (CV-A6) was identified in all the typed cases (n=11). As atypical forms of HFMD associated with CV-A6 are likely to emerge throughout Europe, clinicians should be alert to unusual clinical presentations of HFMD and virologists aware of effective diagnostic testing and enterovirus typing methods.

Harvala H, Calvert J, Van Nguyen D, Clasper L, Gadsby N, Molyneaux P, Templeton K, McWilliams Leitch C, Simmonds P. 2014. Comparison of diagnostic clinical samples and environmental sampling for enterovirus and parechovirus surveillance in Scotland, 2010 to 2012. Euro Surveill, 19 (15), pp. 14-22. | Show Abstract

Human enteroviruses (EV) and parechoviruses (HPeV) within the family Picornaviridae are the most common causes of viral central nervous system (CNS)-associated infections including meningitis and neonatal sepsis-like disease. The frequencies of EV and HPeV types identified in clinical specimens collected in Scotland over an eight-year period were compared to those identified in sewage surveillance established in Edinburgh. Of the 35 different EV types belonging to four EV species (A to D) and the four HPeV types detected in this study, HPeV3 was identified as the most prevalent picornavirus in cerebrospinal fluid samples, followed by species B EV. Interestingly, over half of EV and all HPeV CNS-associated infections were observed in young infants (younger than three months). Detection of species A EV including coxsackievirus A6 and EV71 in clinical samples and sewage indicates that these viruses are already widely circulating in Scotland. Furthermore, species C EV were frequently identified EV in sewage screening but they were not present in any of 606 EV-positive clinical samples studied, indicating their likely lower pathogenicity. Picornavirus surveillance is important not only for monitoring the changing epidemiology of these infections but also for the rapid identification of spread of emerging EV and/or HPeV types.

Tulloch F, Atkinson NJ, Evans DJ, Ryan MD, Simmonds P. 2014. RNA virus attenuation by codon pair deoptimisation is an artefact of increases in CpG/UpA dinucleotide frequencies. Elife, 3 pp. e04531. | Show Abstract | Read more

Mutating RNA virus genomes to alter codon pair (CP) frequencies and reduce translation efficiency has been advocated as a method to generate safe, attenuated virus vaccines. However, selection for disfavoured CPs leads to unintended increases in CpG and UpA dinucleotide frequencies that also attenuate replication. We designed and phenotypically characterised mutants of the picornavirus, echovirus 7, in which these parameters were independently varied to determine which most influenced virus replication. CpG and UpA dinucleotide frequencies primarily influenced virus replication ability while no fitness differences were observed between mutants with different CP usage where dinucleotide frequencies were kept constant. Contrastingly, translation efficiency was unaffected by either CP usage or dinucleotide frequencies. This mechanistic insight is critical for future rational design of live virus vaccines and their safety evaluation; attenuation is mediated through enhanced innate immune responses to viruses with elevated CpG/UpA dinucleotide frequencies rather the viruses themselves being intrinsically defective.

Harvala H, Griffiths M, Solomon T, Simmonds P. 2014. Distinct systemic and central nervous system disease patterns in enterovirus and parechovirus infected children. J Infect, 69 (1), pp. 69-74. | Show Abstract | Read more

OBJECTIVES: Enteroviruses (EV) and human parechoviruses (HPeV) infections are increasingly identified in neonates and young children with sepsis, meningitis and encephalitis. We investigated EV and HPeV viral loads in plasma and cerebrospinal fluid (CSF) among those presenting with sepsis or central nervous system (CNS) disease to gain understanding of the nature of these infections. METHODS: Detections frequencies and viral loads of EV and HPeV RNA were compared in plasma and CSF obtained from infected children originally identified on sepsis or CNS screening. RESULTS: Two distinct infection profiles were identified; 11 subjects with CNS disease, showed higher or similar viral loads in CSF than in plasma (median plasma:CSF ratio 0.5), whereas 14 children with sepsis showed low or undetectable viral loads in CSF and high viral loads in plasma (mean ratio 5700). HPeV type 3 and one EV serotype (coxsackievirus B2) were primarily associated with the latter presentation. CONCLUSIONS: Simple detection of EV or HPeV RNA in CSF is not predictive of CNS disease, especially in the absence of clinical markers (i.e. pleocytosis). Screening of plasma can identify EV and HPeV RNA in a substantial proportion of sepsis cases, some of which will be missed if CSF samples alone are screened.

Nishiyama S, Dutia BM, Stewart JP, Meredith AL, Shaw DJ, Simmonds P, Sharp CP. 2014. Identification of novel anelloviruses with broad diversity in UK rodents. J Gen Virol, 95 (Pt 7), pp. 1544-1553. | Show Abstract | Read more

Anelloviruses are a family of small circular ssDNA viruses with a vast genetic diversity. Human infections with the prototype anellovirus, torque teno virus (TTV), are ubiquitous and related viruses have been described in a number of other mammalian hosts. Despite over 15 years of investigation, there is still little known about the pathogenesis and possible disease associations of anellovirus infections, arising in part due to the lack of a robust cell culture system for viral replication or tractable small-animal model. We report the identification of diverse anelloviruses in several species of wild rodents. The viruses are highly prevalent in wood mice (Apodemus sylvaticus) and field voles (Microtus agrestis), detectable at a low frequency in bank voles (Myodes glareolus), but absent from house mice (Mus musculus). The viruses identified have a genomic organization consistent with other anelloviruses, but form two clear phylogenetic groups that are as distinct from each other as from defined genera.

Cabrerizo M, Trallero G, Simmonds P. 2014. Recombination and evolutionary dynamics of human echovirus 6. J Med Virol, 86 (5), pp. 857-864. | Show Abstract | Read more

Enterovirus (EV) infections are associated with a wide array of often severe disease presentations including aseptic meningitis, encephalitis, and acute flaccid paralysis. Surveillance for polioviruses and other EVs is therefore important as a public health measure both for patient management and epidemiological studies. From 1988 to 2008, echovirus (E) 30 was the predominant genotype in Spain (33.7% of the total typed EVs). E6 was also endemic throughout this period although isolated less frequently (12.5%). In 2009, however, a substantial increase in the incidence of E6 was detected (60%), displacing E30 type (2%). To investigate the evolution and recombination in the epidemiology and transmission of E6 in Spain, a genetic analysis in VP1 and 3Dpol regions of 67 Spanish strains collected during the period 2004-2010 was performed. All VP1 sequences clustered monophyletically in the assigned genogroup C, subgroup 9, currently the predominant circulating strains identified in Europe and elsewhere in the last 10 years. 3Dpol sequences were interspersed with other species B EVs resulting from several recombination events that generated at least 12 different recombinant forms (RFs) among study samples. These showed typically minimal divergence in VP1. The co-circulation of different lineages of E6 in the same geographical area associated with its mainly endemic pattern of transmission may have contributed to the extremely short estimated half-life of E6 RFs (0.87 years). This pattern contrasts markedly with other species B EVs and EV71 where VP1 lineage expansion and extinction occurred in step with defined recombination events and periodic changes in incidence.

Servant-Delmas A, Laperche S, Lionnet F, Sharp C, Simmonds P, Lefrère JJ. 2014. Human parvovirus 4 infection in low- and high-risk French individuals. Transfusion, 54 (3), pp. 744-745. | Read more

Lyons S, Kapoor A, Schneider BS, Wolfe ND, Culshaw G, Corcoran B, Durham AE, Burden F, McGorum BC, Simmonds P. 2014. Viraemic frequencies and seroprevalence of non-primate hepacivirus and equine pegiviruses in horses and other mammalian species. J Gen Virol, 95 (Pt 8), pp. 1701-1711. | Show Abstract | Read more

Non-primate hepacivirus (NPHV), equine pegivirus (EPgV) and Theiler's disease associated virus (TDAV) are newly discovered members of two genera in the Flaviviridae family, Hepacivirus and Pegivirus respectively, that include human hepatitis C virus (HCV) and human pegivirus (HPgV). To investigate their epidemiology, persistence and clinical features of infection, large cohorts of horses and other mammalian species were screened for NPHV, EPgV and TDAV viraemia and for past exposure through serological assays for NPHV and EPgV-specific antibodies. NPHV antibodies were detected in 43% of 328 horses screened for antibodies to NS3 and core antibodies, of which three were viraemic by PCR. All five horses that were stablemates of a viraemic horse were seropositive, as was a dog on the same farm. With this single exception, all other species were negative for NPHV antibodies and viraemia: donkeys (n=100), dogs (n=112), cats (n=131), non-human primates (n=164) and humans (n=362). EPgV antibodies to NS3 were detected in 66.5% of horses, including 10 of the 12 horses that had EPgV viraemia. All donkey samples were negative for EPgV antibody and RNA. All horse and donkey samples were negative for TDAV RNA. By comparing viraemia frequencies in horses with and without liver disease, no evidence was obtained that supported an association between active NPHV and EPgV infections with hepatopathy. The study demonstrates that NPHV and EPgV infections are widespread and enzootic in the study horse population and confirms that NPHV and potentially EPgV have higher frequencies of viral clearance than HCV and HPgV infections in humans.

Siebrasse EA, Nguyen NL, Smith C, Simmonds P, Wang D. 2014. Immunohistochemical detection of KI polyomavirus in lung and spleen. Virology, 468-470 pp. 178-184. | Show Abstract | Read more

Little is known about the tissue tropism of KI polyomavirus (KIPyV), and there are no studies to date describing any specific cell types it infects. The limited knowledge of KIPyV tropism has hindered study of this virus and understanding of its potential pathogenesis in humans. We describe tissues from two immunocompromised patients that stained positive for KIPyV antigen using a newly developed immunohistochemical assay targeting the KIPyV VP1 (KVP1) capsid protein. In the first patient, a pediatric bone marrow transplant recipient, KVP1 was detected in lung tissue. Double immunohistochemical staining demonstrated that approximately 50% of the KVP1-positive cells were CD68-positive cells of the macrophage/monocyte lineage. In the second case, an HIV-positive patient, KVP1 was detected in spleen and lung tissues. These results provide the first identification of a specific cell type in which KVP1 can be detected and expand our understanding of basic properties and in vivo tropism of KIPyV.

Smith DB, Simmonds P, International Committee on Taxonomy of Viruses Hepeviridae Study Group, Jameel S, Emerson SU, Harrison TJ, Meng XJ, Okamoto H, Van der Poel WH, Purdy MA. 2014. Consensus proposals for classification of the family Hepeviridae. J Gen Virol, 95 (Pt 10), pp. 2223-2232. | Show Abstract | Read more

The family Hepeviridae consists of positive-stranded RNA viruses that infect a wide range of mammalian species, as well as chickens and trout. A subset of these viruses infects humans and can cause a self-limiting acute hepatitis that may become chronic in immunosuppressed individuals. Current published descriptions of the taxonomical divisions within the family Hepeviridae are contradictory in relation to the assignment of species and genotypes. Through analysis of existing sequence information, we propose a taxonomic scheme in which the family is divided into the genera Orthohepevirus (all mammalian and avian hepatitis E virus (HEV) isolates) and Piscihepevirus (cutthroat trout virus). Species within the genus Orthohepevirus are designated Orthohepevirus A (isolates from human, pig, wild boar, deer, mongoose, rabbit and camel), Orthohepevirus B (isolates from chicken), Orthohepevirus C (isolates from rat, greater bandicoot, Asian musk shrew, ferret and mink) and Orthohepevirus D (isolates from bat). Proposals are also made for the designation of genotypes within the human and rat HEVs. This hierarchical system is congruent with hepevirus phylogeny, and the three classification levels (genus, species and genotype) are consistent with, and reflect discontinuities in the ranges of pairwise distances between amino acid sequences. Adoption of this system would include the avoidance of host names in taxonomic identifiers and provide a logical framework for the assignment of novel variants.

Harvala H, Smith D, Salvatierra K, Gunson R, von Wissmann B, Reynolds A, Frew C, MacLean A, Hunt A, Yirrell D et al. 2014. Burden of influenza B virus infections in Scotland in 2012/13 and epidemiological investigations between 2000 and 2012. Euro Surveill, 19 (37), pp. 6-12. | Show Abstract

© 2014, European Centre for Disease Prevention and Control (ECDC). All rights reserved. We describe the burden of influenza B infections in Scotland during a 13-year study period. Influenza A and B viruses cocirculated throughout the period, with numbers of influenza B cases approaching or exceeding those of influenza A during six influenza seasons. Influenza B viruses of both Victoria and Yamagata lineage were detected in two of six seasons investigated. For the 2012/13 season, influenza B accounted for 44.4% of all influenzas, with the highest incidence in those under the age of five years. Influenza B virus infections led to fewer admissions to an intensive care unit (ICU) and a lower mortality rate than influenza A (37 vs 81 ICU admissions and three vs 29 deaths) during the 2012/13 season. However, a quarter of those admitted to ICU with influenza B had not been immunised and 60% had not received specific influenza antiviral therapy. This highlights the need for consistent influenza vaccination and prompt usage of antiviral treatment f or identified risk groups. Combining the newly introduced vaccination programme for children with the use of a tetravalent vaccine may provide the opportunity to improve the control of influenza B in those with the highest influenza B burden, children and young adolescents.

Wami WM, Nausch N, Bauer K, Midzi N, Gwisai R, Simmonds P, Mduluza T, Woolhouse M, Mutapi F. 2014. Comparing parasitological vs serological determination of Schistosoma haematobium infection prevalence in preschool and primary school-aged children: implications for control programmes. Parasitology, 141 (14), pp. 1962-1970. | Show Abstract | Read more

To combat schistosomiasis, the World Health Organization (WHO) recommends that infection levels are determined prior to designing and implementing control programmes, as the treatment regimens depend on the population infection prevalence. However, the sensitivity of the parasitological infection diagnostic method is less reliable when infection levels are low. The aim of this study was to compare levels of Schistosoma haematobium infection obtained by the parasitological method vs serological technique. Infection levels in preschool and primary school-aged children and their implications for control programmes were also investigated. Infection prevalence based on serology was significantly higher compared with that based on parasitology for both age groups. The difference between infection levels obtained using the two methods increased with age. Consequentially, in line with the WHO guidelines, the serological method suggested a more frequent treatment regimen for this population compared with that implied by the parasitological method. These findings highlighted the presence of infection in children aged ⩽5 years, further reiterating the need for their inclusion in control programmes. Furthermore, this study demonstrated the importance of using sensitive diagnostic methods as this has implications on the required intervention controls for the population.

Van Dung N, Anh PH, Van Cuong N, Hoa NT, Carrique-Mas J, Hien VB, Campbell J, Baker S, Farrar J, Woolhouse ME et al. 2014. Prevalence, genetic diversity and recombination of species G enteroviruses infecting pigs in Vietnam. J Gen Virol, 95 (Pt 3), pp. 549-556. | Show Abstract | Read more

Picornaviruses infecting pigs, described for many years as 'porcine enteroviruses', have recently been recognized as distinct viruses within three distinct genera (Teschovirus, Sapelovirus and Enterovirus). To better characterize the epidemiology and genetic diversity of members of the Enterovirus genus, faecal samples from pigs from four provinces in Vietnam were screened by PCR using conserved enterovirus (EV)-specific primers from the 5' untranslated region (5' UTR). High rates of infection were recorded in pigs on all farms, with detection frequencies of approximately 90% in recently weaned pigs but declining to 40% in those aged over 1 year. No differences in EV detection rates were observed between pigs with and without diarrhoea [74% (n = 70) compared with 72% (n = 128)]. Genetic analysis of consensus VP4/VP2 and VP1 sequences amplified from a subset of EV-infected pigs identified species G EVs in all samples. Among these, VP1 sequence comparisons identified six type 1 and seven type 6 variants, while four further VP1 sequences failed to group with any previously identified EV-G types. These have now been formally assigned as EV-G types 8-11 by the Picornavirus Study Group. Comparison of VP1, VP4/VP2, 3D(pol) and 5' UTRs of study samples and those available on public databases showed frequent, bootstrap-supported differences in their phylogenies indicative of extensive within-species recombination between genome regions. In summary, we identified extremely high frequencies of infection with EV-G in pigs in Vietnam, substantial genetic diversity and recombination within the species, and evidence for a much larger number of circulating EV-G types than currently described.

Ramalingam S, Smith D, Wellington L, Vanek J, Simmonds P, MacGilchrist A, Bathgate A, Simpson K, Johannessen I. 2013. Autochthonous hepatitis E in Scotland. J Clin Virol, 58 (4), pp. 619-623. | Show Abstract | Read more

BACKGROUND: Hepatitis E virus is well recognized cause of acute hepatitis. Traditionally hepatitis E virus (HEV) infections were generally associated with travel to Asia and Africa. Autochthonous hepatitis E is recognized as a major cause acute hepatitis in England and Wales. However, autochthonous hepatitis E has never been documented in Scotland. OBJECTIVES: We attempted to determine if autochthonous HEV occurred in Scotland. STUDY DESIGN: Samples from 377 individuals in the South-East of Scotland presenting with acute hepatitis were tested over six years. Acute hepatitis E was confirmed by detecting viraemia or documenting seroconversion and ORF-2 region sequenced. Structured interviews were carried out to identify risk factors for infection. RESULTS: Sixteen individuals (4.2%) had evidence of past HEV infection. Twelve (3.2%) had acute HEV infection, 10 of whom had viraemia (genotype 1=3; genotype 3=7). Of these seven with genotype 3 infection, three had not travelled outside Scotland within the incubation period, while four had travelled to Spain (n=3) or Turkey (n=1). All three individuals with genotype 1 infection had travelled to the Indian subcontinent. CONCLUSIONS: A significant proportion of HEV genotype 3 infections was autochthonous (43%). HEV screening should hence be an integral part of acute hepatitis screening in Scotland, irrespective of the travel history.

Stewart H, Walter C, Jones D, Lyons S, Simmonds P, Harris M. 2013. The non-primate hepacivirus 5' untranslated region possesses internal ribosomal entry site activity. J Gen Virol, 94 (Pt 12), pp. 2657-2663. | Show Abstract | Read more

The 5' untranslated region (5'UTR) of the recently described non-primate hepacivirus (NPHV) contains a region with sequence homology to the internal ribosomal entry site (IRES) of hepatitis C virus (HCV) and GB virus B (GBV-B). Here, we demonstrated internal translation initiation by the NPHV 5'UTR in a bicistronic vector. An RNA stem-loop upstream of the NPHV IRES was structurally distinct from corresponding regions in HCV and GBV-B, and was not required for IRES function. Insertion of the NPHV stem-loop into the corresponding region of the HCV 5'UTR within the HCV subgenomic replicon significantly impaired RNA replication, indicating that long-range interactions between the 5'UTR and cis-acting downstream elements within the NPHV genome are not interchangeable with those of HCV. Despite similarities in IRES structure and function between hepaciviruses, replication elements in the NPHV 5'UTR appear functionally distinct from those of HCV.

Phan TG, Vo NP, Simmonds P, Samayoa E, Naccache S, Chiu CY, Delwart E. 2013. Rosavirus: the prototype of a proposed new genus of the Picornaviridae family. Virus Genes, 47 (3), pp. 556-558. | Show Abstract | Read more

We describe a 8,724-nucleotide-long picornavirus genome encoding a single 2,470-aa polyprotein obtained from the feces of a wild mouse. Rosavirus is genetically closest to the double ORF Dicipivirus found in canine feces that is currently the only picornavirus with a second internal ribosome entry site (IRES). Of note, a section of rosavirus' 5'UTR showed strong sequence and structural conservation with the type II IRES from the Parechovirus and Hungarovirus genera possibly reflecting exchange of genetic modules between genera. Based on genetic distance criteria rosavirus qualifies as prototype of a new genus of the Picornaviridae family.

Iles JC, Abby Harrison GL, Lyons S, Djoko CF, Tamoufe U, Lebreton M, Schneider BS, Fair JN, Tshala FM, Kayembe PK et al. 2013. Hepatitis C virus infections in the Democratic Republic of Congo exhibit a cohort effect. Infect Genet Evol, 19 pp. 386-394. | Show Abstract | Read more

The prevalence and genetic diversity of hepatitis C virus (HCV) and human pegivirus (HPgV) in many regions of sub-Saharan Africa is poorly characterized, including in the Democratic Republic of Congo--the largest country in the region and one of the most populous. To address this situation we conducted a molecular epidemiological survey of HCV and HPgV (previously named GB Virus C or hepatitis G virus) in samples collected in 2007 from 299 males from the DRC, whose ages ranged from 21 to 71 years old. Samples were tested for the presence of HCV antibodies by ELISA and reactive samples were subsequently tested for HCV RNA using RT-PCR in which both the HCV Core and NS5B genome regions were amplified. Remaining samples were tested for HPgV RNA and the HPgV NS3 genome region of positive samples was amplified. For HCV, 13.7% of the samples were seropositive (41/299) but only 3.7% were viremic (11/299). HPgV RNA was found in 12.7% (33/259) of samples. HCV viremia was strongly associated with age; the percentage of samples that contained detectable HCV RNA was ~0.5% in those younger than 50 and 13% in those older than 50. Our study represents the first systematic survey of HCV genetic diversity in the DRC. HCV sequences obtained belonged to diverse lineages of genotype 4, including subtypes 4c, 4 k, 4 l and 4r, plus one unclassified lineage that may constitute a new subtype. These data suggest that HCV in the DRC exhibits an age 'cohort effect', as has been recently reported in neighbouring countries, and are consistent with the hypothesis that HCV transmission rates were higher in the mid-twentieth century, possibly as a result of parenteral, iatrogenic, or other unidentified factors. Different HCV subtypes were associated with individuals of different ages, implying that HCV infection in the DRC may have arisen through multiple separate HCV epidemics with different causes.

Shah A, Connolly M, Clasper L, McIntyre C, Harvala H, Hale A, Whitaker P, Simmonds P, Peckham D. 2013. Frequency and pathogenicity of rhinovirus associated pulmonary exacerbations in patients with cystic fibrosis EUROPEAN RESPIRATORY JOURNAL, 42

McIntyre CL, Knowles NJ, Simmonds P. 2013. Proposals for the classification of human rhinovirus species A, B and C into genotypically assigned types. J Gen Virol, 94 (Pt 8), pp. 1791-1806. | Show Abstract | Read more

Human rhinoviruses (HRVs) frequently cause mild upper respiratory tract infections and more severe disease manifestations such as bronchiolitis and asthma exacerbations. HRV is classified into three species within the genus Enterovirus of the family Picornaviridae. HRV species A and B contain 75 and 25 serotypes identified by cross-neutralization assays, although the use of such assays for routine HRV typing is hampered by the large number of serotypes, replacement of virus isolation by molecular methods in HRV diagnosis and the poor or absent replication of HRV species C in cell culture. To address these problems, we propose an alternative, genotypic classification of HRV-based genetic relatedness analogous to that used for enteroviruses. Nucleotide distances between 384 complete VP1 sequences of currently assigned HRV (sero)types identified divergence thresholds of 13, 12 and 13 % for species A, B and C, respectively, that divided inter- and intra-type comparisons. These were paralleled by 10, 9.5 and 10 % thresholds in the larger dataset of >3800 VP4 region sequences. Assignments based on VP1 sequences led to minor revisions of existing type designations (such as the reclassification of serotype pairs, e.g. A8/A95 and A29/A44, as single serotypes) and the designation of new HRV types A101-106, B101-103 and C34-C51. A protocol for assignment and numbering of new HRV types using VP1 sequences and the restriction of VP4 sequence comparisons to type identification and provisional type assignments is proposed. Genotypic assignment and identification of HRV types will be of considerable value in the future investigation of type-associated differences in disease outcomes, transmission and epidemiology.

McIntyre CL, Savolainen-Kopra C, Hovi T, Simmonds P. 2013. Recombination in the evolution of human rhinovirus genomes. Arch Virol, 158 (7), pp. 1497-1515. | Show Abstract | Read more

Human rhinoviruses (HRV) are highly prevalent human respiratory pathogens that belong to the genus Enterovirus. Although recombination within the coding region is frequent in other picornavirus groups, most evidence of recombination in HRV has been restricted to the 5' untranslated region. We analysed the occurrence of recombination within published complete genome sequences of members of all three HRV species and additionally compared sequences from HRV strains spanning 14 years. HRV-B and HRV-C showed very little evidence of recombination within the coding region. In contrast, HRV-A sequences appeared to have undergone a large number of recombination events, typically involving whole type groups. This suggests that HRV-A may have been subject to extensive recombination during the period of diversification into types. This study demonstrates the rare and sporadic nature of contemporary recombination of HRV strains and contrasts with evidence of extensive recombination within HRV-A and between members of different species during earlier stages in its evolutionary diversification.

McFadden N, Arias A, Dry I, Bailey D, Witteveldt J, Evans DJ, Goodfellow I, Simmonds P. 2013. Influence of genome-scale RNA structure disruption on the replication of murine norovirus--similar replication kinetics in cell culture but attenuation of viral fitness in vivo. Nucleic Acids Res, 41 (12), pp. 6316-6331. | Show Abstract | Read more

Mechanisms by which certain RNA viruses, such as hepatitis C virus, establish persistent infections and cause chronic disease are of fundamental importance in viral pathogenesis. Mammalian positive-stranded RNA viruses establishing persistence typically possess genome-scale ordered RNA secondary structure (GORS) in their genomes. Murine norovirus (MNV) persists in immunocompetent mice and provides an experimental model to functionally characterize GORS. Substitution mutants were constructed with coding sequences in NS3/4- and NS6/7-coding regions replaced with sequences with identical coding and (di-)nucleotide composition but disrupted RNA secondary structure (F1, F2, F1/F2 mutants). Mutants replicated with similar kinetics to wild-type (WT) MNV3 in RAW264.7 cells and primary macrophages, exhibited similar (highly restricted) induction and susceptibility to interferon-coupled cellular responses and equal replication fitness by serial passaging of co-cultures. In vivo, both WT and F1/F2 mutant viruses persistently infected mice, although F1, F2 and F1/F2 mutant viruses were rapidly eliminated 1-7 days post-inoculation in competition experiments with WT. F1/F2 mutants recovered from tissues at 9 months showed higher synonymous substitution rates than WT and nucleotide substitutions that potentially restored of RNA secondary structure. GORS plays no role in basic replication of MNV but potentially contributes to viral fitness and persistence in vivo.

Newman RM, Kuntzen T, Weiner B, Berical A, Charlebois P, Kuiken C, Murphy DG, Simmonds P, Bennett P, Lennon NJ et al. 2013. Whole genome pyrosequencing of rare hepatitis C virus genotypes enhances subtype classification and identification of naturally occurring drug resistance variants. J Infect Dis, 208 (1), pp. 17-31. | Show Abstract | Read more

BACKGROUND:  Infection with hepatitis C virus (HCV) is a burgeoning worldwide public health problem, with 170 million infected individuals and an estimated 20 million deaths in the coming decades. While 6 main genotypes generally distinguish the global geographic diversity of HCV, a multitude of closely related subtypes within these genotypes are poorly defined and may influence clinical outcome and treatment options. Unfortunately, the paucity of genetic data from many of these subtypes makes time-consuming primer walking the limiting step for sequencing understudied subtypes. METHODS:  Here we combined long-range polymerase chain reaction amplification with pyrosequencing for a rapid approach to generate the complete viral coding region of 31 samples representing poorly defined HCV subtypes. RESULTS:  Phylogenetic classification based on full genome sequences validated previously identified HCV subtypes, identified a recombinant sequence, and identified a new distinct subtype of genotype 4. Unlike conventional sequencing methods, use of deep sequencing also facilitated characterization of minor drug resistance variants within these uncommon or, in some cases, previously uncharacterized HCV subtypes. CONCLUSIONS:  These data aid in the classification of uncommon HCV subtypes while also providing a high-resolution view of viral diversity within infected patients, which may be relevant to the development of therapeutic regimens to minimize drug resistance.




Smith DB, Purdy MA, Simmonds P. 2013. Genetic Variability and the Classification of Hepatitis E Virus (vol 87, pg 4161, 2013) JOURNAL OF VIROLOGY, 87 (13), pp. 7787-7787. | Read more

Jacka B, Lamoury F, Simmonds P, Dore GJ, Grebely J, Applegate T. 2013. Sequencing of the Hepatitis C Virus: A Systematic Review. PLoS One, 8 (6), pp. e67073. | Show Abstract | Read more

Since the identification of hepatitis C virus (HCV), viral sequencing has been important in understanding HCV classification, epidemiology, evolution, transmission clustering, treatment response and natural history. The length and diversity of the HCV genome has resulted in analysis of certain regions of the virus, however there has been little standardisation of protocols. This systematic review was undertaken to map the location and frequency of sequencing on the HCV genome in peer reviewed publications, with the aim to produce a database of sequencing primers and amplicons to inform future research. Medline and Scopus databases were searched for English language publications based on keyword/MeSH terms related to sequence analysis (9 terms) or HCV (3 terms), plus "primer" as a general search term. Exclusion criteria included non-HCV research, review articles, duplicate records, and incomplete description of HCV sequencing methods. The PCR primer locations of accepted publications were noted, and purpose of sequencing was determined. A total of 450 studies were accepted from the 2099 identified, with 629 HCV sequencing amplicons identified and mapped on the HCV genome. The most commonly sequenced region was the HVR-1 region, often utilised for studies of natural history, clustering/transmission, evolution and treatment response. Studies related to genotyping/classification or epidemiology of HCV genotype generally targeted the 5'UTR, Core and NS5B regions, while treatment response/resistance was assessed mainly in the NS3-NS5B region with emphasis on the Interferon sensitivity determining region (ISDR) region of NS5A. While the sequencing of HCV is generally constricted to certain regions of the HCV genome there is little consistency in the positioning of sequencing primers, with the exception of a few highly referenced manuscripts. This study demonstrates the heterogeneity of HCV sequencing, providing a comprehensive database of previously published primer sets to be utilised in future sequencing studies.

Kapoor A, Simmonds P, Cullen JM, Scheel TK, Medina JL, Giannitti F, Nishiuchi E, Brock KV, Burbelo PD, Rice CM, Lipkin WI. 2013. Identification of a pegivirus (GB virus-like virus) that infects horses. J Virol, 87 (12), pp. 7185-7190. | Show Abstract | Read more

The recent identification of nonprimate hepaciviruses in dogs and then in horses prompted us to look for pegiviruses (GB virus-like viruses) in these species. Although none were detected in canines, we found widespread natural infection of horses by a novel pegivirus. Unique genomic features and phylogenetic analyses confirmed that the tentatively named equine pegivirus (EPgV) represents a novel species within the Pegivirus genus. We also determined that EPgV causes persistent viremia whereas its clinical significance is undetermined.

Monk SL, Simmonds P, Matthews KR. 2013. A short bifunctional element operates to positively or negatively regulate ESAG9 expression in different developmental forms of Trypanosoma brucei. J Cell Sci, 126 (Pt 10), pp. 2294-2304. | Show Abstract | Read more

In their mammalian host trypanosomes generate 'stumpy' forms from proliferative 'slender' forms as an adaptation for transmission to their tsetse fly vector. This transition is characterised by the repression of many genes while quiescent stumpy forms accumulate during each wave of parasitaemia. However, a subset of genes are upregulated either as an adaptation for transmission or to sustain infection chronicity. Among this group are ESAG9 proteins, whose genes were originally identified as a component of some telomeric variant surface glycoprotein gene expression sites, although many members of this diverse family are also transcribed elsewhere in the genome. ESAG9 genes are among the most highly regulated genes in transmissible stumpy forms, encoding a group of secreted proteins of cryptic function. To understand their developmental silencing in slender forms and activation in stumpy forms, the post-transcriptional control signals for a well conserved ESAG9 gene have been mapped. This identified a precise RNA sequence element of 34 nucleotides that contributes to gene expression silencing in slender forms but also acts positively, activating gene expression in stumpy forms. We predict that this bifunctional RNA sequence element is targeted by competing negative and positive regulatory factors in distinct developmental forms of the parasite. Analysis of the 3'UTR regulatory regions flanking the highly diverse ESAG9 family reveals that the linear regulatory sequence is not highly conserved, suggesting that RNA structure is important for interactions with regulatory proteins.

Simmonds P. 2013. The origin of hepatitis C virus. Curr Top Microbiol Immunol, 369 pp. 1-15. | Show Abstract | Read more

The origin of hepatitis C virus (HCV) can be conceptualised at several levels. Firstly, origins might refer to its dramatic spread throughout the Western world and developing countries throughout the twentieth century. As a blood-borne virus, this epidemic was fuelled by new parenteral transmission routes associated with medical treatments, immunisation, blood transfusion and more recently injecting drug use. At another level, however, origins might refer to the immediate sources of HCV associated with its pandemic spread, now identified as areas in Central and West sub-Saharan Africa and South and South East Asia where genetically diverse variants of HCV appear to have circulated for hundreds of years. Going back a final step to the actual source of HCV infection in these endemic areas, non-human primates have been long suspected as harbouring viruses related to HCV with potential cross-species transmission of variants corresponding to the 7 main genotypes into humans. Although there is tempting analogy between this and the clearly zoonotic origin of HIV-1 from chimpanzees in Central Africa, no published evidence to date has been obtained for infection of HCV-like viruses in either apes or Old World monkey species. Indeed, a radical re-think of both the host range and host-specificity of hepaciviruses is now required following the very recent findings of a non-primate hepacivirus (NPHV) in horses and potentially in dogs. Further research on a much wider range of mammals is needed to better understand the true genetic diversity of HCV-like viruses and their host ranges in the search for the ultimate origin of HCV in humans.

Smith DB, Purdy MA, Simmonds P. 2013. Genetic variability and the classification of hepatitis E virus. J Virol, 87 (8), pp. 4161-4169. | Show Abstract | Read more

The classification of hepatitis E virus (HEV) variants is currently in transition without agreed definitions for genotypes and subtypes or for deeper taxonomic groupings into species and genera that could incorporate more recently characterized viruses assigned to the Hepeviridae family that infect birds, bats, rodents, and fish. These conflicts arise because of differences in the viruses and genomic regions compared and in the methodology used. We have reexamined published sequences and found that synonymous substitutions were saturated in comparisons between and within virus genotypes. Analysis of complete genome sequences or concatenated ORF1/ORF2 amino acid sequences indicated that HEV variants most closely related to those infecting humans can be consistently divided into six genotypes (types 1 to 4 and two additional genotypes from wild boar). Variants isolated from rabbits, closely related to genotype 3, occupy an intermediate position. No consistent criteria could be defined for the assignment of virus subtypes. Analysis of amino acid sequences from these viruses with the more divergent variants from chickens, bats, and rodents in three conserved subgenomic regions (residues 1 to 452 or 974 to 1534 of ORF1 or residues 105 to 458 of ORF2) provided consistent support for a division into 4 groups, corresponding to HEV variants infecting humans and pigs, those infecting rats and ferrets, those from bats, and those from chickens. This approach may form the basis for a future genetic classification of HEV into four species, with the more divergent HEV-like virus from fish (cutthroat trout virus) representing a second genus.

Smith DB, Vanek J, Wellington L, Johannessen I, Ramalingam S, Simmonds P. 2013. Hepatitis E virus mixed infection in immunocompetent patient. Emerg Infect Dis, 19 (3), pp. 468-470. | Show Abstract | Read more

We detected 2 hepatitis E virus (HEV) strains in an acutely infected immunocompetent patient. Two populations of genotype 3 virus were observed in the hypervariable regions and open reading frames 2 and 3, indicating multiple infection with hepatitis E virus. Persons with mixed infections may provide the opportunity for virus recombination.

Kapoor A, Simmonds P, Scheel TK, Hjelle B, Cullen JM, Burbelo PD, Chauhan LV, Duraisamy R, Sanchez Leon M, Jain K et al. 2013. Identification of rodent homologs of hepatitis C virus and pegiviruses. MBio, 4 (2), pp. e00216-e00213. | Show Abstract | Read more

UNLABELLED: Hepatitis C virus (HCV) and human pegivirus (HPgV or GB virus C) are globally distributed and infect 2 to 5% of the human population. The lack of tractable-animal models for these viruses, in particular for HCV, has hampered the study of infection, transmission, virulence, immunity, and pathogenesis. To address this challenge, we searched for homologous viruses in small mammals, including wild rodents. Here we report the discovery of several new hepaciviruses (HCV-like viruses) and pegiviruses (GB virus-like viruses) that infect wild rodents. Complete genome sequences were acquired for a rodent hepacivirus (RHV) found in Peromyscus maniculatus and a rodent pegivirus (RPgV) found in Neotoma albigula. Unique genomic features and phylogenetic analyses confirmed that these RHV and RPgV variants represent several novel virus species in the Hepacivirus and Pegivirus genera within the family Flaviviridae. The genetic diversity of the rodent hepaciviruses exceeded that observed for hepaciviruses infecting either humans or non-primates, leading to new insights into the origin, evolution, and host range of hepaciviruses. The presence of genes, encoded proteins, and translation elements homologous to those found in human hepaciviruses and pegiviruses suggests the potential for the development of new animal systems with which to model HCV pathogenesis, vaccine design, and treatment. IMPORTANCE: The genetic and biological characterization of animal homologs of human viruses provides insights into the origins of human infections and enhances our ability to study their pathogenesis and explore preventive and therapeutic interventions. Horses are the only reported host of nonprimate homologs of hepatitis C virus (HCV). Here, we report the discovery of HCV-like viruses in wild rodents. The majority of HCV-like viruses were found in deer mice (Peromyscus maniculatus), a small rodent used in laboratories to study viruses, including hantaviruses. We also identified pegiviruses in rodents that are distinct from the pegiviruses found in primates, bats, and horses. These novel viruses may enable the development of small-animal models for HCV, the most common infectious cause of liver failure and hepatocellular carcinoma after hepatitis B virus, and help to explore the health relevance of the highly prevalent human pegiviruses.

Cosgrove C, Ussher JE, Rauch A, Gärtner K, Kurioka A, Hühn MH, Adelmann K, Kang YH, Fergusson JR, Simmonds P et al. 2013. Early and nonreversible decrease of CD161++ /MAIT cells in HIV infection. Blood, 121 (6), pp. 951-961. | Show Abstract | Read more

HIV infection is associated with immune dysfunction, perturbation of immune-cell subsets and opportunistic infections. CD161++ CD8+ T cells are a tissue-infiltrating population that produce IL17A, IL22, IFN, and TNFα, cytokines important in mucosal immunity. In adults they dominantly express the semi-invariant TCR Vα7.2, the canonical feature of mucosal associated invariant T (MAIT) cells and have been recently implicated in host defense against pathogens. We analyzed the frequency and function of CD161++ /MAIT cells in peripheral blood and tissue from patients with early stage or chronic-stage HIV infection. We show that the CD161++ /MAIT cell population is significantly decreased in early HIV infection and fails to recover despite otherwise successful treatment. We provide evidence that CD161++ /MAIT cells are not preferentially infected but may be depleted through diverse mechanisms including accumulation in tissues and activation-induced cell death. This loss may impact mucosal defense and could be important in susceptibility to specific opportunistic infections in HIV.

Cosgrove C, Ussher JE, Rauch A, Gaertner K, Kurioka A, Huehn MH, Adelmann K, Kang Y-H, Fergusson JR, Simmonds P et al. 2013. EARLY AND NON-REVERSIBLE DECREASE OF CD161++/MUCOSAL ASSOCIATED INVARIANT T CELLS IN HIV INFECTION JOURNAL OF INFECTION, 67 (4), pp. 342-343. | Read more

Simmons R, Sharp C, Levine J, Bowness P, Simmonds P, Cox A, Klenerman P. 2013. Evolution of CD8+ T cell responses after acute PARV4 infection. J Virol, 87 (6), pp. 3087-3096. | Show Abstract | Read more

PARV4 is a small DNA human virus that is strongly associated with hepatitis C virus (HCV) and HIV infections. The immunologic control of acute PARV4 infection has not been previously described. We define the acute onset of PARV4 infection and the characteristics of the acute-phase and memory immune responses to PARV4 in a group of HCV- and HIV-negative, active intravenous drug users. Ninety-eight individuals at risk of blood-borne infections were tested for PARV4 IgG. Gamma interferon enzyme-linked immunosorbent spot assays, intracellular cytokine staining, and a tetrameric HLA-A2-peptide complex were used to define the T cell populations responding to PARV4 peptides in those individuals who acquired infection during the study. Thirty-five individuals were found to be PARV4 seropositive at the end of the study, eight of whose baseline samples were found to be seronegative. Persistent and functional T cell responses were detected in the acute infection phase. These responses had an active, mature, and cytotoxic phenotype and were maintained several years after infection. Thus, PARV4 infection is common in individuals exposed to blood-borne infections, independent of their HCV or HIV status. Since PARV4 elicits strong, broad, and persistent T cell responses, understanding of the processes responsible may prove useful for future vaccine design.

Simmonds P, Xia W, Baillie JK, McKinnon K. 2013. Modelling mutational and selection pressures on dinucleotides in eukaryotic phyla--selection against CpG and UpA in cytoplasmically expressed RNA and in RNA viruses. BMC Genomics, 14 (1), pp. 610. | Show Abstract | Read more

BACKGROUND: Loss of CpG dinucleotides in genomic DNA through methylation-induced mutation is characteristic of vertebrates and plants. However, these and other eukaryotic phyla show a range of other dinucleotide frequency biases with currently uncharacterized underlying mutational or selection mechanisms. We developed a parameterized Markov process to identify what neighbour context-dependent mutations best accounted for patterns of dinucleotide frequency biases in genomic and cytoplasmically expressed mRNA sequences of different vertebrates, other eukaryotic groups and RNA viruses that infect them. RESULTS: Consistently, 11- to 14-fold greater frequencies of the methylation-associated mutation of C to T upstream of G (depicted as C→T,G) than other transitions best modelled dinucleotide frequencies in mammalian genomic DNA. However, further mutations such as G→T,T (5-fold greater than the default transversion rate) were required to account for the full spectrum of dinucleotide frequencies in mammalian sequence datasets. Consistent with modeling predictions for these two mutations, instability of both CpG and CpT dinucleotides was identified through SNP frequency analysis of human DNA sequences. Different sets of context-dependent mutations were modelled in other eukaryotes with non-methylated genomic DNA. In contrast to genomic DNA, best-fit models of dinucleotide frequencies in transcribed RNA sequences expressed in the cytoplasm from all organisms were dominated by mutations that eliminated UpA dinucleotides, observations consistent with cytoplasmically driven selection for mRNA stability. Surprisingly, mRNA sequences from organisms with methylated genomes showed evidence for additional selection against CpG through further context-dependent mutations (eg. C→A,G). Similar mutation or selection processes were identified among single-stranded mammalian RNA viruses; these potentially account for their previously described but unexplained under-representations of CpG and UpA dinucleotides. CONCLUSIONS: Methods we have developed identify mutational processes and selection pressures in organisms that provide new insights into nucleotide compositional constraints and a wealth of biochemical and evolutionarily testable predictions for the future.

McAllister G, Holmes A, Garcia L, Cameron F, Cloy K, Danial J, Cepeda JA, Simmonds P, Templeton KE. 2012. Molecular epidemiology of norovirus in Edinburgh healthcare facilities, Scotland 2007-2011. Epidemiol Infect, 140 (12), pp. 2273-2281. | Show Abstract | Read more

Norovirus (NoV) is a leading cause of outbreaks of gastroenteritis worldwide, and a major burden for healthcare facilities. This study investigated the NoV genotypes responsible for outbreaks in Edinburgh healthcare facilities between June 2008 and July 2011, and studied their temporal distribution to enable a better understanding of the epidemiology of the outbreaks. A total of 287 samples positive for NoV genogroup II (GII) RNA by reverse transcription-polymerase chain reaction (RT-PCR) during routine diagnostic testing were investigated. Nested RT-PCR (nRT-PCR) and sequencing was used to genotype the NoV strains. Overall, a total of 69 NoV strains belonging to six different genoclusters (GII.1, GII.2, GII.3, GII.4, GII.6, GII.13) were detected. The predominant genotype was GII.4 that included four variants, GII.4 2006a, GII.4 2006b, GII.4 2007 and GII.4 2010. Importantly, increases in NoV activity coincided with the emergence of new GII.4 strains, highlighting the need for an active surveillance system to allow the rapid identification of new strains.

Lyons S, Kapoor A, Sharp C, Schneider BS, Wolfe ND, Culshaw G, Corcoran B, McGorum BC, Simmonds P. 2012. Nonprimate hepaciviruses in domestic horses, United kingdom. Emerg Infect Dis, 18 (12), pp. 1976-1982. | Show Abstract | Read more

Although the origin of hepatitis C virus infections in humans remains undetermined, a close homolog of this virus, termed canine hepacivirus (CHV) and found in respiratory secretions of dogs, provides evidence for a wider distribution of hepaciviruses in mammals. We determined frequencies of active infection among dogs and other mammals in the United Kingdom. Samples from dogs (46 respiratory, 99 plasma, 45 autopsy samples) were CHV negative by PCR. Screening of 362 samples from cats, horses, donkeys, rodents, and pigs identified 3 (2%) positive samples from 142 horses. These samples were genetically divergent from CHV and nonprimate hepaciviruses that horses were infected with during 2012 in New York state, USA. Investigation of infected horses demonstrated nonprimate hepacivirus persistence, high viral loads in plasma (10(5)-10(7) RNA copies/mL), and liver function test results usually within reference ranges, although several values ranged from high normal to mildly elevated. Disease associations and host range of nonprimate hepaciviruses warrant further investigation.

Smith DB, Vanek J, Ramalingam S, Johannessen I, Templeton K, Simmonds P. 2012. Evolution of the hepatitis E virus hypervariable region. J Gen Virol, 93 (Pt 11), pp. 2408-2418. | Show Abstract | Read more

The presence of a hypervariable (HVR) region within the genome of hepatitis E virus (HEV) remains unexplained. Previous studies have described the HVR as a proline-rich spacer between flanking functional domains of the ORF1 polyprotein. Others have proposed that the region has no function, that it reflects a hypermutable region of the virus genome, that it is derived from the insertion and evolution of host sequences or that it is subject to positive selection. This study attempts to differentiate between these explanations by documenting the evolutionary processes occurring within the HVR. We have measured the diversity of HVR sequences within acutely infected individuals or amongst sequences derived from epidemiologically linked samples and, surprisingly, find relative homogeneity amongst these datasets. We found no evidence of positive selection for amino acid substitution in the HVR. Through an analysis of published sequences, we conclude that the range of HVR diversity observed within virus genotypes can be explained by the accumulation of substitutions and, to a much lesser extent, through deletions or duplications of this region. All published HVR amino acid sequences display a relative overabundance of proline and serine residues that cannot be explained by a local bias towards cytosine in this part of the genome. Although all published HVRs contain one or more SH3-binding PxxP motifs, this motif does not occur more frequently than would be expected from the proportion of proline residues in these sequences. Taken together, these observations are consistent with the hypothesis that the HVR has a structural role that is dependent upon length and amino acid composition, rather than a specific sequence.

Ng TF, Marine R, Wang C, Simmonds P, Kapusinszky B, Bodhidatta L, Oderinde BS, Wommack KE, Delwart E. 2012. High variety of known and new RNA and DNA viruses of diverse origins in untreated sewage. J Virol, 86 (22), pp. 12161-12175. | Show Abstract | Read more

Deep sequencing of untreated sewage provides an opportunity to monitor enteric infections in large populations and for high-throughput viral discovery. A metagenomics analysis of purified viral particles in untreated sewage from the United States (San Francisco, CA), Nigeria (Maiduguri), Thailand (Bangkok), and Nepal (Kathmandu) revealed sequences related to 29 eukaryotic viral families infecting vertebrates, invertebrates, and plants (BLASTx E score, <10(-4)), including known pathogens (>90% protein identities) in numerous viral families infecting humans (Adenoviridae, Astroviridae, Caliciviridae, Hepeviridae, Parvoviridae, Picornaviridae, Picobirnaviridae, and Reoviridae), plants (Alphaflexiviridae, Betaflexiviridae, Partitiviridae, Sobemovirus, Secoviridae, Tombusviridae, Tymoviridae, Virgaviridae), and insects (Dicistroviridae, Nodaviridae, and Parvoviridae). The full and partial genomes of a novel kobuvirus, salivirus, and sapovirus are described. A novel astrovirus (casa astrovirus) basal to those infecting mammals and birds, potentially representing a third astrovirus genus, was partially characterized. Potential new genera and families of viruses distantly related to members of the single-stranded RNA picorna-like virus superfamily were genetically characterized and named Picalivirus, Secalivirus, Hepelivirus, Nedicistrovirus, Cadicistrovirus, and Niflavirus. Phylogenetic analysis placed these highly divergent genomes near the root of the picorna-like virus superfamily, with possible vertebrate, plant, or arthropod hosts inferred from nucleotide composition analysis. Circular DNA genomes distantly related to the plant-infecting Geminiviridae family were named Baminivirus, Nimivirus, and Niminivirus. These results highlight the utility of analyzing sewage to monitor shedding of viral pathogens and the high viral diversity found in this common pollutant and provide genetic information to facilitate future studies of these newly characterized viruses.

Harvala H, Gaunt E, McIntyre C, Roddie H, Labonte S, Curran E, Othieno R, Simmonds P, Bremner J. 2012. Epidemiology and clinical characteristics of parainfluenza virus 3 outbreak in a Haemato-oncology unit. J Infect, 65 (3), pp. 246-254. | Show Abstract | Read more

OBJECTIVES: We describe molecular investigations of a large hospital outbreak of parainfluenza virus type 3 (PIV3), in which 32 patients became infected. We outline infection control measures that successfully limited further spread of PIV3 in a Haemato-oncology unit. METHODS: Clinical retrospective review of infected haemato-oncology patients was undertaken. PIV3 haemagglutinin sequences from each case (n = 32) and local epidemiologically unlinked controls (n = 53) were compared to identify potential linkage. RESULTS: PIV3-infected patients presented with upper (n = 18) and lower (n = 11) respiratory tract infections, 3 showed pyrexia only and one was asymptomatic. All symptomatic patients received antibiotics; bacterial co-infection was confirmed in eleven patients. PIV3 infections were associated with lower mortality than documented previously; three of the PIV3-infected patients died (3/32; 9%). All deaths were associated with relapsed malignancies, and PIV3 was not believed to be the primary cause of death in any of these patients. Sequences from 27 cases clustered closely together, consistent with nosocomial infections from PIV3 circulating within the ward. Factors favouring transmission were high patient turnaround between the day treatment unit and in-patient ward, and limited isolation facilities for immunocompromised and infected patients, especially within the day treatment unit. New infections reduced to baseline levels three days after enhanced infection control interventions were introduced. CONCLUSIONS: Molecular epidemiological analysis provided evidence for nosocomial transmission of PIV3 infection that facilitated effective implementation of infection control measures. These were instrumental in restricting further spread of the virus among high-risk patients.

McLeish NJ, Witteveldt J, Clasper L, McIntyre C, McWilliam Leitch EC, Hardie A, Bennett S, Gunson R, Carman WF, Feeney SA et al. 2012. Development and assay of RNA transcripts of enterovirus species A to D, rhinovirus species a to C, and human parechovirus: assessment of assay sensitivity and specificity of real-time screening and typing methods. J Clin Microbiol, 50 (9), pp. 2910-2917. | Show Abstract | Read more

Nucleic acid amplification methods such as the PCR have had a major impact on the diagnosis of viral infections, often achieving greater sensitivities and shorter turnaround times than conventional assays and an ability to detect viruses refractory to conventional isolation methods. Their effectiveness is, however, significantly influenced by assay target sequence variability due to natural diversity and rapid sequence changes in viruses that prevent effective binding of primers and probes. This was investigated for a diverse range of enteroviruses (EVs; species A to D), human rhinoviruses (HRVs; species A to C), and human parechovirus (HPeV) in a multicenter assay evaluation using a series of full-length prequantified RNA transcripts. RNA concentrations were quantified by absorption (NanoDrop) and fluorescence methods (RiboGreen) prior to dilution in buffer supplemented with RNase inhibitors and carrier RNA. RNA transcripts were extremely stable, showing minimal degradation after prolonged storage at temperatures between ambient and -20°C and after multiple freeze-thaw cycles. Transcript dilutions distributed to six referral laboratories were screened by real-time reverse transcriptase PCR assays using different primers and probes. All of the laboratories reported high assay sensitivities for EV and HPeV transcripts approaching single copies and similar amplification kinetics for all four EV species. HRV detection sensitivities were more variable, often with substantially impaired detection of HRV species C. This could be accounted for in part by the placement of primers and probes to genetically variable target regions. Transcripts developed in this study provide reagents for the ongoing development of effective diagnostics that accommodate increasing knowledge of genetic heterogeneity of diagnostic targets.

Tuplin A, Struthers M, Simmonds P, Evans DJ. 2012. A twist in the tail: SHAPE mapping of long-range interactions and structural rearrangements of RNA elements involved in HCV replication. Nucleic Acids Res, 40 (14), pp. 6908-6921. | Show Abstract | Read more

The RNA structure and long-range interactions of the SL9266 cis-acting replication element located within the NS5B coding region of hepatitis C virus (HCV) were determined using selective 2'-hydroxyl acylation analysed by primer extension. Marked differences were found in the long-range interactions of SL9266 when the two widely used genotype 2a JFH-1 (HCVcc) and genotype 1b Con1b sub-genomic replicon systems were compared. In both genomes, there was evidence for interaction of the sub-terminal bulge loop of SL9266 and sequences around nucleotide 9110, though the replication phenotype of genomes bearing mutations that disrupted this interaction was fundamentally different. In contrast, a 'kissing loop' interaction between the terminal loop of SL9266 and sequences in the 3'-untranslated X-tail was only detectable in JFH-1-based genomes. In the latter, where both long-range interactions are present, they were independent, implying that SL9266 forms the core of an extended pseudoknot. The presence of the 'kissing loop' interaction inhibited the formation of SL9571 in the 3'-X-tail, an RNA structure implicated in genome replication. We propose that, SL9266 may contribute a switch function that modulates the mutually incompatible translation and replication events that must occur for replication of the positive-strand RNA genome of HCV.

Sharp CP, Lail A, Donfield S, Gomperts ED, Simmonds P. 2012. Virologic and clinical features of primary infection with human parvovirus 4 in subjects with hemophilia: frequent transmission by virally inactivated clotting factor concentrates. Transfusion, 52 (7), pp. 1482-1489. | Show Abstract | Read more

BACKGROUND: Human parvovirus 4 (PARV4) is a newly discovered parvovirus prevalent in injecting drug users and other groups with histories of parenteral exposure including persons with hemophilia exposed to non-virally inactivated clotting factor concentrates. To investigate its potential ongoing transmission to persons with hemophilia treated with plasma-derived, virally inactivated clotting factors, we screened a large cohort of persons with hemophilia for antibody seroconversion to PARV4 over a 5-year observation period. STUDY DESIGN AND METHODS: Samples from 195 persons with hemophilia enrolled in the Hemophilia Growth and Development Study cohort were screened for PARV4 antibodies at the start and end of a 5-year period of treatment with exclusively virally inactivated clotting factor concentrates. Samples collected at intermediate time points from subjects seroconverting over the study period were screened to narrow down the seroconversion time and investigate immunoglobulin (Ig)M responses, duration of acute viremia, and clinical presentations. RESULTS: PARV4 seroprevalence at the outset of the study was 44%. Over the observation period, nine subjects (seven human immunodeficiency virus positive) seroconverted for anti-PARV4 (incidence, 1.7%/year). Infected subjects showed relatively prolonged durations of viremia (mean, 7 months) and weak, transient IgM responses during acute infections. Clotting factors inactivated by solvent/detergent or by wet or dry heat were infectious. The most common clinical presentations were rashes and exacerbation of hepatitis. CONCLUSION: This study identifies PARV4 as a transfusion-transmissible agent that is resistant to viral inactivation. Of concern, infections may still regularly occur in those exposed to plasma-derived blood products. Urgent evaluation of the incidence of PARV4 in treated individuals and disease associations of PARV4 infections is required.

Simmonds P, Sharp CP, Donfield S, Gomperts ED. 2012. Assessing causality in the transmission of viruses by blood products reply TRANSFUSION, 52 (7), pp. 1598-1599. | Read more

Lycett S, McLeish NJ, Robertson C, Carman W, Baillie G, McMenamin J, Rambaut A, Simmonds P, Woolhouse M, Leigh Brown AJ. 2012. Origin and fate of A/H1N1 influenza in Scotland during 2009. J Gen Virol, 93 (Pt 6), pp. 1253-1260. | Show Abstract | Read more

The spread of influenza has usually been described by a 'density' model, where the largest centres of population drive the epidemic within a country. An alternative model emphasizing the role of air travel has recently been developed. We have examined the relative importance of the two in the context of the 2009 H1N1 influenza epidemic in Scotland. We obtained genome sequences of 70 strains representative of the geographical and temporal distribution of H1N1 influenza during the summer and winter phases of the pandemic in 2009. We analysed these strains, together with another 128 from the rest of the UK and 292 globally distributed strains, using maximum-likelihood phylogenetic and bayesian phylogeographical methods. This revealed strikingly different epidemic patterns within Scotland in the early and late parts of 2009. The summer epidemic in Scotland was characterized by multiple independent introductions from both international and other UK sources, followed by major local expansion of a single clade that probably originated in Birmingham. The winter phase, in contrast, was more diverse genetically, with several clades of similar size in different locations, some of which had no particularly close phylogenetic affinity to strains sampled from either Scotland or England. Overall there was evidence to support both models, with significant links demonstrated between North American sequences and those from England, and between England and East Asia, indicating that major air-travel routes played an important role in the pattern of spread of the pandemic, both within the UK and globally.

Burbelo PD, Dubovi EJ, Simmonds P, Medina JL, Henriquez JA, Mishra N, Wagner J, Tokarz R, Cullen JM, Iadarola MJ et al. 2012. Serology-enabled discovery of genetically diverse hepaciviruses in a new host. J Virol, 86 (11), pp. 6171-6178. | Show Abstract | Read more

Genetic and biological characterization of new hepaciviruses infecting animals contributes to our understanding of the ultimate origins of hepatitis C virus (HCV) infection in humans and dramatically enhances our ability to study its pathogenesis using tractable animal models. Animal homologs of HCV include a recently discovered canine hepacivirus (CHV) and GB virus B (GBV-B), both viruses with largely undetermined natural host ranges. Here we used a versatile serology-based approach to determine the natural host of the only known nonprimate hepacivirus (NPHV), CHV, which is also the closest phylogenetic relative of HCV. Recombinant protein expressed from the helicase domain of CHV NS3 was used as antigen in the luciferase immunoprecipitation system (LIPS) assay to screen several nonprimate animal species. Thirty-six samples from 103 horses were immunoreactive, and viral genomic RNA was present in 8 of the 36 seropositive animals and none of the seronegative animals. Complete genome sequences of these 8 genetically diverse NPHVs showed 14% (range, 6.4% to 17.2%) nucleotide sequence divergence, with most changes occurring at synonymous sites. RNA secondary structure prediction of the 383-base 5' untranslated region of NPHV was refined and extended through mapping of polymorphic sites to unpaired regions or (semi)covariant pairings. Similar approaches were adopted to delineate extensive RNA secondary structures in the coding region of the genome, predicted to form 27 regularly spaced, thermodynamically stable stem-loops. Together, these findings suggest a promising new nonprimate animal model and provide a database that will aid creation of functional NPHV cDNA clones and other novel tools for hepacivirus studies.

Simmons R, Sharp C, McClure CP, Rohrbach J, Kovari H, Frangou E, Simmonds P, Irving W, Rauch A, Bowness P et al. 2012. Parvovirus 4 infection and clinical outcome in high-risk populations. J Infect Dis, 205 (12), pp. 1816-1820. | Show Abstract | Read more

Parvovirus 4 (PARV4) is a DNA virus frequently associated with human immunodeficiency virus (HIV) and hepatitis C virus (HCV) infections, but its clinical significance is unknown. We studied the prevalence of PARV4 antibodies in 2 cohorts of HIV- and HCV-infected individuals (n = 469) and the correlations with disease status. We found that PARV4 infection frequently occurred in individuals exposed to bloodborne viruses (95% in HCV-HIV coinfected intravenous drug users [IDUs]). There were no correlations between PARV4 serostatus and HCV outcomes. There was, however, a significant association with early HIV-related symptoms, although because this was tightly linked to both HCV status and clinical group (IDU), the specific role of PARV4 is not yet clear.

Gonzalez-Perez MP, O'Connell O, Lin R, Sullivan WM, Bell J, Simmonds P, Clapham PR. 2012. Independent evolution of macrophage-tropism and increased charge between HIV-1 R5 envelopes present in brain and immune tissue. Retrovirology, 9 (1), pp. 20. | Show Abstract | Read more

BACKGROUND: Transmitted HIV-1 clade B or C R5 viruses have been reported to infect macrophages inefficiently, while other studies have described R5 viruses in late disease with either an enhanced macrophage-tropism or carrying envelopes with an increased positive charge and fitness. In contrast, our previous data suggested that viruses carrying non-macrophage-tropic R5 envelopes were still predominant in immune tissue of AIDS patients. To further investigate the tropism and charge of HIV-1 viruses in late disease, we evaluated the properties of HIV-1 envelopes amplified from immune and brain tissues of AIDS patients with neurological complications. RESULTS: Almost all envelopes amplified were R5. There was clear compartmentalization of envelope sequences for four of the five subjects. However, strong compartmentalization of macrophage-tropism in brain was observed even when brain and immune tissue envelope sequences were not segregated. R5 envelopes from immune tissue of four subjects carried a higher positive charge compared to brain envelopes. We also confirm a significant correlation between macrophage tropism and sensitivity to soluble CD4, a weak association with sensitivity to the CD4 binding site antibody, b12, but no clear relationship with maraviroc sensitivity. CONCLUSIONS: Our study shows that non-macrophage-tropic R5 envelopes carrying gp120s with an increased positive charge were predominant in immune tissue in late disease. However, highly macrophage-tropic variants with lower charged gp120s were nearly universal in the brain. These results are consistent with HIV-1 R5 envelopes evolving gp120s with an increased positive charge in immune tissue or sites outside the brain that likely reflect an adaptation for increased replication or fitness for CD4+ T-cells. Our data are consistent with the presence of powerful pressures in brain and in immune tissues selecting for R5 envelopes with very different properties; high macrophage-tropism, sCD4 sensitivity and low positive charge in brain and non-macrophage-tropism, sCD4 resistance and high positive charge in immune tissue.

McWilliam Leitch EC, Cabrerizo M, Cardosa J, Harvala H, Ivanova OE, Koike S, Kroes AC, Lukashev A, Perera D, Roivainen M et al. 2012. The association of recombination events in the founding and emergence of subgenogroup evolutionary lineages of human enterovirus 71. J Virol, 86 (5), pp. 2676-2685. | Show Abstract | Read more

Enterovirus 71 (EV71) is responsible for frequent large-scale outbreaks of hand, foot, and mouth disease worldwide and represent a major etiological agent of severe, sometimes fatal neurological disease. EV71 variants have been classified into three genogroups (GgA, GgB, and GgC), and the latter two are further subdivided into subgenogroups B1 to B5 and C1 to C5. To investigate the dual roles of recombination and evolution in the epidemiology and transmission of EV71 worldwide, we performed a large-scale genetic analysis of isolates (n = 308) collected from 19 countries worldwide over a 40-year period. A series of recombination events occurred over this period, which have been identified through incongruities in sequence grouping between the VP1 and 3Dpol regions. Eleven 3Dpol clades were identified, each specific to EV71 and associated with specific subgenogroups but interspersed phylogenetically with clades of coxsackievirus A16 and other EV species A serotypes. The likelihood of recombination increased with VP1 sequence divergence; mean half-lives for EV71 recombinant forms (RFs) of 6 and 9 years for GgB and GgC overlapped with those observed for the EV-B serotypes, echovirus 9 (E9), E30, and E11, respectively (1.3 to 9.8 years). Furthermore, within genogroups, sporadic recombination events occurred, such as the linkage of two B4 variants to RF-W instead of RF-A and of two C4 variants to RF-H. Intriguingly, recombination events occurred as a founding event of most subgenogroups immediately preceding their lineage expansion and global emergence. The possibility that recombination contributed to their subsequent spread through improved fitness requires further biological and immunological characterization.

Harvala H, McIntyre CL, McLeish NJ, Kondracka J, Palmer J, Molyneaux P, Gunson R, Bennett S, Templeton K, Simmonds P. 2012. High detection frequency and viral loads of human rhinovirus species A to C in fecal samples; diagnostic and clinical implications. J Med Virol, 84 (3), pp. 536-542. | Show Abstract | Read more

Human rhinoviruses (HRVs) can be divided into three species; HRV-A to HRV-C. Up to 148 different HRV (sero)types have been identified to date. Because of sequence similarity between 5'-NCR of HRVs and enteroviruses (EVs), it is problematic to design EV-specific RT-PCR assays. The aims of this study were to assess the rate of false-detection of different rhinoviruses by EV RT-PCR, and to evaluate the diagnostic and clinical significance of such cross-reactivity. In vitro RNA transcripts of HRV A-C created from cDNA templates were quantified spectrophotometrically. Six hundred twenty-one stool samples screened as part of routine diagnostic for EV, 17 EV-positive stool samples referred for typing, 288 stool samples submitted for gastroenteritis investigations, and 1,500 CSF samples were included in the study. EV-specific RT-PCR detected RNA transcripts of HRV-A1b, HRV-B14, and HRV-Crpat18 but with 10-1,000 reduced sensitivity compared to EV transcripts. Screening fecal samples by EV RT-PCR identified 13 positive samples identified subsequently as rhinoviruses; a further 26 HRV-positive samples were identified by nested HRV RT-PCR. All individuals were hospitalized and presented mostly with diarrhea. A total of 26 HRV types were identified (HRV-A: 46%; HRV-B: 13%; HRV-C: 41%). Results confirm that EV-specific RT-PCR can detect HRVs, and at a practical level, identify potential problems of interpretation if fecal samples are used for surrogate screening in cases of suspected viral meningitis. High detection frequencies (10%) and viral loads in stool samples provide evidence for enteric replication of HRV, and its association with enteric disease requires further etiological studies.

Smith DB, McFadden N, Blundell RJ, Meredith A, Simmonds P. 2012. Diversity of murine norovirus in wild-rodent populations: species-specific associations suggest an ancient divergence. J Gen Virol, 93 (Pt 2), pp. 259-266. | Show Abstract | Read more

A survey of wild-rodent populations has revealed that murine norovirus (MNV) is present and diverse in wild-house mice Mus musculus. This virus is genetically similar to MNV infecting show mice and previously described variants circulating in laboratory mice. The detection of MNV in wild-mouse populations suggests that MNV infection of laboratory mice and show mice (from which laboratory mice are derived) derives from contact with or their origins from wild-mouse progenitors. The survey additionally identified frequent infection of wood mice (Apodemus sylvaticus) with genetically divergent variants of MNV. These viruses are distinct from previously described MNV variants, differing by 22-23 % over the complete genome sequence compared with a maximum of 13 % between M. musculus-derived strains. Comparison with other noroviruses reveals that the Apodemus MNV groups with MNV in genogroup V and shares the same overall genome organization, predicted lengths of proteins encoded by ORFs 1-3 and the existence of a conserved alternative reading frame in VP1 encoding a homologue of the MNV ORF4. Different Apodemus MNV isolates were as variable as MNV isolates and showed evidence for inter-isolate recombination. Our observation of species-specific associations of MNV variants in wild populations suggests that murine noroviruses have an ancient origin, a feature that they may share with other norovirus genogroups.

Harvala H, McIntyre CL, Imai N, Clasper L, Djoko CF, LeBreton M, Vermeulen M, Saville A, Mutapi F, Tamoufé U et al. 2012. High seroprevalence of enterovirus infections in apes and old world monkeys. Emerg Infect Dis, 18 (2), pp. 283-286. | Show Abstract | Read more

To estimate population exposure of apes and Old World monkeys in Africa to enteroviruses (EVs), we conducted a seroepidemiologic study of serotype-specific neutralizing antibodies against 3 EV types. Detection of species A, B, and D EVs infecting wild chimpanzees demonstrates their potential widespread circulation in primates.

Kapoor A, Mehta N, Dubovi EJ, Simmonds P, Govindasamy L, Medina JL, Street C, Shields S, Lipkin WI. 2012. Characterization of novel canine bocaviruses and their association with respiratory disease. J Gen Virol, 93 (Pt 2), pp. 341-346. | Show Abstract | Read more

We report the first identification, genetic characterization and disease association studies of several novel species of canine bocaviruses (CBoV). Evolutionary analysis confirmed that CBoV are genetically distinct from the only other known canine bocavirus, minute virus of canines, with which they share less than 63, 62 and 64 % protein identity in NS, NP and VP genes, respectively. Comparative genetic analysis of 37 VP gene variants found in diseased and healthy animals showed that these novel viruses are genetically highly diverse and are common in canine respiratory infections that have remained undetected until now. Interestingly, we observed that a CBoV genotype with a unique deletion in the VP2 gene was significantly more prevalent in animals with respiratory diseases compared with healthy animals.

Stapleton JT, Smith DB, Simmonds P. 2012. Evidence against GB virus C infection in dromedary camels. Vet Microbiol, 154 (3-4), pp. 403-406. | Show Abstract | Read more

A recent publication described finding GB virus C (GBV-C) RNA in 4 of 22 dromedary camel sera, and sequence analysis found that these viruses were phylogenetically clustered within human GBV-C isolates. Since all other GB viruses to date form monophyletic groups according to their host species, the close relationship between the sequences generated from camel sera and human GBV-C isolates seemed implausible, leading us to conduct an independent analysis of the sequences. Our investigation found three lines of evidence arguing against GBV-C infection in dromedary camels. First, strong evidence of artifactual sequence generation was identified for some of the sequences. Secondly, the sequence diversity within individual camel sera was 10-152-fold greater than that described for GBV-C within a human host. Finally, GBV-C sequences generated from each camel shared near complete identity with human isolates previously described by the same laboratory. Taken together, these data strongly suggest laboratory contamination. We suggest that additional validation experiments are needed before it is possible to conclude that camels are permissive for GBV-C infection.

Simmonds P. 2012. SSE: a nucleotide and amino acid sequence analysis platform. BMC Res Notes, 5 (1), pp. 50. | Show Abstract | Read more

BACKGROUND: There is an increasing need to develop bioinformatic tools to organise and analyse the rapidly growing amount of nucleotide and amino acid sequence data in organisms ranging from viruses to eukaryotes. FINDING: A simple sequence editor (SSE) was developed to create an integrated environment where sequences can be aligned, annotated, classified and directly analysed by a number of built-in bioinformatic programs. SSE incorporates a sequence editor for the creation of sequence alignments, a process assisted by integrated CLUSTAL/MUSCLE alignment programs and automated removal of indels. Sequences can be fully annotated and classified into groups and annotated of sequences and sequence groups and access to analytical programs that analyse diversity, recombination and RNA secondary structure. Methods for analysing sequence diversity include measures of divergence and evolutionary distances, identity plots to detect regions of nucleotide or amino acid homology, reconstruction of sequence changes, mono-, di- and higher order nucleotide compositional biases and codon usage.Association Index calculations, GroupScans, Bootscanning and TreeOrder scans perform phylogenetic analyses that reconcile group membership with tree branching orders and provide powerful methods for examining segregation of alleles and detection of recombination events. Phylogeny changes across alignments and scoring of branching order differences between trees using the Robinson-Fould algorithm allow effective visualisation of the sites of recombination events.RNA secondary and tertiary structures play important roles in gene expression and RNA virus replication. For the latter, persistence of infection is additionally associated with pervasive RNA secondary structure throughout viral genomic RNA that modulates interactions with innate cell defences. SSE provides several programs to scan alignments for RNA secondary structure through folding energy thermodynamic calculations and phylogenetic methods (detection of co-variant changes, and structure conservation between divergent sequences). These analyses complement methods based on detection of sequence constraints, such as suppression of synonymous site variability.For each program, results can be plotted in real time during analysis through an integrated graphics package, providing publication quality graphs. Results can be also directed to tabulated datafiles for import into spreadsheet or database programs for further analysis. CONCLUSIONS: SSE combines sequence editor functions with analytical tools in a comprehensive and user-friendly package that assists considerably in bioinformatic and evolution research.

Lyons S, Sharp C, LeBreton M, Djoko CF, Kiyang JA, Lankester F, Bibila TG, Tamoufé U, Fair J, Wolfe ND, Simmonds P. 2012. Species association of hepatitis B virus (HBV) in non-human apes; evidence for recombination between gorilla and chimpanzee variants. PLoS One, 7 (3), pp. e33430. | Show Abstract | Read more

Hepatitis B virus (HBV) infections are widely distributed in humans, infecting approximately one third of the world's population. HBV variants have also been detected and genetically characterised from Old World apes; Gorilla gorilla (gorilla), Pan troglodytes (chimpanzee), Pongo pygmaeus (orang-utan), Nomascus nastusus and Hylobates pileatus (gibbons) and from the New World monkey, Lagothrix lagotricha (woolly monkey). To investigate species-specificity and potential for cross species transmission of HBV between sympatric species of apes (such as gorillas and chimpanzees in Central Africa) or between humans and chimpanzees or gorillas, variants of HBV infecting captive wild-born non-human primates were genetically characterised. 9 of 62 chimpanzees (11.3%) and two from 11 gorillas (18%) were HBV-infected (15% combined frequency), while other Old world monkey species were negative. Complete genome sequences were obtained from six of the infected chimpanzee and both gorillas; those from P. t .ellioti grouped with previously characterised variants from this subspecies. However, variants recovered from P. t. troglodytes HBV variants also grouped within this clade, indicative of transmission between sub-species, forming a paraphyletic clade. The two gorilla viruses were phylogenetically distinct from chimpanzee and human variants although one showed evidence for a recombination event with a P.t.e.-derived HBV variant in the partial X and core gene region. Both of these observations provide evidence for circulation of HBV between different species and sub-species of non-human primates, a conclusion that differs from the hypothesis if of strict host specificity of HBV genotypes.

Lavoie M, Sharp CP, Pépin J, Pennington C, Foupouapouognigni Y, Pybus OG, Njouom R, Simmonds P. 2012. Human parvovirus 4 infection, Cameroon. Emerg Infect Dis, 18 (4), pp. 680-683. | Show Abstract | Read more

In a post hoc analysis of samples collected in 2009, we determined seroprevalence of parvovirus 4 (PARV4) among elderly Cameroonians. PARV4 seropositivity was associated with receipt of intravenous antimalarial drugs, intramuscular streptomycin, or an intramuscular contraceptive, but not hepatitis C virus seropositivity. Findings suggest parenteral acquisition of some PARV4 infections.

Boros Á, Pankovics P, Simmonds P, Reuter G. 2011. Novel positive-sense, single-stranded RNA (+ssRNA) virus with di-cistronic genome from intestinal content of freshwater carp (Cyprinus carpio). PLoS One, 6 (12), pp. e29145. | Show Abstract | Read more

A novel positive-sense, single-stranded RNA (+ssRNA) virus (Halastavi árva RNA virus, HalV; JN000306) with di-cistronic genome organization was serendipitously identified in intestinal contents of freshwater carps (Cyprinus carpio) fished by line-fishing from fishpond "Lőrinte halastó" located in Veszprém County, Hungary. The complete nucleotide (nt) sequence of the genomic RNA is 9565 nt in length and contains two long--non-in-frame--open reading frames (ORFs), which are separated by an intergenic region. The ORF1 (replicase) is preceded by an untranslated sequence of 827 nt, while an untranslated region of 139 nt follows the ORF2 (capsid proteins). The deduced amino acid (aa) sequences of the ORFs showed only low (less than 32%) and partial similarity to the non-structural (2C-like helicase, 3C-like cystein protease and 3D-like RNA dependent RNA polymerase) and structural proteins (VP2/VP4/VP3) of virus families in Picornavirales especially to members of the viruses with dicistronic genome. Halastavi árva RNA virus is present in intestinal contents of omnivorous freshwater carps but the origin and the host species of this virus remains unknown. The unique viral sequence and the actual position indicate that Halastavi árva RNA virus seems to be the first member of a new di-cistronic ssRNA virus. Further studies are required to investigate the specific host species (and spectrum), ecology and role of Halastavi árva RNA virus in the nature.

McFadden N, Bailey D, Carrara G, Benson A, Chaudhry Y, Shortland A, Heeney J, Yarovinsky F, Simmonds P, Macdonald A, Goodfellow I. 2011. Norovirus regulation of the innate immune response and apoptosis occurs via the product of the alternative open reading frame 4. PLoS Pathog, 7 (12), pp. e1002413. | Show Abstract | Read more

Small RNA viruses have evolved many mechanisms to increase the capacity of their short genomes. Here we describe the identification and characterization of a novel open reading frame (ORF4) encoded by the murine norovirus (MNV) subgenomic RNA, in an alternative reading frame overlapping the VP1 coding region. ORF4 is translated during virus infection and the resultant protein localizes predominantly to the mitochondria. Using reverse genetics we demonstrated that expression of ORF4 is not required for virus replication in tissue culture but its loss results in a fitness cost since viruses lacking the ability to express ORF4 restore expression upon repeated passage in tissue culture. Functional analysis indicated that the protein produced from ORF4 antagonizes the innate immune response to infection by delaying the upregulation of a number of cellular genes activated by the innate pathway, including IFN-Beta. Apoptosis in the RAW264.7 macrophage cell line was also increased during virus infection in the absence of ORF4 expression. In vivo analysis of the WT and mutant virus lacking the ability to express ORF4 demonstrated an important role for ORF4 expression in infection and virulence. STAT1-/- mice infected with a virus lacking the ability to express ORF4 showed a delay in the onset of clinical signs when compared to mice infected with WT virus. Quantitative PCR and histopathological analysis of samples from these infected mice demonstrated that infection with a virus not expressing ORF4 results in a delayed infection in this system. In light of these findings we propose the name virulence factor 1, VF1 for this protein. The identification of VF1 represents the first characterization of an alternative open reading frame protein for the calicivirus family. The immune regulatory function of the MNV VF1 protein provide important perspectives for future research into norovirus biology and pathogenesis.

Sharp PM, Simmonds P. 2011. Evaluating the evidence for virus/host co-evolution. Curr Opin Virol, 1 (5), pp. 436-441. | Show Abstract | Read more

There is currently much debate about the timescales of virus evolution. Some viruses may have co-evolved with human populations for tens of thousands of years, or even with our primate ancestors over many millions of years. However, calibrations of the rate of short-term virus evolution lead to estimates of dates for viral ancestors that are orders of magnitude more recent, and a number of the proposed host-virus co-divergence scenarios have been questioned. Other considerations indicate that the proposed recent timescales for virus evolution are implausible, that co-divergence has been rejected prematurely, and that long-term evolutionary rates are very much slower than short-term rates. There is a need to understand the biological basis of this discrepancy and to develop evolutionary models that can accommodate this.

Shan T, Li L, Simmonds P, Wang C, Moeser A, Delwart E. 2011. The fecal virome of pigs on a high-density farm. J Virol, 85 (22), pp. 11697-11708. | Show Abstract | Read more

Swine are an important source of proteins worldwide but are subject to frequent viral outbreaks and numerous infections capable of infecting humans. Modern farming conditions may also increase viral transmission and potential zoonotic spread. We describe here the metagenomics-derived virome in the feces of 24 healthy and 12 diarrheic piglets on a high-density farm. An average of 4.2 different mammalian viruses were shed by healthy piglets, reflecting a high level of asymptomatic infections. Diarrheic pigs shed an average of 5.4 different mammalian viruses. Ninety-nine percent of the viral sequences were related to the RNA virus families Picornaviridae, Astroviridae, Coronaviridae, and Caliciviridae, while 1% were related to the small DNA virus families Circoviridae, and Parvoviridae. Porcine RNA viruses identified, in order of decreasing number of sequence reads, consisted of kobuviruses, astroviruses, enteroviruses, sapoviruses, sapeloviruses, coronaviruses, bocaviruses, and teschoviruses. The near-full genomes of multiple novel species of porcine astroviruses and bocaviruses were generated and phylogenetically analyzed. Multiple small circular DNA genomes encoding replicase proteins plus two highly divergent members of the Picornavirales order were also characterized. The possible origin of these viral genomes from pig-infecting protozoans and nematodes, based on closest sequence similarities, is discussed. In summary, an unbiased survey of viruses in the feces of intensely farmed animals revealed frequent coinfections with a highly diverse set of viruses providing favorable conditions for viral recombination. Viral surveys of animals can readily document the circulation of known and new viruses, facilitating the detection of emerging viruses and prospective evaluation of their pathogenic and zoonotic potentials.

Gaunt ER, Harvala H, McIntyre C, Templeton KE, Simmonds P. 2011. Disease burden of the most commonly detected respiratory viruses in hospitalized patients calculated using the disability adjusted life year (DALY) model. J Clin Virol, 52 (3), pp. 215-221. | Show Abstract | Read more

BACKGROUND: The most common acute infections occur in the respiratory tract. Recent discoveries of several novel viruses have markedly increased the repertoire of agents understood to cause presentations of acute respiratory disease. OBJECTIVES: Further understanding is needed of the relative importance of newly discovered pathogens in the clinical setting to provide clinicians with an indication of appropriate diagnostic and therapeutic targets. To address this, quantification of the disease burden of respiratory viruses in hospitalized patients was undertaken. STUDY DESIGN: Disease burden caused by respiratory viruses in hospitalized patients was quantified using the World Health Organization endorsed DALY model. Diagnostic testing results from samples collected over three years for adenovirus (AdV), influenzas A and B, parainfluenza viruses 1, 2 and 3 (PIV-1, -2 and -3), respiratory syncytial virus (HRSV), and previously published retrospective screening for human metapneumovirus, rhinoviruses, and four respiratory coronaviruses were applied to the DALY model. Disability weights were calculated per 1000 hospitalized patients in age banded groups. RESULTS: Strikingly different disease burden profiles were observed in children and adults. Adenoviruses were among the leading cause of respiratory presentations in children but not adults. HRSV and influenza A were consistently one of the greatest causes of disease regardless of sampled population. Rhinoviruses and PIV-3 were significant pathogens in all groups except those aged 16-64 years. In immunocompromised patients rhinoviruses were the leading viral cause of disease. CONCLUSIONS: These analyses provide a framework which can be used to identify where finite resources should be directed in respiratory therapeutics and vaccine development.

Kapoor A, Simmonds P, Dubovi EJ, Qaisar N, Henriquez JA, Medina J, Shields S, Lipkin WI. 2011. Characterization of a canine homolog of human Aichivirus. J Virol, 85 (21), pp. 11520-11525. | Show Abstract | Read more

Many of our fatal "civilization" infectious diseases have arisen from domesticated animals. Although picornaviruses infect most mammals, infection of a companion animal is not known. Here we describe the identification and genomic characterization of the first canine picornavirus. Canine kobuvirus (CKoV), identified in stool samples from dogs with diarrhea, has a genomic organization typical of a picornavirus and encodes a 2,469-amino-acid polyprotein flanked by 5' and 3' untranslated regions. Comparative phylogenetic analysis using various structural and nonstructural proteins of CKoV confirmed it as the animal virus homolog most closely related to human Aichivirus (AiV). Bayesian Markov chain Monte Carlo analysis suggests a mean recent divergence time of CKoV and AiV within the past 20 to 50 years, well after the domestication of canines. The discovery of CKoV provides new insights into the origin and evolution of AiV and the species specificity and pathogenesis of kobuviruses.

Simmonds P, Domingo E. 2011. Virus evolution. Curr Opin Virol, 1 (5), pp. 410-412. | Read more

Benjamin LA, Lewthwaite P, Vasanthapuram R, Zhao G, Sharp C, Simmonds P, Wang D, Solomon T. 2011. Human parvovirus 4 as potential cause of encephalitis in children, India. Emerg Infect Dis, 17 (8), pp. 1484-1487. | Show Abstract | Read more

To investigate whether uncharacterized infectious agents were associated with neurologic disease, we analyzed cerebrospinal fluid specimens from 12 children with acute central nervous system infection. A high-throughput pyrosequencing screen detected human parvovirus 4 DNA in cerebrospinal fluid of 2 children with encephalitis of unknown etiology.

Leitch ECM, Harvala H, Robertson I, Ubillos I, Templeton K, Simmonds P. 2011. Direct identification of human enterovirus serotypes in cerebrospinal fluid by amplification and sequencing of the VP1 region (vol 44, pg 119, 2009) JOURNAL OF CLINICAL VIROLOGY, 51 (4), pp. 286-286. | Read more

Kapoor A, Simmonds P, Gerold G, Qaisar N, Jain K, Henriquez JA, Firth C, Hirschberg DL, Rice CM, Shields S, Lipkin WI. 2011. Characterization of a canine homolog of hepatitis C virus. Proc Natl Acad Sci U S A, 108 (28), pp. 11608-11613. | Show Abstract | Read more

An estimated 3% of the world's population is chronically infected with hepatitis C virus (HCV). Although HCV was discovered more than 20 y ago, its origin remains obscure largely because no closely related animal virus homolog has been identified; furthermore, efforts to understand HCV pathogenesis have been hampered by the absence of animal models other than chimpanzees for human disease. Here we report the identification in domestic dogs of a nonprimate hepacivirus. Comparative phylogenetic analysis of the canine hepacivirus (CHV) confirmed it to be the most genetically similar animal virus homolog of HCV. Bayesian Markov chains Monte Carlo and associated time to most recent common ancestor analyses suggest a mean recent divergence time of CHV and HCV clades within the past 500-1,000 y, well after the domestication of canines. The discovery of CHV may provide new insights into the origin and evolution of HCV and a tractable model system with which to probe the pathogenesis, prevention, and treatment of diseases caused by hepacivirus infection.

Bennett S, Harvala H, Witteveldt J, McWilliam Leitch EC, McLeish N, Templeton K, Gunson R, Carman WF, Simmonds P. 2011. Rapid simultaneous detection of enterovirus and parechovirus RNAs in clinical samples by one-step real-time reverse transcription-PCR assay. J Clin Microbiol, 49 (7), pp. 2620-2624. | Show Abstract | Read more

Enteroviruses (EVs) are recognized as the major etiological agent in meningitis in children and young adults. The use of molecular techniques, such as PCR, has substantially improved the sensitivity of enterovirus detection compared to that of virus culture methods. PCR-based methods also can detect a much wider range of EV variants, including those within species A, as well as human parechoviruses (HPeVs) that often grow poorly in vitro and which previously have been underdiagnosed by traditional methods. To exploit these developments, we developed a real-time one-step reverse transcription-PCR (RT-PCR) for the rapid and sensitive detection of EV and HPeV in clinical specimens. Two commercially available RT-PCR kits were used (method I, Platinum one-step kit; method II, Express qPCR one-step kit) with primers and probes targeting the EV and HPeV 5'-untranslated regions (5'UTR). Amplification dynamics (threshold cycle [C(T)]values and efficiencies) of absolutely quantified full-length RNA transcripts representative of EV species A to D and HPeV were similar, demonstrating the effectiveness of both assays across the range of currently described human EV and HPeV variants. Probit analysis of multiple endpoint replicates demonstrated comparable sensitivities of the assays for EV and HPeV (method I, approximately 10 copies per reaction for both targets; method II, 20 copies per reaction). C(T) values were highly reproducible on repeat testing of positive controls within assays and between assay runs. Considering the sample turnaround time of less than 3 h, the multiplexed one-step RT-PCR method provides rapid diagnostic testing for EV and HPeV in cases of suspected central nervous system infections in a clinically relevant time frame.

Harvala H, Sharp CP, Ngole EM, Delaporte E, Peeters M, Simmonds P. 2011. Detection and genetic characterization of enteroviruses circulating among wild populations of chimpanzees in Cameroon: relationship with human and simian enteroviruses. J Virol, 85 (9), pp. 4480-4486. | Show Abstract | Read more

Enteroviruses (EVs), members of the family Picornaviridae, are a genetically and antigenically diverse range of viruses causing acute infections in humans and several Old World monkey (OWM) species. Despite their known wide distribution in primates, nothing is currently known about the occurrence, frequency, and genetic diversity of enteroviruses infecting apes. To investigate this, 27 chimpanzee and 27 gorilla fecal samples collected from undisturbed jungle areas with minimal human contact in Cameroon were screened for EVs. Four chimpanzee samples were positive, but none of the gorilla samples were positive. Genetic characterization of the VP1, VP4, and partial VP2 genes, the 5' untranslated region, and partial 3Dpol sequences enabled chimpanzee-derived EVs to be identified as (i) the species A type, EV76, (ii) a new species D type assigned as EV111, along with a human isolate from the Democratic Republic of Congo previously described by the International Committee on the Taxonomy of Viruses, and (iii) a new species B type (assigned as EV110) most closely related to, although a distinct type from, the SA5 isolate recovered from a vervet monkey. The identification of EVs infecting chimpanzees related to those circulating in human and OWM populations provides evidence for cross-species transmission of EVs between primates. However, the direction of transfer and the existence of primate sources of zoonotic enterovirus infections in humans require further investigation of population exposure and more extensive characterization of EVs circulating in wild ape populations.

Harvala H, McLeish N, Kondracka J, McIntyre CL, McWilliam Leitch EC, Templeton K, Simmonds P. 2011. Comparison of human parechovirus and enterovirus detection frequencies in cerebrospinal fluid samples collected over a 5-year period in edinburgh: HPeV type 3 identified as the most common picornavirus type. J Med Virol, 83 (5), pp. 889-896. | Show Abstract | Read more

Human enteroviruses (EVs) and more recently parechoviruses (HPeVs) have been identified as the principal viral causes of neonatal sepsis-like disease and meningitis. The relative frequencies of specific EV and HPeV types were determined over a 5-year surveillance period using highly sensitive EV and HPeV PCR assays for screening 4,168 cerebrospinal fluid (CSF) specimens collected from hospitalized individuals between 2005 and 2010 in Edinburgh. Positive CSF samples were typed by sequencing of VP1. From the 201 EV and 31 HPeV positive (uncultured) CSF samples on screening, a high proportion of available samples could be directly typed (176/182, 97%). Highest frequencies of EV infections occurred in young adults (n = 43; 8.6%) although a remarkably high proportion of positive samples (n = 98; 46%) were obtained from young infants (<3 months). HPeV infections were seen exclusively in children under the age of 3 months (31/1,105; 2.8%), and confined to spring on even-numbered years (22% in March 2006, 25% in April 2008, and 22% in March 2010). In contrast, EV infections were distributed widely across the years. Twenty different EV serotypes were detected; E9, E6, and CAV9 being found most frequently, whereas all but one HPeVs were type 3. Over this period, HPeV3 was identified as the most prevalent picornavirus type in CNS-related infections with similarly high incidences of EV infection frequencies in very young children. The highly sensitive virus typing methods applied in this study will assist further EV and HPeV screening of sepsis and meningitis cases as well as in future molecular epidemiological studies and population surveillance.

Simmons R, Sharp C, Sims S, Kloverpris H, Goulder P, Simmonds P, Bowness P, Klenerman P. 2011. High frequency, sustained T cell responses to PARV4 suggest viral persistence in vivo. J Infect Dis, 203 (10), pp. 1378-1387. | Show Abstract | Read more

BACKGROUND: Parvovirus 4 (PARV4) is a recently identified human virus that has been found in livers of patients infected with hepatitis C virus (HCV) and in bone marrow of individuals infected with human immunodeficiency virus (HIV). T cells are important in controlling viruses but may also contribute to disease pathogenesis. The interaction of PARV4 with the cellular immune system has not been described. Consequently, we investigated whether T cell responses to PARV4 could be detected in individuals exposed to blood-borne viruses. METHODS: Interferon γ (IFN-γ) enzyme-linked immunospot assay, intracellular cytokine staining, and a tetrameric HLA-A*0201-peptide complex were used to define the lymphocyte populations responding to PARV4 NS peptides in 88 HCV-positive and 13 HIV-positive individuals. Antibody responses were tested using a recently developed PARV4 enzyme-linked immunosorbent assay. RESULTS: High-frequency T cell responses against multiple PARV4 NS peptides and antibodies were observed in 26% of individuals. Typical responses to the NS pools were >1000 spot-forming units per million peripheral blood mononuclear cells. CONCLUSIONS: PARV4 infection is common in individuals exposed to blood-borne viruses and elicits strong T cell responses, a feature typically associated with persistent, contained infections such as cytomegalovirus. Persistence of PARV4 viral antigen in tissue in HCV-positive and HIV-positive individuals and/or the associated activated antiviral T cell response may contribute to disease pathogenesis.

Imhof I, Simmonds P. 2011. Genotype differences in susceptibility and resistance development of hepatitis C virus to protease inhibitors telaprevir (VX-950) and danoprevir (ITMN-191). Hepatology, 53 (4), pp. 1090-1099. | Show Abstract | Read more

UNLABELLED: Protease inhibitors (PIs) have proven to be effective adjuncts to interferon/ribavirin treatment of hepatitis C virus (HCV) infections. Little clinical or in vitro data exists, however, on their effectiveness for nontype 1 genotypes that predominate in Europe, the Middle East, Africa, and most of Asia. NS3 protease and NS4A genes from genotypes 1-6 were inserted into the JFH clone to generate replication-competent intergenotype chimeras. Susceptibility to PIs was determined by replication and infectivity assays. To study resistance development, chimeras were cultured in subinhibitory concentrations of PIs and mutations phenotypically characterized. Marked differences in susceptibility of different genotypes to danoprevir (ITMN-191) and telaprevir (VX-950) were observed. Genotypes 1, 4, and 6 showed median inhibitory concentration (IC(50) ) values of 2-3 nM, >100-fold lower than genotypes 2/3/5 (250-750 nM). Telaprevir susceptibilities varied over a 4-fold range, with genotypes 1 and 2 being most susceptible and genotypes 4 and 5 most resistant. Culture of genotypes 1-6 in PIs induced numerous mutations in the NS3 protease domain, highly variable between genotypes. Introduction of danoprevir and BILN 2061-induced mutations into the original clones by site-directed mutagenesis (n = 29) all conferred resistant phenotypes, with particularly large increases (1-2 log greater IC(50) values) in the initially susceptible genotypes 1/4/6. Most introduced mutations and showed little or no effect on replicative fitness. CONCLUSION: Major differences were found between genotypes in their susceptibility and resistance development to PIs. However, equal sensitivities of genotypes 1, 4, and 6 to danoprevir and a broader efficacy range of telaprevir between genotypes than initially conceptualized provide strong evidence that PIs might be effectively used beyond their genotype 1 target group.

Siu RW, Fragkoudis R, Simmonds P, Donald CL, Chase-Topping ME, Barry G, Attarzadeh-Yazdi G, Rodriguez-Andres J, Nash AA, Merits A et al. 2011. Antiviral RNA interference responses induced by Semliki Forest virus infection of mosquito cells: characterization, origin, and frequency-dependent functions of virus-derived small interfering RNAs. J Virol, 85 (6), pp. 2907-2917. | Show Abstract | Read more

RNA interference (RNAi) is an important mosquito defense mechanism against arbovirus infection. In this paper we study the processes underlying antiviral RNAi in Aedes albopictus-derived U4.4 mosquito cells infected with Semliki Forest virus (SFV) (Togaviridae; Alphavirus). The production of virus-derived small interfering RNAs (viRNAs) from viral double-stranded RNA (dsRNA) is a key event in this host response. dsRNA could be formed by RNA replication intermediates, by secondary structures in RNA genomes or antigenomes, or by both. Which of these dsRNAs is the substrate for the generation of viRNAs is a fundamental question. Here we used deep sequencing of viRNAs and bioinformatic analysis of RNA secondary structures to gain insights into the characteristics and origins of viRNAs. An asymmetric distribution of SFV-derived viRNAs with notable areas of high-level viRNA production (hot spots) and no or a low frequency of viRNA production (cold spots) along the length of the viral genome with a slight bias toward the production of genome-derived viRNAs over antigenome-derived viRNAs was observed. Bioinformatic analysis suggests that hot spots of viRNA production are rarely but not generally associated with putative secondary structures in the SFV genome, suggesting that most viRNAs are derived from replicative dsRNA. A pattern of viRNAs almost identical to those of A. albopictus cells was observed for Aedes aegypti-derived Aag2 cells, suggesting common mechanisms that lead to viRNA production. Hot-spot viRNAs were found to be significantly less efficient at mediating antiviral RNAi than cold-spot viRNAs, pointing toward a nucleic acid-based viral decoy mechanism to evade the RNAi response.

Stapleton JT, Foung S, Muerhoff AS, Bukh J, Simmonds P. 2011. The GB viruses: a review and proposed classification of GBV-A, GBV-C (HGV), and GBV-D in genus Pegivirus within the family Flaviviridae. J Gen Virol, 92 (Pt 2), pp. 233-246. | Show Abstract | Read more

In 1967, it was reported that experimental inoculation of serum from a surgeon (G.B.) with acute hepatitis into tamarins resulted in hepatitis. In 1995, two new members of the family Flaviviridae, named GBV-A and GBV-B, were identified in tamarins that developed hepatitis following inoculation with the 11th GB passage. Neither virus infects humans, and a number of GBV-A variants were identified in wild New World monkeys that were captured. Subsequently, a related human virus was identified [named GBV-C or hepatitis G virus (HGV)], and recently a more distantly related virus (named GBV-D) was discovered in bats. Only GBV-B, a second species within the genus Hepacivirus (type species hepatitis C virus), has been shown to cause hepatitis; it causes acute hepatitis in experimentally infected tamarins. The other GB viruses have however not been assigned to a genus within the family Flaviviridae. Based on phylogenetic relationships, genome organization and pathogenic features of the GB viruses, we propose to classify GBV-A-like viruses, GBV-C and GBV-D as members of a fourth genus in the family Flaviviridae, named Pegivirus (pe, persistent; g, GB or G). We also propose renaming 'GB' viruses within the tentative genus Pegivirus to reflect their host origin.

Gaunt ER, Jansen RR, Poovorawan Y, Templeton KE, Toms GL, Simmonds P. 2011. Molecular epidemiology and evolution of human respiratory syncytial virus and human metapneumovirus. PLoS One, 6 (3), pp. e17427. | Show Abstract | Read more

Human respiratory syncytial virus (HRSV) and human metapneumovirus (HMPV) are ubiquitous respiratory pathogens of the Pneumovirinae subfamily of the Paramyxoviridae. Two major surface antigens are expressed by both viruses; the highly conserved fusion (F) protein, and the extremely diverse attachment (G) glycoprotein. Both viruses comprise two genetic groups, A and B. Circulation frequencies of the two genetic groups fluctuate for both viruses, giving rise to frequently observed switching of the predominantly circulating group. Nucleotide sequence data for the F and G gene regions of HRSV and HMPV variants from the UK, The Netherlands, Bangkok and data available from Genbank were used to identify clades of both viruses. Several contemporary circulating clades of HRSV and HMPV were identified by phylogenetic reconstructions. The molecular epidemiology and evolutionary dynamics of clades were modelled in parallel. Times of origin were determined and positively selected sites were identified. Sustained circulation of contemporary clades of both viruses for decades and their global dissemination demonstrated that switching of the predominant genetic group did not arise through the emergence of novel lineages each respiratory season, but through the fluctuating circulation frequencies of pre-existing lineages which undergo proliferative and eclipse phases. An abundance of sites were identified as positively selected within the G protein but not the F protein of both viruses. For HRSV, these were discordant with previously identified residues under selection, suggesting the virus can evade immune responses by generating diversity at multiple sites within linear epitopes. For both viruses, different sites were identified as positively selected between genetic groups.

McLeish NJ, Simmonds P, Robertson C, Handel I, McGilchrist M, Singh BK, Kerr S, Chase-Topping ME, Sinka K, Bronsvoort M et al. 2011. Sero-prevalence and incidence of A/H1N1 2009 influenza infection in Scotland in winter 2009-2010. PLoS One, 6 (6), pp. e20358. | Show Abstract | Read more

BACKGROUND: Sero-prevalence is a valuable indicator of prevalence and incidence of A/H1N1 2009 infection. However, raw sero-prevalence data must be corrected for background levels of cross-reactivity (i.e. imperfect test specificity) and the effects of immunisation programmes. METHODS AND FINDINGS: We obtained serum samples from a representative sample of 1563 adults resident in Scotland between late October 2009 and April 2010. Based on a microneutralisation assay, we estimate that 44% (95% confidence intervals (CIs): 40-47%) of the adult population of Scotland were sero-positive for A/H1N1 2009 influenza by 1 March 2010. Correcting for background cross-reactivity and for recorded vaccination rates by time and age group, we estimated that 34% (27-42%) were naturally infected with A/H1N1 2009 by 1 March 2010. The central estimate increases to >40% if we allow for imperfect test sensitivity. Over half of these infections are estimated to have occurred during the study period and the incidence of infection in late October 2009 was estimated at 4.3 new infections per 1000 people per day (1.2 to 7.2), falling close to zero by April 2010. The central estimate increases to over 5.0 per 1000 if we allow for imperfect test specificity. The rate of infection was higher for younger adults than older adults. Raw sero-prevalences were significantly higher in more deprived areas (likelihood ratio trend statistic = 4.92,1 df, P = 0.03) but there was no evidence of any difference in vaccination rates. CONCLUSIONS: We estimate that almost half the adult population of Scotland were sero-positive for A/H1N1 2009 influenza by early 2010 and that the majority of these individuals (except in the oldest age classes) sero-converted as a result of natural infection with A/H1N1 2009. Public health planning should consider the possibility of higher rates of infection with A/H1N1 2009 influenza in more deprived areas.

Lahtinen A, Kivelä P, Hedman L, Kumar A, Kantele A, Lappalainen M, Liitsola K, Ristola M, Delwart E, Sharp C et al. 2011. Serodiagnosis of primary infections with human parvovirus 4, Finland. Emerg Infect Dis, 17 (1), pp. 79-82. | Show Abstract | Read more

To determine the prevalence of parvovirus 4 infection and its clinical and sociodemographic correlations in Finland, we used virus-like particle-based serodiagnostic procedures (immunoglobulin [Ig] G, IgM, and IgG avidity) and PCR. We found 2 persons with parvovirus 4 primary infection who had mild or asymptomatic clinical features among hepatitis C virus-infected injection drug users.

Kapoor A, Simmonds P, Lipkin WI. 2010. Discovery and characterization of mammalian endogenous parvoviruses. J Virol, 84 (24), pp. 12628-12635. | Show Abstract | Read more

Public databases of nucleotide sequences contain exponentially increasing amounts of sequence data from mammalian genomes. Through the use of large-scale bioinformatic screening for sequences homologous to exogenous mammalian viruses, we found several sequences related to human and animal parvoviruses (PVs) in the Parvovirus and Dependovirus genera within genomes of several mammals, including rats, wallabies, opossums, guinea pigs, hedgehogs, African elephants, and European rabbits. However, phylogenetic analysis of these endogenous parvovirus (EnPV) sequences demonstrated substantial genetic divergence from exogenous mammalian PVs characterized to date. Entire nonstructural and capsid gene sequences of a novel EnPV were amplified and genetically characterized from rat (Rattus norvegicus) genomic DNA. Rat EnPV sequences were most closely related to members of the genus Parvovirus, with >70% and 65% amino acid identities to nonstructural and capsid proteins of canine parvovirus, respectively. Integration of EnPV into chromosome 5 of rats was confirmed by PCR cloning and sequence analysis of the viral and chromosomal junctions. Using inverse PCR, we determined that the rat genome contains a single copy of rat EnPV. Considering mammalian phylogeny, we estimate that EnPV integrated into the rat genome less than 30 million years ago. Comparative phylogenetic analysis done using all known representative exogenous parvovirus (ExPV) and EnPV sequences showed two major genetic groups of EnPVs, one genetically more similar to genus Parvovirus and the other genetically more similar to the genus Dependovirus. The full extent of the genetic diversity of parvoviruses that have undergone endogenization during evolution of mammals and other vertebrates will be recognized only once complete genomic sequences from a wider range of classes, orders, and species of animals become available.

Chieochansin T, Simmonds P, Poovorawan Y. 2010. Determination and analysis of complete coding sequence regions of new discovered human bocavirus types 2 and 3. Arch Virol, 155 (12), pp. 2023-2028. | Show Abstract | Read more

In this study, two human bocaviruses (HBoV), HBoV2 and HBoV3, that were detected previously in enteric samples were characterized genetically. Nearly complete genome sequences of three HBoV2 variants and one HBoV3 variant originating from Thailand and the UK were compared to published HBoV sequences. HBoV2 showed divergence from HBoV1 throughout the genome, while the HBoV3 sequence grouped phylogenetically with HBoV1 in the non-structural region and with HBoV2 sequences in the structural gene, consistent with its proposed recombinant origin. Compared to HBoV1 and HBoV3, HBoV2 shows substantially greater intra-species diversity, consistent with a longer period of human circulation.

Simmonds P, McIntyre C, Savolainen-Kopra C, Tapparel C, Mackay IM, Hovi T. 2010. Proposals for the classification of human rhinovirus species C into genotypically assigned types. J Gen Virol, 91 (Pt 10), pp. 2409-2419. | Show Abstract | Read more

Human rhinoviruses (HRVs) are common respiratory pathogens associated with mild upper respiratory tract infections, but also increasingly recognized in the aetiology of severe lower respiratory tract disease. Wider use of molecular diagnostics has led to a recent reappraisal of HRV genetic diversity, including the discovery of HRV species C (HRV-C), which is refractory to traditional virus isolation procedures. Although it is heterogeneous genetically, there has to date been no attempt to classify HRV-C into types analogous to the multiple serotypes identified for HRV-A and -B and among human enteroviruses. Direct investigation of cross-neutralization properties of HRV-C is precluded by the lack of methods for in vitro culture, but sequences from the capsid genes (VP1 and partial VP4/VP2) show evidence for marked phylogenetic clustering, suggesting the possibility of a genetically based system comparable to that used for the assignment of new enterovirus types. We propose a threshold of 13% divergence for VP1 nucleotide sequences for type assignment, a level that classifies the current dataset of 86 HRV-C VP1 sequences into a total of 33 types. We recognize, however, that most HRV-C sequence data have been collected in the VP4/VP2 region (currently 701 sequences between positions 615 and 1043). We propose a subsidiary classification of variants showing > 10% divergence in VP4/VP2, but lacking VP1 sequences, to 28 provisionally assigned types (subject to confirmation once VP1 sequences are determined). These proposals will assist in future epidemiological and clinical studies of HRV-C conducted by different groups worldwide, and provide the foundation for future exploration of type-associated differences in clinical presentations and biological properties.

McIntyre CL, McWilliam Leitch EC, Savolainen-Kopra C, Hovi T, Simmonds P. 2010. Analysis of genetic diversity and sites of recombination in human rhinovirus species C. J Virol, 84 (19), pp. 10297-10310. | Show Abstract | Read more

Human rhinoviruses (HRVs) are a highly prevalent and diverse group of respiratory viruses. Although HRV-A and HRV-B are traditionally detected by virus isolation, a series of unculturable HRV variants have recently been described and assigned as a new species (HRV-C) within the picornavirus Enterovirus genus. To investigate their genetic diversity and occurrence of recombination, we have performed comprehensive phylogenetic analysis of sequences from the 5' untranslated region (5' UTR), VP4/VP2, VP1, and 3Dpol regions amplified from 89 HRV-C-positive respiratory samples and available published sequences. Branching orders of VP4/VP2, VP1, and 3Dpol trees were identical, consistent with the absence of intraspecies recombination in the coding regions. However, numerous tree topology changes were apparent in the 5' UTR, where >60% of analyzed HRV-C variants showed recombination with species A sequences. Two recombination hot spots in stem-loop 5 and the polypyrimidine tract in the 5' UTR were mapped using the program GroupingScan. Available HRV-C sequences showed evidence for additional interspecies recombination with HRV-A in the 2A gene, with breakpoints mapping precisely to the boundaries of the C-terminal domain of the encoded proteinase. Pairwise distances between HRV-C variants in VP1 and VP4/VP2 regions fell into two separate distributions, resembling inter- and intraserotype distances of species A and B. These observations suggest that, without serological cross-neutralization data, HRV-C genetic groups may be equivalently classified into types using divergence thresholds derived from distance distributions. The extensive sequence data from multiple genome regions of HRV-C and analyses of recombination in the current study will assist future formulation of consensus criteria for HRV-C type assignment and identification.

Sharp CP, Vermeulen M, Nébié Y, Djoko CF, LeBreton M, Tamoufe U, Rimoin AW, Kayembe PK, Carr JK, Servant-Delmas A et al. 2010. Changing epidemiology of human parvovirus 4 infection in sub-Saharan Africa. Emerg Infect Dis, 16 (10), pp. 1605-1607. | Show Abstract | Read more

Human parvovirus 4 infections are primarily associated with parenteral exposure in western countries. By ELISA, we demonstrate frequent seropositivity for antibody to parvovirus 4 viral protein 2 among adult populations throughout sub-Saharan Africa (Burkina Faso, 37%; Cameroon, 25%; Democratic Republic of the Congo, 35%; South Africa, 20%), which implies existence of alternative transmission routes.

Sharp CP, LeBreton M, Kantola K, Nana A, Diffo JLED, Djoko CF, Tamoufe U, Kiyang JA, Babila TG, Ngole EM et al. 2010. Widespread infection with homologues of human parvoviruses B19, PARV4, and human bocavirus of chimpanzees and gorillas in the wild. J Virol, 84 (19), pp. 10289-10296. | Show Abstract | Read more

Infections with human parvoviruses B19 and recently discovered human bocaviruses (HBoVs) are widespread, while PARV4 infections are transmitted parenterally and prevalent specifically in injecting drug users and hemophiliacs. To investigate the exposure and circulation of parvoviruses related to B19 virus, PARV4, and HBoV in nonhuman primates, plasma samples collected from 73 Cameroonian wild-caught chimpanzees and gorillas and 91 Old World monkey (OWM) species were screened for antibodies to recombinant B19 virus, PARV4, and HBoV VP2 antigens by enzyme-linked immunosorbent assay (ELISA). Moderate to high frequencies of seroreactivity to PARV4 (63% and 18% in chimpanzees and gorillas, respectively), HBoV (73% and 36%), and B19 virus (8% and 27%) were recorded for apes, while OWMs were uniformly negative (for PARV4 and B19 virus) or infrequently reactive (3% for HBoV). For genetic characterization, plasma samples and 54 fecal samples from chimpanzees and gorillas collected from Cameroonian forest floors were screened by PCR with primers conserved within Erythrovirus, Bocavirus, and PARV4 genera. Two plasma samples (chimpanzee and baboon) were positive for PARV4, while four fecal samples were positive for HBoV-like viruses. The chimpanzee PARV4 variant showed 18% and 15% nucleotide sequence divergence in NS and VP1/2, respectively, from human variants (9% and 7% amino acid, respectively), while the baboon variant was substantially more divergent, mirroring host phylogeny. Ape HBoV variants showed complex sequence relationships with human viruses, comprising separate divergent homologues of HBoV1 and the recombinant HBoV3 species in chimpanzees and a novel recombinant species in gorillas. This study provides the first evidence for widespread circulation of parvoviruses in primates and enables future investigations of their epidemiology, host specificity, and (co)evolutionary histories.

Bailey D, Karakasiliotis I, Vashist S, Chung LMW, Rees J, McFadden N, Benson A, Yarovinsky F, Simmonds P, Goodfellow I. 2010. Functional Analysis of RNA Structures Present at the 3 ' Extremity of the Murine Norovirus Genome: the Variable Polypyrimidine Tract Plays a Role in Viral Virulence (vol 84, pg 2859, 2010) JOURNAL OF VIROLOGY, 84 (20), pp. 10943-10943. | Read more

McWilliam Leitch EC, Cabrerizo M, Cardosa J, Harvala H, Ivanova OE, Kroes AC, Lukashev A, Muir P, Odoom J, Roivainen M et al. 2010. Evolutionary dynamics and temporal/geographical correlates of recombination in the human enterovirus echovirus types 9, 11, and 30. J Virol, 84 (18), pp. 9292-9300. | Show Abstract | Read more

The relationship between virus evolution and recombination in species B human enteroviruses was investigated through large-scale genetic analysis of echovirus type 9 (E9) and E11 isolates (n = 85 and 116) from 16 European, African, and Asian countries between 1995 and 2008. Cluster 1 E9 isolates and genotype D5 and A E11 isolates showed evidence of frequent recombination between the VP1 and 3Dpol regions, the latter falling into 23 (E9) and 43 (E11) clades interspersed phylogenetically with 46 3Dpol clades of E30 and with those of other species B serotypes. Remarkably, only 2 of the 112 3Dpol clades were shared by more than one serotype (E11 and E30), demonstrating an extremely large and genetically heterogeneous recombination pool of species B nonstructural-region variants. The likelihood of recombination increased with geographical separation and time, and both were correlated with VP1 divergence, whose substitution rates allowed recombination half-lives of 1.3, 9.8, and 3.1 years, respectively, for E9, E11, and E30 to be calculated. These marked differences in recombination dynamics matched epidemiological patterns of periodic epidemic cycles of 2 to 3 (E9) and 5 to 6 (E30) years and the longer-term endemic pattern of E11 infections. Phylotemporal analysis using a Bayesian Markov chain Monte Carlo method, which placed recombination events within the evolutionary reconstruction of VP1, showed a close relationship with VP1 lineage expansion, with defined recombination events that correlated with their epidemiological periodicity. Whether recombination events contribute directly to changes in transmissibility that drive epidemic behavior or occur stochastically during periodic population bottlenecks is an unresolved issue vital to future understanding of enterovirus molecular epidemiology and pathogenesis.

Gaunt ER, Hardie A, Claas EC, Simmonds P, Templeton KE. 2010. Epidemiology and clinical presentations of the four human coronaviruses 229E, HKU1, NL63, and OC43 detected over 3 years using a novel multiplex real-time PCR method. J Clin Microbiol, 48 (8), pp. 2940-2947. | Show Abstract | Read more

Four human coronaviruses (HCoV-229E, HCoV-HKU1, HCoV-NL63, and HCoV-OC43) are associated with a range of respiratory outcomes, including bronchiolitis and pneumonia. Their epidemiologies and clinical characteristics are poorly described and are often reliant on case reports. To address these problems, we conducted a large-scale comprehensive screening for all four coronaviruses by analysis of 11,661 diagnostic respiratory samples collected in Edinburgh, United Kingdom, over 3 years between July 2006 and June 2009 using a novel four-way multiplex real-time reverse transcription-PCR (RT-PCR) assay. Coronaviruses were detected in 0.3 to 0.85% of samples in all age groups. Generally, coronaviruses displayed marked winter seasonality between the months of December and April and were not detected in summer months, which is comparable to the pattern seen with influenza viruses. HCoV-229E was the exception; detection was confined to the winter of 2008 and was sporadic in the following year. There were additional longer-term differences in detection frequencies between seasons, with HCoV-OC43 predominant in the first and third seasons and HCoV-HKU1 dominating in the second (see Results for definitions of seasons). A total of 11 to 41% of coronaviruses detected were in samples testing positive for other respiratory viruses, although clinical presentations of coronavirus monoinfections were comparable to those of viruses which have an established role in respiratory disease, such as respiratory syncytial virus, influenza virus, and parainfluenza viruses. The novel multiplex assay for real-time pan-coronavirus detection enhances respiratory virus diagnosis, overcomes potential diagnostic problems arising through seasonal variation in coronavirus frequency, and provides novel insights into the epidemiology and clinical implications of coronaviruses.

Gomperts E, Sharp C, Simmonds P, Donfield S, Lail A, Delwart E. 2010. High frequencies of exposure to the novel human parvovirus, PARV4 in hemophiliacs and injecting drug users detected by a serological assay for PARV4 antibodies HAEMOPHILIA, 16 pp. 65-65.

Harvala H, Wolthers KC, Simmonds P. 2010. Parechoviruses in children: understanding a new infection. Curr Opin Infect Dis, 23 (3), pp. 224-230. | Show Abstract | Read more

PURPOSE OF REVIEW: Human parechoviruses (HPeVs) within the large and growing family of Picornaviridaeare common human pathogens associated with a wide spectrum of disease presentations. Although 10 different HPeV types have been published to date, there is increasing evidence for a specific role of HPeV type 3 (HPeV3) in severe neonatal disease. In this review, we will describe both the disease associations and underlying epidemiological and/or biological basis for the often marked differences in disease outcomes between HPeV types. RECENT FINDINGS: Application of molecular-based diagnostic techniques has revealed an association between neonatal sepsis, encephalitis and hepatitis with HPeV3 but not with other parechovirus types. HPeV3 shows evidence for very recent emergence in human populations as well as inferred differences in cellular receptor usage. SUMMARY: The recently discovered HPeV3 has been shown to play an important role in severe neonatal infections, observations possibly linked to its very recent emergence or possibly different cellular tropism that underlie its targeting of the most susceptible individuals. HPeV infections are currently under-diagnosed and should be considered in the clinical and diagnostic evaluation of severe neonatal disease presentations.

Kapoor A, Simmonds P, Slikas E, Li L, Bodhidatta L, Sethabutr O, Triki H, Bahri O, Oderinde BS, Baba MM et al. 2010. Human bocaviruses are highly diverse, dispersed, recombination prone, and prevalent in enteric infections. J Infect Dis, 201 (11), pp. 1633-1643. | Show Abstract | Read more

A new species of parvovirus, tentatively named human bocavirus 4 (HBoV4), was genetically characterized. Among 641 feces samples obtained from children and adults, the most commonly detected bocavirus species were, in descending order, HBoV2, HBoV3, HBoV4, and HBoV1, with an HBoV2 prevalence of 21% and 26% in Nigerian and Tunisian children, respectively. HBoV3 or HBoV4 species were found in 12 of 192 patients with non-polio acute flaccid paralysis in Tunisia and Nigeria and 0 of 96 healthy Tunisian contacts (P = .01). Evidence of extensive recombination at the NP1 and VP1 gene boundary between and within bocavirus species was found. The high degree of genetic diversity seen among the human bocaviruses found in feces specimens, relative to the highly homogeneous HBoV1, suggest that this worldwide-distributed respiratory pathogen may have recently evolved from an enteric bocavirus after acquiring an expanded tropism favoring the respiratory tract. Elucidating the possible role of the newly identified enteric bocaviruses in human diseases, including acute flaccid paralysis and diarrhea, will require further epidemiological studies.

Calvert J, Chieochansin T, Benschop KS, McWilliam Leitch EC, Drexler JF, Grywna K, da Costa Ribeiro H, Drosten C, Harvala H, Poovorawan Y et al. 2010. Recombination dynamics of human parechoviruses: investigation of type-specific differences in frequency and epidemiological correlates. J Gen Virol, 91 (Pt 5), pp. 1229-1238. | Show Abstract | Read more

Human parechoviruses (HPeVs) are highly prevalent RNA viruses classified in the family Picornaviridae. Several antigenically distinct types circulate in human populations worldwide, whilst recombination additionally contributes to the genetic heterogeneity of the virus. To investigate factors influencing the likelihood of recombination and to compare its dynamics among types, 154 variants collected from four widely geographically separated referral centres (UK, The Netherlands, Thailand and Brazil) were typed by VP3/VP1 amplification/sequencing with recombination groups assigned by analysis of 3Dpol sequences. HPeV1B and HPeV3 were the most frequently detected types in each referral region, but with marked geographical differences in the frequencies of different recombinant forms (RFs) of types 1B, 5 and 6. HPeV1B showed more frequent recombination than HPeV3, in terms both of evolutionary divergence and of temporal/geographical indicators of population separation. HPeV1 variants showing between 10 and 20% divergence in VP3/VP1 almost invariably fell into different recombination groups, compared with only one-third of similarly divergent HPeV3 variants. Substitution rates calculated by beast in the VP3/VP1 region of HPeV1 and HPeV3 allowed half-lives of the RFs of 4 and 20 years, respectively, to be calculated, estimates fitting closely with their observed lifespans based on population sampling. The variability in recombination dynamics between HPeV1B and HPeV3 offers an intriguing link with their markedly different seasonal patterns of transmission, age distributions of infection and clinical outcomes. Future investigation of the epidemiological and biological opportunities and constraints on intertypic recombination will provide more information about its influence on the longer term evolution and pathogenicity of parechoviruses.

Imhof I, Simmonds P. 2010. Development of an intergenotypic hepatitis C virus (HCV) cell culture method to assess antiviral susceptibilities and resistance development of HCV NS3 protease genes from HCV genotypes 1 to 6. J Virol, 84 (9), pp. 4597-4610. | Show Abstract | Read more

Protease inhibitors (PIs) of hepatitis C virus (HCV) provide an additional or alternative therapy for chronic infection. However, assessment of their efficacy and ability to inhibit replication of different genotypes is hampered by the lack of a convenient animal model or a method for in vitro culture of HCV other than the type 1/2-based replicons and the infectious genotype 2a clone JFH1. To address this problem, we constructed a panel of replication-competent chimeric Jc1 (pFK JFH1/J6/C-846) clones containing protease and NS4A coding sequences from all six major genotypes, enabling the determination of replication and the susceptibility to PIs. Chimeras showed substantial variability in replication kinetics, attributable in part to naturally occurring polymorphisms and differing requirements for adaptive mutations in NS3 and NS4A. Through calculation of 50% inhibitory concentrations (IC(50)s) of BILN 2061, measuring reduction in the number of focus-forming units/ml (FFU/ml) and replication inhibition, consistent genotype-associated differences in antiviral susceptibilities were observed. IC(50)s for genotype 1b, 4a, and 6a-derived chimeras (1 to 3 nM) were approximately 100-fold lower than those for genotypes 2a, 3a, and 5a (range, 80 to 720 nM), implying major differences in response to therapy. In vitro passage in increasing concentrations of BILN 2061 rapidly induced resistance-associated mutations at position 168 in chimeras of all 6 genotypes and at position 156 in genotypes 1b and 4a, each with substantial variability in the identity of substituted amino acids. The system will allow future comprehensive phenotypic characterization of naturally occurring and treatment-induced mutations for PIs in trial or entering clinical use.

Harvala H, Gunson R, Simmonds P, Hardie A, Bennett S, Scott F, Roddie H, McKnight J, Walsh T, Rowney D et al. 2010. The emergence of oseltamivir-resistant pandemic influenza A(H1N1) 2009 virus amongst hospitalised immunocompromised patients in Scotland, November-December, 2009 EUROSURVEILLANCE, 15 (14), pp. 2-4.

Bailey D, Karakasiliotis I, Vashist S, Chung LM, Rees J, McFadden N, Benson A, Yarovinsky F, Simmonds P, Goodfellow I. 2010. Functional analysis of RNA structures present at the 3' extremity of the murine norovirus genome: the variable polypyrimidine tract plays a role in viral virulence. J Virol, 84 (6), pp. 2859-2870. | Show Abstract | Read more

Interactions of host cell factors with RNA sequences and structures in the genomes of positive-strand RNA viruses play various roles in the life cycles of these viruses. Our understanding of the functional RNA elements present in norovirus genomes to date has been limited largely to in vitro analysis. However, we recently used reverse genetics to identify evolutionarily conserved RNA structures and sequences required for norovirus replication. We have now undertaken a more detailed analysis of RNA structures present at the 3' extremity of the murine norovirus (MNV) genome. Biochemical data indicate the presence of three stable stem-loops, including two in the untranslated region, and a single-stranded polypyrimidine tract [p(Y)] of variable length between MNV isolates, within the terminal stem-loop structure. The well-characterized host cell pyrimidine binding proteins PTB and PCBP bound the 3'-untranslated region via an interaction with this variable sequence. Viruses lacking the p(Y) tract were viable both in cell culture and upon mouse infection, demonstrating that this interaction was not essential for virus replication. However, competition analysis with wild-type MNV in cell culture indicated that the loss of the p(Y) tract was associated with a fitness cost. Furthermore, a p(Y)-deleted mutant showed a reduction in virulence in the STAT1(-/-) mouse model, highlighting the role of RNA structures in norovirus pathogenesis. This work highlights how, like with other positive-strand RNA viruses, RNA structures present at the termini of the norovirus genome play important roles in virus replication and virulence.

Benschop KS, de Vries M, Minnaar RP, Stanway G, van der Hoek L, Wolthers KC, Simmonds P. 2010. Comprehensive full-length sequence analyses of human parechoviruses: diversity and recombination. J Gen Virol, 91 (Pt 1), pp. 145-154. | Show Abstract | Read more

Human parechoviruses (HPeVs) are highly prevalent pathogens among very young children. Although originally classified into two serologically distinct types, HPeV1 and -2, recent analyses of variants collected worldwide have revealed the existence of 12 further types classified genetically by sequence comparisons of complete genome sequences or the capsid (VP1) gene. To investigate the nature of HPeV evolution, its population dynamics and recombination breakpoints, this study generated 18 full-length genomic sequences of the most commonly circulating genotypes, HPeV1 and -3, collected over a time span of 14 years from The Netherlands. By inclusion of previously published full-length sequences, 35 sequences were analysed in total. Analysis of contemporary strains of HPeV1 and those most similar to the prototype strain (Harris) showed that HPeV1 variants fall into two genetically distinct clusters that are much more divergent from each other than those observed within other HPeV types. Future classification criteria for HPeVs may require modification to accommodate the occurrence of variants with intermediate degrees of diversity within types. Recombination was frequently observed among HPeV1, -4, -5 and -6, but was much more restricted among HPeV3 strains. Favoured sites for recombination were found to flank the capsid region, and further sites were found within the non-structural region, P2. In contrast to other HPeV types, the majority of the HPeV3 sequences remained monophyletic across the genome, a possible reflection of its lower diversity and potentially more recent emergence than other HPeV types, or biological and/or epidemiological constraints that limit opportunities for co-infections with potential recombination partners.

Harvala H, Gunson R, Simmonds P, Hardie A, Bennett S, Scott F, Roddie H, McKnight J, Walsh T, Rowney D et al. 2010. The emergence of oseltamivir-resistant pandemic influenza A (H1N1) 2009 virus amongst hospitalised immunocompromised patients in Scotland, November-December, 2009. Euro Surveill, 15 (14), | Show Abstract

To investigate the frequency of oseltamivir resistance in circulating strains of the 2009 influenza A(H1N1) pandemic virus in Scotland, 1,802 samples from 1,608 infected hospitalised patients were screened by the H275Y discriminatory RT-PCR. Among these, we identified 10 patients who developed the H275Y mutation. All of them were immunocompromised and were under treatment or had been treated previously with oseltamivir.

Kapoor A, Simmonds P, Lipkin WI, Zaidi S, Delwart E. 2010. Use of nucleotide composition analysis to infer hosts for three novel picorna-like viruses. J Virol, 84 (19), pp. 10322-10328. | Show Abstract | Read more

Nearly complete genome sequences of three novel RNA viruses were acquired from the stool of an Afghan child. Phylogenetic analysis indicated that these viruses belong to the picorna-like virus superfamily. Because of their unique genomic organization and deep phylogenetic roots, we propose these viruses, provisionally named calhevirus, tetnovirus-1, and tetnovirus-2, as prototypes of new viral families. A newly developed nucleotide composition analysis (NCA) method was used to compare mononucleotide and dinucleotide frequencies for RNA viruses infecting mammals, plants, or insects. Using a large training data set of 284 representative picornavirus-like genomic sequences with defined host origins, NCA correctly identified the kingdom or phylum of the viral host for >95% of picorna-like viruses. NCA predicted an insect host origin for the 3 novel picorna-like viruses. Their presence in human stool therefore likely reflects ingestion of insect-contaminated food. As metagenomic analyses of different environments and organisms continue to yield highly divergent viral genomes NCA provides a rapid and robust method to identify their likely cellular hosts.

Gaunt E, McWilliam-Leitch EC, Templeton K, Simmonds P. 2009. Incidence, molecular epidemiology and clinical presentations of human metapneumovirus; assessment of its importance as a diagnostic screening target. J Clin Virol, 46 (4), pp. 318-324. | Show Abstract | Read more

BACKGROUND: Human metapneumovirus (HMPV) is a recently discovered human paramyxovirus associated with a spectrum of respiratory symptoms from the common cold to pneumonia and bronchiolitis. OBJECTIVES: To assess the clinical significance and epidemiology of HMPV, standardized comparison of frequencies of infection, age profiles and disease associations were made with other respiratory viruses in Scotland. STUDY DESIGN: 7091 respiratory samples collected in Scotland between 1 July 2006 and 30 June 2008 from 4282 individuals were screened by multiplex RT-PCR for respiratory syncytial virus (HRSV), adenovirus (AdV), parainfluenza viruses 1-3 (PIV-1, -2 and -3), influenza A and B and by nested RT-PCR for HMPV. RESULTS: HMPV was the fifth most prevalent virus (2.0% of samples), found predominantly in young children in winter months. In the 2006-2007 respiratory season, 70% of HMPV isolates were genotype A, but a switch to predominantly type B infections occurred next winter. For samples with information on clinical presentations, 26% of HMPV infections were from subjects with lower respiratory tract presentations, lower than recorded for HRSV, but similar to adenovirus, parainfluenza viruses and influenza viruses A and B. Around 13% of HMPV infections were associated with upper respiratory tract symptoms or disease, comparable with other respiratory virus infections. CONCLUSIONS: Numerically and through its association with respiratory disease, HMPV represents a diagnostically significant target that should be included in PCR-based routine screening of respiratory samples. Understanding the biological basis of observed rapid turnover of HMPV variants, including the observed HMPV genotype change between respiratory seasons requires further longitudinal studies.

Wisdom A, Leitch EC, Gaunt E, Harvala H, Simmonds P. 2009. Screening respiratory samples for detection of human rhinoviruses (HRVs) and enteroviruses: comprehensive VP4-VP2 typing reveals high incidence and genetic diversity of HRV species C. J Clin Microbiol, 47 (12), pp. 3958-3967. | Show Abstract | Read more

Rhinovirus infections are the most common cause of viral illness in humans, and there is increasing evidence of their etiological role in severe acute respiratory tract infections (ARTIs). Human rhinoviruses (HRVs) are classified into two species, species A and B, which contain over 100 serotypes, and a recently discovered genetically heterogeneous third species (HRV species C). To investigate their diversity and population turnover, screening for the detection and the genetic characterization of HRV variants in diagnostic respiratory samples was performed by using nested primers for the efficient amplification of the VP4-VP2 region of HRV (and enterovirus) species and serotype identification. HRV species A, B, and C variants were detected in 14%, 1.8%, and 6.8%, respectively, of 456 diagnostic respiratory samples from 345 subjects (6 samples also contained enteroviruses), predominantly among children under age 10 years. HRV species A and B variants were remarkably heterogeneous, with 22 and 6 different serotypes, respectively, detected among 73 positive samples. Similarly, by using a pairwise distance threshold of 0.1, species C variants occurring worldwide were provisionally assigned to 47 different types, of which 15 were present among samples from Edinburgh, United Kingdom. There was a rapid turnover of variants, with only 5 of 43 serotypes detected during both sampling periods. By using divergence thresholds and phylogenetic analysis, several species A and C variants could provisionally be assigned to new types. An initial investigation of the clinical differences between rhinovirus species found HRV species C to be nearly twice as frequently associated with ARTIs than other rhinovirus species, which matches the frequencies of detection of respiratory syncytial virus. The study demonstrates the extraordinary genetic diversity of HRVs, their rapid population turnover, and their extensive involvement in childhood respiratory disease.

Sharp CP, Lail A, Donfield S, Simmons R, Leen C, Klenerman P, Delwart E, Gomperts ED, Simmonds P. 2009. High frequencies of exposure to the novel human parvovirus PARV4 in hemophiliacs and injection drug users, as detected by a serological assay for PARV4 antibodies. J Infect Dis, 200 (7), pp. 1119-1125. | Show Abstract | Read more

BACKGROUND: PARV4 is a human parvovirus that was first detected in and cloned from an individual with a human immunodeficiency virus (HIV) seroconversion-like illness and that subsequently persisted in the lymphoid tissue and bone marrow. In contrast to human parvovirus B19 infections, PARV4 infections are most frequently detected in injection drug users (IDUs), particularly those who are coinfected with HIV type 1 (HIV-1). To investigate the routes of transmission of PARV4 and to ascertain whether infections are acquired through plasma-derived blood products, we developed a novel anti-PARV4 enzyme-linked immunosorbent assay (ELISA) to determine its seroprevalence in subjects with parenteral exposure. METHODS: PARV4 viral protein 2 (VP2) was expressed and used as antigen in an indirect ELISA, to detect anti-PARV4 immunoglobulin G. RESULTS: All 50 adult control subjects who were nonparenterally exposed to PARV4 were anti-PARV4 negative, in contrast to HIV-infected and HIV-uninfected IDUs, who had antibody frequencies of 67% and 33%, respectively. Predominantly parenteral transmission was confirmed by the finding of similar frequencies of infection among HIV-coinfected and HIV-uninfected hemophiliacs (11 of 20 individuals and 4 of 15 individuals, respectively) who were treated with nonvirally inactivated factor VIII/factor IX, whereas all but 1 of the 35 nonhemophiliac siblings of these siblings were found to be seronegative (despite having close household contact). CONCLUSIONS: The present study provides convincing evidence that PARV4 is primarily transmitted parenterally. Evidence for widespread infection of hemophiliacs treated with nonvirally inactivated clotting factor creates fresh safety concerns for plasma-derived blood products, particularly because parvoviruses are relatively resistant to virus inactivation.

Chieochansin T, Kapoor A, Delwart E, Poovorawan Y, Simmonds P. 2009. Absence of detectable replication of human bocavirus species 2 in respiratory tract. Emerg Infect Dis, 15 (9), pp. 1503-1505. | Show Abstract | Read more

Human bocavirus (HBoV) commonly infects young children and is associated with respiratory disease; disease associations of the divergent HBoV-2 species are unknown. Frequent HBoV-2 detection in fecal samples indicated widespread circulation in the United Kingdom and Thailand, but its lack of detection among 6,524 respiratory samples indicates likely differences from HBoV-1 in tropism/pathogenesis.

Harvala H, Robertson I, Chieochansin T, McWilliam Leitch EC, Templeton K, Simmonds P. 2009. Specific association of human parechovirus type 3 with sepsis and fever in young infants, as identified by direct typing of cerebrospinal fluid samples. J Infect Dis, 199 (12), pp. 1753-1760. | Show Abstract | Read more

BACKGROUND: Human parechoviruses (HPeVs), along with human enteroviruses (HEVs), are associated with neonatal sepsis and meningitis. We determined the relative importance of these viruses and the specific HPeV types involved in the development of central nervous system-associated disease. METHODS: A total of 1575 cerebrospinal fluid (CSF) samples obtained during 2006-2008 were screened for HPeV by means of nested polymerase chain reaction. All samples for which results were positive were typed by sequencing of viral protein (VP) 3/VP1. Screening for HEV was performed in parallel, as was detection of HPeV in respiratory and fecal surveillance samples, to identify virus types circulating in the general population. RESULTS: HPeV was detected in 14 CSF samples obtained exclusively from young infants (age, <3 months) with sepsis or pyrexia. The frequency of detection of HPeVs varied greatly by year, with the highest frequency (7.2%) noted in 2008 exceeding that of HEVs. Direct typing of CSF samples revealed that all infections were caused by HPeV type 3, a finding that is in contrast to the predominant circulation of HPeV1 in contemporary respiratory and fecal surveillance samples. CONCLUSION: HPeV was a significant cause of severe sepsis and fever with central nervous system involvement in young infants, rivaling enteroviruses. The specific targeting of young infants by HPeV type 3 may reflect a difference in tissue tropism between virus types or a lack of protection of young infants by maternal antibody consequent to the recent emergence of HPeV.

Harvala H, Simmonds P. 2009. Human parechoviruses: biology, epidemiology and clinical significance. J Clin Virol, 45 (1), pp. 1-9. | Show Abstract | Read more

Human parechoviruses (HPeVs) are members of the large and growing family of Picornaviridae. Although originally described as echovirus 22 and 23 within human enteroviruses because of their clinical and morphological properties, they have since been shown to be distinct from this and other picornavirus groups in several features of their genome organisation, structure and replication. Human parechoviruses show genetic and antigenic heterogeneity and a number of distinct HPeV types are known to circulate widely in human populations throughout the world. Although the majority of HPeV infections occur early in life without specific symptoms, disease manifestations associated with many of the currently described types have been described, ranging from gastroenteritis and respiratory infections to neurological disease, particularly in neonates. Although HPeV diagnosis has historically been made by virus isolation, a new generation of sensitive and specific molecular tests for HPeV RNA promises to greatly improve the effectiveness of HPeV detection and type identification, as well as providing a greater understanding its molecular epidemiology. By this means, we will learn much more about the clinical relevance of HPeVs and the association of different HPeV types with specific disease presentations.

Simmonds P. 2009. Virus evolution Microbiology Today, 36 (2), pp. 96-99.

Ninomiya M, Takahashi M, Hoshino Y, Ichiyama K, Simmonds P, Okamoto H. 2009. Analysis of the entire genomes of torque teno midi virus variants in chimpanzees: infrequent cross-species infection between humans and chimpanzees. J Gen Virol, 90 (Pt 2), pp. 347-358. | Show Abstract | Read more

Humans are frequently infected with three anelloviruses which have circular DNA genomes of 3.6-3.9 kb [Torque teno virus (TTV)], 2.8-2.9 kb [Torque teno mini virus (TTMV)] and 3.2 kb [a recently discovered anellovirus named Torque teno midi virus (TTMDV)]. Unexpectedly, human TTMDV DNA was not detectable in any of 74 chimpanzees tested, although all but one tested positive for both human TTV and TTMV DNA. Using universal primers for anelloviruses, novel variants of TTMDV that are phylogenetically clearly separate from human TTMDV were identified from chimpanzees, and over the entire genome, three chimpanzee TTMDV variants differed by 17.9-20.3 % from each other and by 40.4-43.6 % from all 18 reported human TTMDVs. A newly developed PCR assay that uses chimpanzee TTMDV-specific primers revealed the high prevalence of chimpanzee TTMDV in chimpanzees (63/74, 85 %) but low prevalence in humans (1/100). While variants of TTV and TTMV from chimpanzees and humans were phylogenetically interspersed, those of TTMDV were monophyletic for each species, with sequence diversity of <33 and <20 % within the 18 human and three chimpanzee TTMDV variants, respectively. Maximum within-group divergence values for TTV and TTMV were 51 and 57 %, respectively; both of these values were substantially greater than the maximum divergence among TTMDV variants (44 %), consistent with a later evolutionary emergence of TTMDV. However, substantiation of this hypothesis will require further analysis of genetic diversity using an expanded dataset of TTMDV variants in humans and chimpanzees. Similarly, the underlying mechanism of observed infrequent cross-species infection of TTMDV between humans and chimpanzees deserves further analysis.

McWilliam Leitch EC, Bendig J, Cabrerizo M, Cardosa J, Hyypiä T, Ivanova OE, Kelly A, Kroes AC, Lukashev A, MacAdam A et al. 2009. Transmission networks and population turnover of echovirus 30. J Virol, 83 (5), pp. 2109-2118. | Show Abstract | Read more

Globally, echovirus 30 (E30) is one of the most frequently identified enteroviruses and a major cause of meningitis. Despite its wide distribution, little is known about its transmission networks or the dynamics of its recombination and geographical spread. To address this, we have conducted an extensive molecular epidemiology and evolutionary study of E30 isolates collected over 8 years from a geographically wide sample base (11 European countries, Asia, and Australia). 3Dpol sequences fell into several distinct phylogenetic groups, interspersed with other species B serotypes, enabling E30 isolates to be classified into 38 recombinant forms (RFs). Substitutions in VP1 and 3Dpol regions occurred predominantly at synonymous sites (ratio of nonsynonymous to synonymous substitutions, 0.05) with VP1 showing a rapid substitution rate of 8.3 x 10(-3) substitutions per site per year. Recombination frequency was tightly correlated with VP1 divergence; viruses differing by evolutionary distances of >0.1 (or 6 years divergent evolution) almost invariably (>97%) had different 3Dpol groups. Frequencies of shared 3Dpol groups additionally correlated with geographical distances, with Europe and South Asia showing turnover of entirely distinct virus populations. Population turnover of E30 was characterized by repeated cycles of emergence, dominance, and disappearance of individual RFs over periods of 3 to 5 years, although the existence and nature of evolutionary selection underlying these population replacements remain unclear. The occurrence of frequent "sporadic" recombinants embedded within VP1 groupings of other RFs and the much greater number of 3Dpol groups than separately identifiable VP1 lineages suggest frequent recombination with an external diverse reservoir of non-E30 viruses.

Sharp CP, Norja P, Anthony I, Bell JE, Simmonds P. 2009. Reactivation and mutation of newly discovered WU, KI, and Merkel cell carcinoma polyomaviruses in immunosuppressed individuals. J Infect Dis, 199 (3), pp. 398-404. | Show Abstract | Read more

BACKGROUND: Infection with the human polyomaviruses BK (BKV) and JC (JCV) is almost ubiquitous, asymptomatic, and lifelong. However, reactivation during immunosuppression, associated with mutations in the transcriptional control region (TCR) that up-regulates viral replication, can cause life-threatening disease. In this study, we investigated whether the recently discovered WU and KI polyomaviruses (WUPyV and KIPyV) and Merkel cell polyomavirus (MCPyV) could, like BKV and JCV, persist, mutate, and reactivate in immunodeficient subjects. METHODS: Autopsy samples of lymphoid tissue from 42 AIDS-immunosuppressed subjects and 55 control samples were screened by polymerase chain reaction for all 5 polyomaviruses. TCR sequences from KIPyV and WUPyV recovered from both immunosuppressed and nonimmunosuppressed subjects were compared. RESULTS: Combined polyomavirus detection frequencies were much higher for the immunosuppressed group, compared with the nonimmunosuppressed group (35.7% vs. 3.6%), with viral loads in lymphoid tissues ranging from < or = 8.4 x 10(5) to > 1.5 x 10(5) viral genome copies per 10(6) cells. MCPyV was recovered from only 1 HIV-negative study subject. TCR sequences from reactivated WUPyV and KIPyV variants showed a number of point mutations and insertions that were absent in viruses recovered from respiratory tract specimens obtained from nonimmunosuppressed subjects. CONCLUSIONS: KIPyV and WUPyV show reactivation frequencies comparable to those of BKV and JCV during immunosuppression. TCR changes that potentially lead to transcriptional dysregulation may have pathogenic consequences equivalent in severity to those observed for JCV and BKV.

Leitch EC, Harvala H, Robertson I, Ubillos I, Templeton K, Simmonds P. 2009. Direct identification of human enterovirus serotypes in cerebrospinal fluid by amplification and sequencing of the VP1 region. J Clin Virol, 44 (2), pp. 119-124. | Show Abstract | Read more

BACKGROUND: Human enteroviruses (HEV) are a major cause of meningitis and other neurological disease. Identification of HEV serotypes in clinical cases is important for monitoring emergence of more pathogenic variants, epidemiological surveillance and investigating sources of infection. Serotype identification is currently problematic following the widespread adoption of polymerase chain reaction (PCR)-based methods for HEV detection in place of virus culture. OBJECTIVES: To develop a reliable, sensitive method to identify species A and B serotypes directly from cerebrospinal fluid (CSF) specimens. STUDY DESIGN: A nested-PCR was used to amplify VP1 region sequences of HEV species A and B, that enabled unambiguous serotype identification by comparison with reference strains. RESULTS: 62 from 64 diagnostic CSF samples collected over a 19-month study period were successfully amplified (97% sensitivity), compared with 9/22 (41%) identified by virus culture of co-referred faecal and throat swab samples. Among these, 60 samples contained species B and 2 samples contained species A serotypes (coxsackievirus A6 and enterovirus 71) were identified. Rapid changes in serotype frequencies and diversity were observed; echovirus (E) type 9 infections predominated in early 2007, to be replaced by E30 later in the year and followed by a diverse range of eight different species B serotypes in 2008. CONCLUSIONS: The availability of a simple and rapid method for identification of serotypes and individual HEV strains or clusters directly from CSF will be of substantial value in surveillance, understanding more about serotype-associated differences in disease and monitoring the global spread of pathogenic variants such as enterovirus 71.

Drexler JF, Kupfer B, Petersen N, Grotto RM, Rodrigues SM, Grywna K, Panning M, Annan A, Silva GF, Douglas J et al. 2009. A novel diagnostic target in the hepatitis C virus genome. PLoS Med, 6 (2), pp. e31. | Show Abstract | Read more

BACKGROUND: Detection and quantification of hepatitis C virus (HCV) RNA is integral to diagnostic and therapeutic regimens. All molecular assays target the viral 5'-noncoding region (5'-NCR), and all show genotype-dependent variation of sensitivities and viral load results. Non-western HCV genotypes have been under-represented in evaluation studies. An alternative diagnostic target region within the HCV genome could facilitate a new generation of assays. METHODS AND FINDINGS: In this study we determined by de novo sequencing that the 3'-X-tail element, characterized significantly later than the rest of the genome, is highly conserved across genotypes. To prove its clinical utility as a molecular diagnostic target, a prototype qualitative and quantitative test was developed and evaluated multicentrically on a large and complete panel of 725 clinical plasma samples, covering HCV genotypes 1-6, from four continents (Germany, UK, Brazil, South Africa, Singapore). To our knowledge, this is the most diversified and comprehensive panel of clinical and genotype specimens used in HCV nucleic acid testing (NAT) validation to date. The lower limit of detection (LOD) was 18.4 IU/ml (95% confidence interval, 15.3-24.1 IU/ml), suggesting applicability in donor blood screening. The upper LOD exceeded 10(-9) IU/ml, facilitating viral load monitoring within a wide dynamic range. In 598 genotyped samples, quantified by Bayer VERSANT 3.0 branched DNA (bDNA), X-tail-based viral loads were highly concordant with bDNA for all genotypes. Correlation coefficients between bDNA and X-tail NAT, for genotypes 1-6, were: 0.92, 0.85, 0.95, 0.91, 0.95, and 0.96, respectively; X-tail-based viral loads deviated by more than 0.5 log10 from 5'-NCR-based viral loads in only 12% of samples (maximum deviation, 0.85 log10). The successful introduction of X-tail NAT in a Brazilian laboratory confirmed the practical stability and robustness of the X-tail-based protocol. The assay was implemented at low reaction costs (US$8.70 per sample), short turnover times (2.5 h for up to 96 samples), and without technical difficulties. CONCLUSION: This study indicates a way to fundamentally improve HCV viral load monitoring and infection screening. Our prototype assay can serve as a template for a new generation of viral load assays. Additionally, to our knowledge this study provides the first open protocol to permit industry-grade HCV detection and quantification in resource-limited settings.

Kapoor A, Slikas E, Simmonds P, Chieochansin T, Naeem A, Shaukat S, Alam MM, Sharif S, Angez M, Zaidi S, Delwart E. 2009. A newly identified bocavirus species in human stool. J Infect Dis, 199 (2), pp. 196-200. | Show Abstract | Read more

Viral metagenomic analysis was used to identify a previously uncharacterized parvovirus species, "HBoV2," whose closest phylogenetic relative is the human bocavirus (HBoV). HBoV2 has a genomic organization identical to that of HBoV but has only 78%, 67%, and 80% identity, respectively, with the latter's NS1, NP1, and VP1/VP2 proteins. The study used polymerase chain reaction to detect HBoV2 sequences in 5 of 98 stool samples from Pakistani children and in 3 of 699 stool samples from Edinburgh. Nearly-full-length genome sequencing revealed the presence of 3 HBoV2 genotypes and evidence of recombination between genotypes. Further studies are necessary to identify anatomical sites of HBoV2 replication and potential associations with clinical symptoms or disease.

Wisdom A, Kutkowska AE, McWilliam Leitch EC, Gaunt E, Templeton K, Harvala H, Simmonds P. 2009. Genetics, recombination and clinical features of human rhinovirus species C (HRV-C) infections; interactions of HRV-C with other respiratory viruses. PLoS One, 4 (12), pp. e8518. | Show Abstract | Read more

To estimate the frequency, molecular epidemiological and clinical associations of infection with the newly described species C variants of human rhinoviruses (HRV), 3243 diagnostic respiratory samples referred for diagnostic testing in Edinburgh were screened using a VP4-encoding region-based selective polymerase chain reaction (PCR) for HRV-C along with parallel PCR testing for 13 other respiratory viruses. HRV-C was the third most frequently detected behind respiratory syncytial virus (RSV) and adenovirus, with 141 infection episodes detected among 1885 subjects over 13 months (7.5%). Infections predominantly targeted the very young (median age 6-12 months; 80% of infections in those <2 years), occurred throughout the year but with peak incidence in early winter months. HRV-C was detected significantly more frequently among subjects with lower (LRT) and upper respiratory tract (URT) disease than controls without respiratory symptoms; HRV-C mono-infections were the second most frequently detected virus (behind RSV) in both disease presentations (6.9% and 7.8% of all cases respectively). HRV variants were classified by VP4/VP2 sequencing into 39 genotypically defined types, increasing the current total worldwide to 60. Through sequence comparisons of the 5'untranslated region (5'UTR), the majority grouped with species A (n = 96; 68%, described as HRV-Ca), the remainder forming a phylogenetically distinct 5'UTR group (HRV-Cc). Multiple and bidirectional recombination events between HRV-Ca and HRV-Cc variants and with HRV species A represents the most parsimonious explanation for their interspersed phylogeny relationships in the VP4/VP2-encoding region. No difference in age distribution, seasonality or disease associations was identified between HRV-Ca and HRV-Cc variants. HRV-C-infected subjects showed markedly reduced detection frequencies of RSV and other respiratory viruses, providing evidence for a major interfering effect of HRV-C on susceptibility to other respiratory virus infections. HRV-C's disease associations, its prevalence and evidence for interfering effects on other respiratory viruses mandates incorporation of rhinoviruses into future diagnostic virology screening.

Kapoor A, Victoria J, Simmonds P, Slikas E, Chieochansin T, Naeem A, Shaukat S, Sharif S, Alam MM, Angez M et al. 2008. A highly prevalent and genetically diversified Picornaviridae genus in South Asian children. Proc Natl Acad Sci U S A, 105 (51), pp. 20482-20487. | Show Abstract | Read more

Viral metagenomics focused on particle-protected nucleic acids was used on the stools of South Asian children with nonpolio acute flaccid paralysis (AFP). We identified sequences distantly related to Seneca Valley virus and cardioviruses that were then used as genetic footholds to characterize multiple viral species within a previously unreported genus of the Picornaviridae family. The picornaviruses were detected in the stools of >40% of AFP and healthy Pakistani children. A genetically diverse and highly prevalent enteric viral infection, characteristics similar to the Enterovirus genus, was therefore identified substantially expanding the genetic diversity of the RNA viral flora commonly found in children.

Davis M, Sagan SM, Pezacki JP, Evans DJ, Simmonds P. 2008. Bioinformatic and physical characterizations of genome-scale ordered RNA structure in mammalian RNA viruses. J Virol, 82 (23), pp. 11824-11836. | Show Abstract | Read more

By the analysis of thermodynamic RNA secondary structure predictions, we previously obtained evidence for evolutionarily conserved large-scale ordering of RNA virus genomes (P. Simmonds, A. Tuplin, and D. J. Evans, RNA 10:1337-1351, 2004). Genome-scale ordered RNA structure (GORS) was widely distributed in many animal and plant viruses, much greater in extent than RNA structures required for viral translation or replication, but in mammalian viruses was associated with host persistence. To substantiate the existence of large-scale RNA structure differences between viruses, a large set of alignments of mammalian RNA viruses and rRNA sequences as controls were examined by thermodynamic methods (to calculate minimum free energy differences) and by algorithmically independent RNAz and Pfold methods. These methods produced generally concordant results and identified substantial differences in the degrees of evolutionarily conserved, sequence order-dependent RNA secondary structure between virus genera and groups. A probe hybridization accessibility assay was used to investigate the physical nature of GORS. Transcripts of hepatitis C virus (HCV), hepatitis G virus/GB virus-C (HGV/GBV-C), and murine norovirus, which are predicted to be structured, were largely inaccessible to hybridization in solution, in contrast to the almost universal binding of probes to a range of unstructured virus transcripts irrespective of G+C content. Using atomic force microscopy, HCV and HGV/GBV-C RNA was visualized as tightly compacted prolate spheroids, while under the same experimental conditions the predicted unstructured poliovirus and rubella virus RNA were pleomorphic and had extensively single-stranded RNA on deposition. Bioinformatic and physical characterization methods both identified fundamental differences in the configurations of viral genomic RNA that may modify their interactions with host cell defenses and their ability to persist.

Trinks J, Cuestas ML, Tanaka Y, Mathet VL, Minassian ML, Rivero CW, Benetucci JA, Gímenez ED, Segura M, Bobillo MC et al. 2008. Two simultaneous hepatitis B virus epidemics among injecting drug users and men who have sex with men in Buenos Aires, Argentina: characterization of the first D/A recombinant from the American continent. J Viral Hepat, 15 (11), pp. 827-838. | Show Abstract | Read more

Previous studies have revealed that hepatitis B virus (HBV)/D and HBV/F predominate among blood donors from Buenos Aires, Argentina. In the present study, blood samples from two high-risk groups were analysed: 160 corresponding to street- and hospital-recruited injecting drug users [81.2% showing the 'anti-hepatitis B core antigen (anti-HBc) only' serological pattern] and 20 to hepatitis B surface antigen (HBsAg)(+)/anti-HBc(+) men who have sex with men. HBV genotypes were assigned by polymerase chain reaction amplification followed by restriction fragment length polymorphism and confirmed by nucleotide sequencing of two different coding regions. HBV DNA was detected in 27 injecting drug users (16.9%, occult infection prevalence: 7.7%), and 14 men who have sex with men (70%). HBV/A prevailed among injecting drug users (81.8%) while HBV/F was predominant among men who have sex with men (57.1%). The high predominance of HBV/A among injecting drug users is in sharp contrast to its low prevalence among blood donors (P = 0.0006) and men who have sex with men (P = 0.0137). Interestingly, all HBV/A S gene sequences obtained from street-recruited injecting drug users encoded the rare serotype ayw1 and failed to cluster within any of the known A subgenotypes. Moreover, one of the HBV strains from a hospital-recruited injecting drug user was fully sequenced and found to be the first completely characterized D/A recombinant genome from the American continent. Data suggest that two simultaneous and independent HBV epidemics took place in Buenos Aires: one spreading among injecting drug users and another one sexually transmitted among the homosexual and heterosexual population.

Harvala H, Robertson I, McWilliam Leitch EC, Benschop K, Wolthers KC, Templeton K, Simmonds P. 2008. Epidemiology and clinical associations of human parechovirus respiratory infections. J Clin Microbiol, 46 (10), pp. 3446-3453. | Show Abstract | Read more

Infections with human parechoviruses (HPeVs) are prevalent in young children and have been associated with mild gastroenteritis and, less frequently, with meningitis and neonatal sepsis. To investigate the involvement of these viruses in respiratory disease, a highly sensitive nested PCR was used to screen a large archive of respiratory specimens, collected between January and December 2007. Respiratory samples had previously been tested for eight respiratory viruses, including respiratory syncytial virus and adenovirus, by PCR. HPeV was detected in 34 of 3,844 specimens, representing 27 of 2,220 study subjects (1.2%). HPeV types were identified by sequencing the VP3/VP1 junction amplified by PCR directly from clinical specimens. The assay could amplify all HPeV types examined with high sensitivity (types 1 and 3 to 6) and also identified HPeV types in all but one of the screen-positive study specimens (25 HPeV1 and eight HPeV6 specimens). Infections with both HPeV1 and HPeV6 were seasonal, with highest frequencies in July and August, and restricted to children aged between 6 months and 5 years. Other respiratory viruses were frequently codetected in HPeV-positive specimens, with significant overrepresentation of adenovirus coinfections (37%). Most HPeV-positive specimens were referred from emergency departments, although no association with specific respiratory symptoms or disease was found. In summary, the low frequency of detection and lack of clear disease associations indicate that HPeV1 and -6 are not major pathogens in individuals presenting with respiratory disease. However, the screening and typing methods developed will be of value in further HPeV testing, including testing for meningitis cases and other suspected HPeV-associated disease presentations.

Yang Y, Yi M, Evans DJ, Simmonds P, Lemon SM. 2008. Identification of a conserved RNA replication element (cre) within the 3Dpol-coding sequence of hepatoviruses. J Virol, 82 (20), pp. 10118-10128. | Show Abstract | Read more

Internally located, cis-acting RNA replication elements (cre) have been identified within the genomes of viruses representing each of the major picornavirus genera (Enterovirus, Rhinovirus, Aphthovirus, and Cardiovirus) except Hepatovirus. Previous efforts to identify a stem-loop structure with cre function in hepatitis A virus (HAV), the type species of this genus, by phylogenetic analyses or thermodynamic predictions have not succeeded. However, a region of markedly suppressed synonymous codon variability was identified in alignments of HAV sequences near the 5' end of the 3D(pol)-coding sequence of HAV, consistent with noncoding constraints imposed by an underlying RNA secondary structure. Subsequent MFOLD predictions identified a 110-nucleotide (nt) complex stem-loop in this region with a typical AAACA/G cre motif in its top loop. A potentially homologous RNA structure was identified in this region of the avian encephalitis virus genome, despite little nucleotide sequence relatedness between it and HAV. Mutations that disrupted secondary RNA structure or the AAACA/G motif, without altering the amino acid sequence of 3D(pol), ablated replication of a subgenomic HAV replicon in transfected human hepatoma cells. Replication competence could be rescued by reinsertion of the native 110-nt stem-loop structure (but not an abbreviated 45-nt stem-loop) upstream of the HAV coding sequence in the replicon. These results suggest that this stem-loop is functionally similar to cre elements of other picornaviruses and likely involved in templating VPg uridylylation as in other picornaviruses, despite its significantly larger size and lower free folding energy.

Simmonds P, Douglas J, Bestetti G, Longhi E, Antinori S, Parravicini C, Corbellino M. 2008. A third genotype of the human parvovirus PARV4 in sub-Saharan Africa. J Gen Virol, 89 (Pt 9), pp. 2299-2302. | Show Abstract | Read more

PARV4 is a recently discovered human parvovirus widely distributed in injecting drug users in the USA and Europe, particularly in those co-infected with human immunodeficiency virus (HIV). Like parvovirus B19, PARV4 persists in previously exposed individuals. In bone marrow and lymphoid tissue, PARV4 sequences were detected in two sub-Saharan African study subjects with AIDS but without a reported history of parenteral exposure and who were uninfected with hepatitis C virus. PARV4 variants infecting these subjects were phylogenetically distinct from genotypes 1 and 2 (formerly PARV5) that were reported previously. Analysis of near-complete genome sequences demonstrated that they should be classified as a third (equidistant) PARV4 genotype. The availability of a further near-complete genome sequence of this novel genotype facilitated identification of conserved novel open reading frames embedded in the ORF2 coding sequence; one encoded a putative protein with identifiable homology to SAT proteins of members of the genus Parvovirus.

Diviney S, Tuplin A, Struthers M, Armstrong V, Elliott RM, Simmonds P, Evans DJ. 2008. A hepatitis C virus cis-acting replication element forms a long-range RNA-RNA interaction with upstream RNA sequences in NS5B. J Virol, 82 (18), pp. 9008-9022. | Show Abstract | Read more

The genome of hepatitis C virus (HCV) contains cis-acting replication elements (CREs) comprised of RNA stem-loop structures located in both the 5' and 3' noncoding regions (5' and 3' NCRs) and in the NS5B coding sequence. Through the application of several algorithmically independent bioinformatic methods to detect phylogenetically conserved, thermodynamically favored RNA secondary structures, we demonstrate a long-range interaction between sequences in the previously described CRE (5BSL3.2, now SL9266) with a previously predicted unpaired sequence located 3' to SL9033, approximately 200 nucleotides upstream. Extensive reverse genetic analysis both supports this prediction and demonstrates a functional requirement in genome replication. By mutagenesis of the Con-1 replicon, we show that disruption of this alternative pairing inhibited replication, a phenotype that could be restored to wild-type levels through the introduction of compensating mutations in the upstream region. Substitution of the CRE with the analogous region of different genotypes of HCV produced replicons with phenotypes consistent with the hypothesis that both local and long-range interactions are critical for a fundamental aspect of genome replication. This report further extends the known interactions of the SL9266 CRE, which has also been shown to form a "kissing loop" interaction with the 3' NCR (P. Friebe, J. Boudet, J. P. Simorre, and R. Bartenschlager, J. Virol. 79:380-392, 2005), and suggests that cooperative long-range binding with both 5' and 3' sequences stabilizes the CRE at the core of a complex pseudoknot. Alternatively, if the long-range interactions were mutually exclusive, the SL9266 CRE may function as a molecular switch controlling a critical aspect of HCV genome replication.

Kuntzen T, Berical A, Ndjomou J, Bennett P, Schneidewind A, Lennon N, Birren BW, Kuiken C, Henn MR, Simmonds P, Allen TM. 2008. A set of reference sequences for the hepatitis C genotypes 4d, 4f, and 4k covering the full open reading frame. J Med Virol, 80 (8), pp. 1370-1378. | Show Abstract | Read more

Infection with genotype 4 of the Hepatitis C virus is common in Africa and the Mediterranean area, but has also been found at increasing frequencies in injection drug users in Europe and North America. Full length viral sequences to characterize viral diversity and structure have recently become available mostly for subtype 4a, and studies in Egypt and Saudi Arabia, where high proportions of subtype 4a infected patients exist, have begun to establish optimized treatment regimens. However knowledge about other subtype variants of genotype 4 present in less developed African states is lacking. In this study the full coding region from so far poorly characterized variants of HCV genotype 4 was amplified and sequenced using a long range PCR technique. Sequences were analyzed with respect to phylogenetic relationship, possible recombination and prominent sequence characteristics compared to other known HCV strains. We present for the first time two full-length sequences from the HCV genotype 4k, in addition to five strains from HCV genotypes 4d and 4f. Reference sequences for accurate HCV genotyping are required for optimized treatment, and a better knowledge of the global viral sequence diversity is needed to guide vaccines or new drugs effective in the world wide epidemic.

Kurbanov F, Tanaka Y, Kramvis A, Simmonds P, Mizokami M. 2008. When should "I" consider a new hepatitis B virus genotype? J Virol, 82 (16), pp. 8241-8242. | Read more

Norja P, Eis-Hübinger AM, Söderlund-Venermo M, Hedman K, Simmonds P. 2008. Rapid sequence change and geographical spread of human parvovirus B19: comparison of B19 virus evolution in acute and persistent infections. J Virol, 82 (13), pp. 6427-6433. | Show Abstract | Read more

Parvovirus B19 is a common human pathogen maintained by horizontal transmission between acutely infected individuals. However, B19 virus can also be detected in tissues throughout the life of the host, although little is understood about the nature of such persistence. In the current study, we created large VP1/2 sequence data sets of plasma- and tissue (autopsy)-derived variants of B19 virus with known sample dates to compare the rates of sequence change in exogenous virus populations with those in persistently infected individuals. By using linear regression and likelihood-based methods (such as the BEAST program), we found that plasma-derived B19 virus showed a substitution rate of 4 x 10(-4) and an unconstrained (synonymous)-substitution rate of 18 x 10(-4) per site per year, several times higher than previously estimated and within the range of values for mammalian RNA viruses. The underlying high mutation frequency implied by these substitution rates may enable rapid adaptive changes that are more commonly ascribed to RNA virus populations. These revised estimates predict that the last common ancestor for currently circulating genotype 1 variants of B19 virus existed around 1956 to 1959, fitting well with previous analyses of the B19 virus "bioportfolio" that support a complete cessation of genotype 2 infections and their replacement by genotype 1 infections in the 1960s. In contrast, the evolution of B19 virus amplified from tissue samples was best modeled by using estimated dates of primary infection rather than sample dates, consistent with slow or absent sequence change during persistence. Determining what epidemiological or biological factors led to such a complete and geographically extensive population replacement over this short period is central to further understanding the nature of parvovirus evolution.

Allander T, de Lamballerie X, Simmonds P. 2008. A new arenavirus in transplantation. N Engl J Med, 358 (24), pp. 2638. | Read more

Simmonds P, Karakasiliotis I, Bailey D, Chaudhry Y, Evans DJ, Goodfellow IG. 2008. Bioinformatic and functional analysis of RNA secondary structure elements among different genera of human and animal caliciviruses. Nucleic Acids Res, 36 (8), pp. 2530-2546. | Show Abstract | Read more

The mechanism and role of RNA structure elements in the replication and translation of Caliciviridae remains poorly understood. Several algorithmically independent methods were used to predict secondary structures within the Norovirus, Sapovirus, Vesivirus and Lagovirus genera. All showed profound suppression of synonymous site variability (SSSV) at genomic 5' ends and the start of the sub-genomic (sg) transcript, consistent with evolutionary constraints from underlying RNA structure. A newly developed thermodynamic scanning method predicted RNA folding mapping precisely to regions of SSSV and at the genomic 3' end. These regions contained several evolutionarily conserved RNA secondary structures, of variable size and positions. However, all caliciviruses contained 3' terminal hairpins, and stem-loops in the anti-genomic strand invariably six bases upstream of the sg transcript, indicating putative roles as sg promoters. Using the murine norovirus (MNV) reverse-genetics system, disruption of 5' end stem-loops produced approximately 15- to 20-fold infectivity reductions, while disruption of the RNA structure in the sg promoter region and at the 3' end entirely destroyed replication ability. Restoration of infectivity by repair mutations in the sg promoter region confirmed a functional role for the RNA secondary structure, not the sequence. This study provides comprehensive bioinformatic resources for future functional studies of MNV and other caliciviruses.

Benschop KS, Williams CH, Wolthers KC, Stanway G, Simmonds P. 2008. Widespread recombination within human parechoviruses: analysis of temporal dynamics and constraints. J Gen Virol, 89 (Pt 4), pp. 1030-1035. | Show Abstract | Read more

Human parechoviruses (HPeVs), members of the family Picornaviridae, are classified into six types. To investigate the dynamics and likelihood of recombination among HPeVs, we compared phylogenies of two distant regions (VP1 and 3Dpol) of 37 HPeV isolates (types 1 and 3-5) and prototype sequences (types 1-6). Evidence for frequent recombination between HPeV1, 4, 5 and 6 was found. The likelihood of recombination was correlated with the degree of VP1 divergence and differences in isolation dates, both indicative of evolutionary times of divergence. These temporal dynamics were found to be most similar to those of human enterovirus species B variants. In contrast, HPeV3 remained phylogenetically distinct from other types throughout the genome. As HPeV3 is equally divergent in nucleotide sequence from the other HPeV types, its genetic isolation may reflect different biology and changed cellular tropisms, arising from the deletion of the RGD motif, and likely use of a non-integrin receptor.

Simmonds P. 2008. Steps towards serological diagnosis of human bocavirus infections. Clin Infect Dis, 46 (4), pp. 547-549. | Read more

Peters PJ, Duenas-Decamp MJ, Sullivan WM, Brown R, Ankghuambom C, Luzuriaga K, Robinson J, Burton DR, Bell J, Simmonds P et al. 2008. Variation in HIV-1 R5 macrophage-tropism correlates with sensitivity to reagents that block envelope: CD4 interactions but not with sensitivity to other entry inhibitors. Retrovirology, 5 (1), pp. 5. | Show Abstract | Read more

BACKGROUND: HIV-1 R5 viruses cause most of the AIDS cases worldwide and are preferentially transmitted compared to CXCR4-using viruses. Furthermore, R5 viruses vary extensively in capacity to infect macrophages and highly macrophage-tropic variants are frequently identified in the brains of patients with dementia. Here, we investigated the sensitivity of R5 envelopes to a range of inhibitors and antibodies that block HIV entry. We studied a large panel of R5 envelopes, derived by PCR amplification without culture from brain, lymph node, blood and semen. These R5 envelopes conferred a wide range of macrophage tropism and included highly macrophage-tropic variants from brain and non-macrophage-tropic variants from lymph node. RESULTS: R5 macrophage-tropism correlated with sensitivity to inhibition by reagents that inhibited gp120:CD4 interactions. Thus, increasing macrophage-tropism was associated with increased sensitivity to soluble CD4 and to IgG-CD4 (PRO 542), but with increased resistance to the anti-CD4 monoclonal antibody (mab), Q4120. These observations were highly significant and are consistent with an increased affinity of envelope for CD4 for macrophage-tropic envelopes. No overall correlations were noted between R5 macrophage-tropism and sensitivity to CCR5 antagonists or to gp41 specific reagents. Intriguingly, there was a relationship between increasing macrophage-tropism and increased sensitivity to the CD4 binding site mab, b12, but decreased sensitivity to 2G12, a mab that binds a glycan complex on gp120. CONCLUSION: Variation in R5 macrophage-tropism is caused by envelope variation that predominantly influences sensitivity to reagents that block gp120:CD4 interactions. Such variation has important implications for therapy using viral entry inhibitors and for the design of envelope antigens for vaccines.

Hughes GJ, Cochrane A, Leen C, Morris S, Bell JE, Simmonds P. 2008. HIV-1-infected CD8+CD4+ T cells decay in vivo at a similar rate to infected CD4 T cells during HAART. AIDS, 22 (1), pp. 57-65. | Show Abstract | Read more

OBJECTIVES: To investigate the potential for CD4+CD8+ T cells [CD8 double positive (CD8 DP)] T cells to form a reservoir of HIV-1 following HAART through measurement of the rate of decay of infected CD4/CD8 DP T cells. METHODS: HIV-1 proviral loads in highly pure CD4 and CD8 DP T cells were determined for study subjects before and after 200-400 days of therapy and HIV-1 DNA decay rates were calculated. RESULTS: Before therapy, HIV-1 proviral load in CD8 DP correlated negatively with CD4 cell count. Decay rates of HIV-1-infected CD4 and CD8 DP T cells were similar. Rates for CD8 DP T cells correlated with the time to suppression of viral replication, whereas no such relationship was true for CD4 cell decay rates. A significant reduction in activated cells was observed for both cell types. The action of HAART on HIV-1 replication was similar for both CD4 cells and CD8 DP T cells, although the rate of clearance of infected CD8 DP T cells appeared more critical for a rapid reduction in plasma viral load. Although the size of the CD8 DP T cell reservoir in peripheral blood was smaller relative to that of CD4 cells, HAART did not completely clear HIV-1 infection from this cell subset. CONCLUSION: This study confirmed that CD8 DP T cells are a major reservoir for HIV-1 in vivo and, therefore, represent a potential reservoir for HIV-1 during HAART, in a manner analogous to that of CD4 T cells.

Kapoor A, Victoria J, Simmonds P, Wang C, Shafer RW, Nims R, Nielsen O, Delwart E. 2008. A highly divergent picornavirus in a marine mammal. J Virol, 82 (1), pp. 311-320. | Show Abstract | Read more

Nucleic acids from an unidentified virus from ringed seals (Phoca hispida) were amplified using sequence-independent PCR, subcloned, and then sequenced. The full genome of a novel RNA virus was derived, identifying the first sequence-confirmed picornavirus in a marine mammal. The phylogenetic position of the tentatively named seal picornavirus 1 (SePV-1) as an outlier to the grouping of parechoviruses was found consistently in alignable regions of the genome. A mean protein sequence identity of only 19.3 to 30.0% was found between the 3D polymerase gene sequence of SePV-1 and those of other picornaviruses. The predicted secondary structure of the short 506-base 5'-untranslated region showed some attributes of a type IVB internal ribosome entry site, and the polyprotein lacked an apparent L peptide, both properties associated with the Parechovirus genus. The presence of two SePV-1 2A genes and of the canonical sequence required for cotranslational cleavage resembled the genetic organization of Ljungan virus. Minor genetic variants were detected in culture supernatants derived from 8 of 108 (7.4%) seals collected in 2000 to 2002, indicating a high prevalence of SePV-1 in this hunted seal population. The high level of genetic divergence of SePV-1 compared to other picornaviruses and its mix of characteristics relative to its closest relatives support the provisional classification of SePV-1 as the prototype for a new genus in the family Picornaviridae.

Anthony IC, Arango JC, Stephens B, Simmonds P, Bell JE. 2008. The effects of illicit drugs on the HIV infected brain. Front Biosci, 13 (13), pp. 1294-1307. | Show Abstract | Read more

Evidence accumulating from clinical observations, neuroimaging and neuropathological studies suggests that illicit drug abuse accentuates the adverse effects of HIV on the central nervous system (CNS). Experimental investigation in cell culture models supports this conclusion. Injecting drug abuse is also a risk factor for the acquisition of HIV infection, the incidence of which continues to rise in intravenous drug users (IVDU) even in countries with access to effective therapy. In order to understand the interactions of drug abuse and HIV infection, it is necessary to examine the effects of each insult in isolation before looking for their combined effects. This review traces progress in understanding the pathogenesis of HIV related CNS disorders before the introduction of effective therapy and compares the state of our knowledge now that effective therapy has significantly modified disease progression. The additional impact of intravenous drug abuse on HIV-associated brain disease, then and now, is also reviewed. Predictions for the future are discussed, based on what is known at present and on recently emerging data.

Norja P, Ubillos I, Templeton K, Simmonds P. 2007. No evidence for an association between infections with WU and KI polyomaviruses and respiratory disease. J Clin Virol, 40 (4), pp. 307-311. | Show Abstract | Read more

BACKGROUND: WU virus (WUV) and KI polyomavirus (KIPyV) are newly discovered related human polyomaviruses detected in respiratory samples. To investigate their potential role in respiratory disease, we determined their frequencies of detection, clinical presentations and epidemiological characteristics among samples referred for diagnostic respiratory virus testing. METHODS: Anonymised samples and accompanying study subject information were obtained from the Edinburgh respiratory specimen archive. Samples were screened by nested PCR using two sets of primers conserved between WUV and KIPyV, as well for other respiratory viruses (respiratory syncytial virus [RSV], adenoviruses [AdV], influenza A/B and parainfluenza viruses 1-3, human bocavirus, B19). RESULTS AND CONCLUSIONS: WUV and KIPyV were detected in 10 and 14 samples, respectively from 983 specimens (from 9 to 10 different individuals from 612 study subjects). Infections occurred in two types of study subject; those who were young (<2 years) with lower respiratory tract infections (n=8), and almost invariably co-infected with other respiratory viruses (RSV, AdV), and a second, generally older group either without respiratory disease (n=6) or with mild upper respiratory tract infections (n=5) but who were generally clinically severely immunosuppressed from leukaemia or transplant therapy. Findings from either group do not support an aetiological link between infection with WUV or KIPyV and respiratory disease.

Brown KE, Simmonds P. 2007. Parvoviruses and blood transfusion. Transfusion, 47 (10), pp. 1745-1750. | Read more

Simmonds P, Manning A, Kenneil R, Carnie FW, Bell JE. 2007. Parenteral transmission of the novel human parvovirus PARV4. Emerg Infect Dis, 13 (9), pp. 1386-1388. | Show Abstract | Read more

Transmission routes of PARV4, a newly discovered human parvovirus, were investigated by determining frequencies of persistent infections using autopsy samples from different risk groups. Predominantly parenteral routes of transmission were demonstrated by infection restricted to injection drug users and persons with hemophilia and absence of infection in homosexual men with AIDS and low-risk controls.

Hughes GJ, Willey SJ, Cochrane A, Leen C, Bell JE, Simmonds P. 2007. Virus immunocapture provides evidence of CD8 lymphocyte-derived HIV-1 in vivo. AIDS, 21 (12), pp. 1507-1513. | Show Abstract | Read more

OBJECTIVES: To demonstrate that HIV-1 immunocapture with an antibody against CD8 specifically captures virions derived from infected CD8 T cells, and to determine the proportion of HIV-1 derived from CD8 lymphocytes in plasma samples from HIV-infected individuals. METHODS: A virus capture method was developed to enable the detection of HIV-1 virions based upon the presence of certain cell-specific host-derived proteins (CD8, CD3, CD36) within the viral envelope. HIV-1 virions were captured using antibodies against these proteins and levels of bound virus were determined by quantitative reverse transcriptase-polymerase chain reaction. Highly pure CD8 and CD3+CD8- T-cell cultures were used as in-vitro models to determine the specificity of the virus capture technique. RESULTS: The in-vitro model demonstrates that incorporation of the CD8 molecule into released virions is specific to infection of CD8 T cells. Levels of HIV-1 immunocaptured from plasma of infected individuals using the anti-CD8 antibody indicate that up to 15% (range 10-33) of the plasma viral load is derived from CD8 lymphocytes. CONCLUSION: This study demonstrates for the first time that HIV-1-infected CD8 T cells can contribute substantially to levels of circulating virus during the course of infection. Levels of CD8-derived virus did not correlate with the level of infection of circulating CD8 T cells, but do show a significantly good fit to plasma viral loads based on a power model. The extensive infection of CD8 T cells implied by these results may contribute towards immune dysfunction and disease progression to AIDS.

Manning A, Willey SJ, Bell JE, Simmonds P. 2007. Comparison of tissue distribution, persistence, and molecular epidemiology of parvovirus B19 and novel human parvoviruses PARV4 and human bocavirus. J Infect Dis, 195 (9), pp. 1345-1352. | Show Abstract | Read more

BACKGROUND: PARV4 and human bocavirus (HBoV) are newly discovered human parvoviruses with poorly understood epidemiologies and disease associations. We investigated the frequencies of persistence, tissue distribution, and influence of immunosuppression on replication of these viruses. METHODS: At autopsy, bone marrow, lymphoid tissue, and brain tissue from human immunodeficiency virus (HIV)-infected individuals with acquired immunodeficiency syndrome (AIDS) and those without AIDS and from HIV-uninfected individuals were screened for parvovirus B19, PARV4, and HBoV DNA by means of quantitative polymerase chain reaction analyses. RESULTS: B19 DNA was detected both in HIV-infected study subjects (13 of 24) and in HIV-uninfected study subjects (8 of 8), whereas PARV4 DNA was detected only in HIV-infected study subjects (17 of 24). HBoV DNA was not detected in any study subjects. The degree of immunosuppression with HIV infection did not influence B19 or PARV4 viral loads. B19 or PARV4 plasma viremia was not detected in any study subjects (n=76; viral load <25 DNA copies/mL). A significantly older age distribution was found for study subjects infected with B19 genotype 2, compared with those infected with B19 genotype 1. Two genotypes of PARV4 were detected; study subjects carrying prototype PARV4 (genotype 1) were younger (all born after 1958) than those infected with genotype 2 (PARV5; study subjects born between 1949 and 1956). CONCLUSIONS: Tight immune control of replication of B19 and PARV4 was retained despite profound immunosuppression. Recent genotype replacement of PARV4, combined with absent sequence diversity among genotype 1 sequences, suggests a recent, epidemic spread in the United Kingdom, potentially through transmission routes shared by HIV.

Kuiken C, Combet C, Bukh J, Shin-I T, Deleage G, Mizokami M, Richardson R, Sablon E, Yusim K, Pawlotsky JM et al. 2006. A comprehensive system for consistent numbering of HCV sequences, proteins and epitopes. Hepatology, 44 (5), pp. 1355-1361. | Show Abstract | Read more

This numbering proposal, using the AF009606 (isolate H77) sequence as a reference, should be able to unequivocally number all possible mutations in HCV, both natural and manmade. The HCV sequence databases 8 and the Los Alamos HCV immunology database 9 (as well as the Los Alamos HIV database) number positions and epitopes according to this system. Moreover, the databases websites provides tools for finding stretches of sequence by their numbers, for assigning start and end coordinates to a sequence, and for converting between the various numbering systems. Numbering HCV nucleotide sequences is done by analogy to H77. The first step is aligning your sequence to H77. If there is no length variation, the numbering is straightforward; nucleotide numbers run from 1 (start of 5′ UTR) to 9646 (end of 3′ UTR). Insertions relative to H77 are labeled with letters. Protein numbering works like the nucleotide numbering, but starts at the start of the polyprotein. The sequence databases will support both systems, but use polyprotein numbering as a basis. Absolute numbering moves across the coding regions, relative numbering starts over at every coding region. Relative numbering is almost exclusively used for proteins, polyprotein numbering mainly in immunology, protein numbering in drug resistance research. The Los Alamos immunology database uses polyprotein numbering. The 5′ UTR numbering starts at 1 and ends at 341; the Core cds starts at 342. The numbering of the 3′ UTR starts at 9378 (after the stop codon), but complications arise due to the variable length of the PPT. The UTR consists of 3 elements: a variable 5′ region, the PPT, and a conserved 3′ region, often called X. The first region is numbered 9378-9410. The PPT consists almost entirely of T's and therefore cannot b e meaningfully aligned; it is numbered according to its length in H77, 9411-9545. The X region starts at 9546 (regardless of its actual location, which depends on the length of the PPT) and ends at 9646. Copyright © 2006 by the American Association for the Study of Liver Diseases.

Simmonds P. 2006. Recombination and selection in the evolution of picornaviruses and other Mammalian positive-stranded RNA viruses. J Virol, 80 (22), pp. 11124-11140. | Show Abstract | Read more

Picornaviridae are a large virus family causing widespread, often pathogenic infections in humans and other mammals. Picornaviruses are genetically and antigenically highly diverse, with evidence for complex evolutionary histories in which recombination plays a major part. To investigate the nature of recombination and selection processes underlying the evolution of serotypes within different picornavirus genera, large-scale analysis of recombination frequencies and sites, segregation by serotype within each genus, and sequence selection and composition was performed, and results were compared with those for other nonenveloped positive-stranded viruses (astroviruses and human noroviruses) and with flavivirus and alphavirus control groups. Enteroviruses, aphthoviruses, and teschoviruses showed phylogenetic segregation by serotype only in the structural region; lack of segregation elsewhere was attributable to extensive interserotype recombination. Nonsegregating viruses also showed several characteristic sequence divergence and composition differences between genome regions that were absent from segregating virus control groups, such as much greater amino acid sequence divergence in the structural region, markedly elevated ratios of nonsynonymous-to-synonymous substitutions, and differences in codon usage. These properties were shared with other picornavirus genera, such as the parechoviruses and erboviruses. The nonenveloped astroviruses and noroviruses similarly showed high frequencies of recombination, evidence for positive selection, and differential codon use in the capsid region, implying similar underlying evolutionary mechanisms and pressures driving serotype differentiation. This process was distinct from more-recent sequence evolution generating diversity within picornavirus serotypes, in which neutral or purifying selection was prominent. Overall, this study identifies common themes in the diversification process generating picornavirus serotypes that contribute to understanding of their evolution and pathogenicity.

Manning A, Russell V, Eastick K, Leadbetter GH, Hallam N, Templeton K, Simmonds P. 2006. Epidemiological profile and clinical associations of human bocavirus and other human parvoviruses. J Infect Dis, 194 (9), pp. 1283-1290. | Show Abstract | Read more

BACKGROUND: Human bocavirus (HBoV) and PARV4 are newly discovered human parvoviruses. HBoV, which was first detected in respiratory samples, has a potential role in the development of human respiratory disease. The present study compared the frequencies, epidemiological profiles, and clinical backgrounds of HBoV and PARV4 infections with those of other respiratory virus infections, by evaluating diagnostic samples referred to the Specialist Virology Laboratory (SVL) at the Royal Infirmary of Edinburgh (Edinburgh, United Kingdom). METHODS: Anonymized samples and study subject information were obtained from the respiratory sample archive of the SVL. Samples were screened for HBoV, PARV4, B19, respiratory syncytial virus (RSV), adenoviruses, influenza viruses, and parainfluenza viruses by use of nested polymerase chain reaction. RESULTS: HBoV infection was detected in 47 (8.2%) of 574 study subjects, ranking third in prevalence behind RSV infection (15.7%) and adenovirus infection (10.3%). Peak incidences of HBoV were noted among infants and young children (age, 6-24 months) during the midwinter months (December and January) and were specifically associated with lower respiratory tract infections. HBoV infections were frequently accompanied by other respiratory viruses (frequency, 43%), and they were more prevalent among individuals infected with other respiratory viruses (17%), frequently adenovirus or RSV. All respiratory samples were negative for PARV4. CONCLUSIONS: In the present study, HBoV was a frequently detected, potential respiratory pathogen, with a prevalence and an epidemiological profile comparable to those of RSV. Identification of HBoV infections may be clinically important in the future.

Welch J, Bienek C, Gomperts E, Simmonds P. 2006. Resistance of porcine circovirus and chicken anemia virus to virus inactivation procedures used for blood products. Transfusion, 46 (11), pp. 1951-1958. | Show Abstract | Read more

BACKGROUND: Virus inactivation procedures are used to prevent contamination of plasma-derived blood products with viruses. Pasteurization or prolonged dry heat has proven effective against several enveloped and nonenveloped viruses and provides an additional layer of safety for plasma products. STUDY DESIGN AND METHODS: The resistance of porcine circovirus 2 (PCV2) and chicken anemia virus (CAV), two small, nonenveloped viruses, to standard (pasteurization, 10 hr at 60 degrees C; dry heating, 80 degrees C for 72 hr) and more extreme heat inactivation procedures (temperatures up to 120 degrees C) was determined. The ability of these procedures to inactivate PCV2 and CAV was measured by comparison of in vitro infectivity before and after treatment. RESULTS: Infectivity of PCV2 and CAV was reduced by approximately 1.6 and 1.4 log by pasteurization and by 0.75 and 1.25 log by dry-heat treatment, both substantially more resistant than other viruses previously investigated. PCV2 and CAV were additionally almost completely resistant to dry-heat treatment up to 120 degrees C for 30 minutes (mean log infectivity reductions, 1.25 and 0.6), although both were more effectively inactivated when the temperature of wet-heat treatment was increased to 80 degrees C (>3.2 and >3.6 log infectivity reduction). CONCLUSION: Although neither PCV2 nor CAV are known to infect humans, their inactivation properties may represent those of other small DNA viruses known to be present (e.g., TT virus, small anellovirus) or potentially present in human plasma. Findings of extreme thermal resistance demonstrate that recipients of plasma-derived therapeutics may potentially still be exposed to small DNA viruses, despite the implementation of viral inactivation steps.

Peters PJ, Sullivan WM, Duenas-Decamp MJ, Bhattacharya J, Ankghuambom C, Brown R, Luzuriaga K, Bell J, Simmonds P, Ball J, Clapham PR. 2006. Non-macrophage-tropic human immunodeficiency virus type 1 R5 envelopes predominate in blood, lymph nodes, and semen: implications for transmission and pathogenesis. J Virol, 80 (13), pp. 6324-6332. | Show Abstract | Read more

Human immunodeficiency virus type 1 (HIV-1) R5 isolates that predominantly use CCR5 as a coreceptor are frequently described as macrophage tropic. Here, we compare macrophage tropism conferred by HIV-1 R5 envelopes that were derived directly by PCR from patient tissue. This approach avoids potentially selective culture protocols used in virus isolation. Envelopes were amplified (i) from blood and semen of adult patients and (ii) from plasma of pediatric patients. The phenotypes of these envelopes were compared to those conferred by an extended panel of envelopes derived from brain and lymph node that we reported previously. Our results show that R5 envelopes vary by up to 1,000-fold in their capacity to confer infection of primary macrophages. Highly macrophage-tropic envelopes were predominate in brain but were infrequent in semen, blood, and lymph node samples. We also confirmed that the presence of N283 in the C2 CD4 binding site of gp120 is associated with HIV-1 envelopes from the brain but absent from macrophage-tropic envelopes amplified from blood and semen. Finally, we compared infection of macrophages, CD4(+) T cells, and peripheral blood mononuclear cells (PBMCs) conferred by macrophage-tropic and non-macrophage-tropic envelopes in the context of full-length replication competent viral clones. Non-macrophage-tropic envelopes conferred low-level infection of macrophages yet infected CD4(+) T cells and PBMCs as efficiently as highly macrophage-tropic brain envelopes. The lack of macrophage tropism for the majority of the envelopes amplified from lymph node, blood, and semen is striking and contrasts with the current consensus that R5 primary isolates are generally macrophage tropic. The extensive variation in R5 tropism reported here is likely to have an important impact on pathogenesis and on the capacity of HIV-1 to transmit.

Bell JE, Anthony IC, Simmonds P. 2006. Impact of HIV on regional & cellular organisation of the brain. Curr HIV Res, 4 (3), pp. 249-257. | Show Abstract | Read more

There are many excellent reviews of HIV infection of the nervous system. However these all assume that the reader has a working knowledge of the structure and cellular architecture of the brain. It may be that specialised brain vocabulary represents an unwelcome hurdle for those scientists with expert knowledge of the effects of HIV in other cell systems and who wish to extend that interest to the brain. This review provides an introduction to the component structures and cells of the brain and an overview of their involvement in HIV/AIDS. HIV infection leads to death through its capacity to progressively devastate the immune system. Current anti-HIV therapy has achieved considerable success in halting and partially reversing this process. In the absence of treatment, the breakdown of immunity is marked by declining CD4 counts and increasing vulnerability to opportunistic infections. In parallel with these effects on the lymphoid system, the nervous system is frequently the site of an initially stealthy infection which leads ultimately to symptomatic disease in a significant proportion of HIV infected individuals. The most feared manifestation of central nervous system (CNS) involvement is dementia. Unfortunately, serial CD4 counts and measurement of blood viral load do not serve to identify or monitor early infection of brain tissue. Since effective anti-HIV therapy has not achieved eradication of virus from lymphoid tissues, and anti-HIV drugs do not enter the nervous system easily, it is hardly surprising that HIV infection of the nervous system continues to cause clinical problems. Even in treatment-compliant patients, a measurable degree of cognitive impairment may develop, signalling previous or present HIV-related brain injury. The cause of HIV associated dementia and cognitive disability remains poorly understood. Perhaps most significantly, the long-term consequences of clinically occult brain infection are unknown and will require further investigation.

Anthony IC, Ramage SN, Carnie FW, Simmonds P, Bell JE. 2006. Accelerated Tau deposition in the brains of individuals infected with human immunodeficiency virus-1 before and after the advent of highly active anti-retroviral therapy. Acta Neuropathol, 111 (6), pp. 529-538. | Show Abstract | Read more

This study aims to investigate the influence of human immunodeficiency virus (HIV) infection on the neurodegenerative processes normally associated with ageing. We have looked for evidence of beta amyloid and hyperphosphorylated Tau deposition in HIV-infected subjects before and after the advent of highly active anti-retroviral therapy (HAART). In addition we have looked for evidence of axonal damage. We have compared these HIV-positive cases with age-matched controls and with older non-demented controls. We find no evidence of significant premature beta amyloid deposition in HIV-infected cases; however, we do observe elevated levels of hyperphosphorylated Tau in the hippocampus of many HIV-infected subjects, compared with age-matched controls. The greatest levels of hyperphosphorylated Tau are noted in HAART-treated subjects. Axonal damage marked by expression of beta amyloid pre-cursor protein (BAPP) was highly variable in all groups including control subjects. We surmise that HIV infection and/or the use of anti-retroviral therapy may predispose to accelerated neuroageing in the form of hyperphosphorylated Tau deposition in the hippocampus. Within the age groups studied these significant neuropathological changes remained subclinical and were not yet associated with cognitive impairment.

McCrossan M, Marsden M, Carnie FW, Minnis S, Hansoti B, Anthony IC, Brettle RP, Bell JE, Simmonds P. 2006. An immune control model for viral replication in the CNS during presymptomatic HIV infection. Brain, 129 (Pt 2), pp. 503-516. | Show Abstract | Read more

The brain is targeted by human immunodeficiency virus type 1 (HIV-1) during the course of untreated infection, leading to cognitive impairment, neurological damage and HIV encephalitis (HIVE). To study early dynamics of HIV entry into the brain, we examined a unique autopsy series of samples obtained from 15 untreated individuals who died in the presymptomatic stages of infection from non-HIV causes. HIV was detected and quantified by limiting dilution PCR and genetically characterized in the V3 region of env. Limiting dilution was shown to be essential for correct estimation of genetic partitioning between brain- and lymphoid-associated HIV populations. While no actively expressing HIV-infected cells were detected by immunohistochemistry, variable and generally extremely low levels of proviral DNA were detected in presymptomatic brain samples. V3 region sequences were frequently genetically distinct from lymphoid-associated HIV variants, with association index (AI) values similar to those observed in cases of HIVE. Infiltration of CD8 lymphocytes in the brain was strongly associated with expression of activation markers (MHCII; R = 0.619; P < 0.05), the presence of HIV-infected cells (proviral load; R = 0.608; P < 0.05) and genetic segregation of brain variants from populations in lymphoid tissue (AI value, R = -0.528; P approximately 0.05). CD8 lymphocytes may thus limit replication of HIV seeded into the brain in early stages of infection. Neurological complications in AIDS occur when this control breaks down, due to systemic immunosuppression from HIV that destroys CD8 lymphocyte function and/or through the evolution of more aggressive neuropathogenic variants.

Ludlam CA, Powderly WG, Bozzette S, Diamond M, Koerper MA, Kulkarni R, Ritchie B, Siegel J, Simmonds P, Stanley S et al. 2006. Clinical perspectives of emerging pathogens in bleeding disorders. Lancet, 367 (9506), pp. 252-261. | Show Abstract | Read more

As a result of immunological and nucleic-acid screening of plasma donations for transfusion-transmissible viruses, and the incorporation of viral reduction processes during plasma fractionation, coagulation-factor concentrates (CFC) are now judged safe in terms of many known infectious agents, including hepatitis B and C viruses, HIV, and human T-cell lymphotropic virus. However, emerging pathogens could pose future threats, particularly those with blood-borne stages that are resistant to viral-inactivation steps in the manufacturing process, such as non-lipid-coated viruses. As outlined in this Review, better understanding of infectious diseases allows challenges from newly described agents of potential concern in the future to be anticipated, but the processes of zoonotic transmission and genetic selection or modification ensure that plasma-derived products will continue to be subject to infectious concerns. Manufacturers of plasma-derived CFC have addressed the issue of emerging infectious agents by developing recombinant products that limit the need for human plasma during production. Such recombinant products have extended the safety profile of their predecessors by ensuring that all reagents used for cell culture, purification steps, and stabilisation and storage buffers are completely independent of human plasma.

Simmonds P, Welch J. 2006. Frequency and dynamics of recombination within different species of human enteroviruses. J Virol, 80 (1), pp. 483-493. | Show Abstract | Read more

Enteroviruses are members of the family Picornaviridae that cause widespread infections in human and other mammalian populations. Enteroviruses are genetically and antigenically highly variable, and recombination within and between serotypes contributes to their genetic diversity. To investigate the dynamics of the recombination process, sequence phylogenies between three regions of the genome (VP4, VP1, and 3Dpol) were compared among species A and B enterovirus variants detected in a human population-based survey in Scotland between 2000 and 2001, along with contemporary virus isolates collected in the same geographical region. This analysis used novel bioinformatic methods to quantify phylogenetic compatibility and correlations with serotype assignments of evolutionary trees constructed for different regions of the enterovirus genome. Species B enteroviruses showed much more frequent, time-correlated recombination events than those found for species A, despite the equivalence in population sampling, concordant with a linkage analysis of previously characterized enterovirus sequences obtained over longer collection periods. An analysis of recombination among complete genome sequences by computation of a phylogenetic compatibility matrix (PCM) demonstrated sharply defined boundaries between the VP2/VP3/VP1 block and sequences to either side in phylogenetic compatibility. The PCM also revealed equivalent or frequently greater degrees of incompatibility between different parts within the nonstructural region (2A-3D), indicating the occurrence of extensive recombination events in the past evolution of this part of the genome. Together, these findings provide new insights into the dynamics of species A and B enterovirus recombination and evolution and into the contribution of structured sampling to documenting reservoirs, emergence, and spread of novel recombinant forms in human populations.

Simmonds P, Midgley S. 2005. Recombination in the genesis and evolution of hepatitis B virus genotypes. J Virol, 79 (24), pp. 15467-15476. | Show Abstract | Read more

Hepatitis B virus (HBV) infection is widely distributed in both human and ape populations throughout the world and is a major cause of human morbidity and mortality. HBV variants are currently classified into the human genotypes A to H and species-associated chimpanzee and gibbon/orangutan groups. To examine the role of recombination in the evolution of HBV, large-scale data retrieval and automated phylogenetic analysis (TreeOrder scanning) were carried out on all available published complete genome sequences of HBV. We detected a total of 24 phylogenetically independent potential recombinants (different genotype combinations or distinct breakpoints), eight of which were previously undescribed. Instances of intergenotype recombination were observed in all human and ape HBV variants, including evidence for a novel gibbon/genotype C recombinant among HBV variants from Vietnam. By recording sequence positions in trees generated from sequential fragments across the genome, violations of phylogeny between trees also provided evidence for frequent intragenotype recombination between members of genotypes A, D, F/H, and gibbon variants but not in B, C, or the Asian B/C recombinant group. In many cases, favored positions for both inter- and intragenotype recombination matched positions of phylogenetic reorganization between the human and ape genotypes, such as the end of the surface gene and the core gene, where sequence relationships between genotypes changed in the TreeOrder scan. These findings provide evidence for the occurrence of past, extensive recombination events in the evolutionary history of the currently classified genotypes of HBV and potentially in changes in its global epidemiology and associations with human disease.

Simmonds P, Bukh J, Combet C, Deléage G, Enomoto N, Feinstone S, Halfon P, Inchauspé G, Kuiken C, Maertens G et al. 2005. Consensus proposals for a unified system of nomenclature of hepatitis C virus genotypes. Hepatology, 42 (4), pp. 962-973. | Show Abstract | Read more

International standardization and coordination of the nomenclature of variants of hepatitis C virus (HCV) is increasingly needed as more is discovered about the scale of HCV-related liver disease and important biological and antigenic differences that exist between variants. A group of scientists expert in the field of HCV genetic variability, and those involved in development of HCV sequence databases, the Hepatitis Virus Database (Japan), euHCVdb (France), and Los Alamos (United States), met to re-examine the status of HCV genotype nomenclature, resolve conflicting genotype or subtype names among described variants of HCV, and draw up revised criteria for the assignment of new genotypes as they are discovered in the future. A comprehensive listing of all currently classified variants of HCV incorporates a number of agreed genotype and subtype name re-assignments to create consistency in nomenclature. The paper also contains consensus proposals for the classification of new variants into genotypes and subtypes, which recognizes and incorporates new knowledge of HCV genetic diversity and epidemiology. A proposal was made that HCV variants be classified into 6 genotypes (representing the 6 genetic groups defined by phylogenetic analysis). Subtype name assignment will be either confirmed or provisional, depending on the availability of complete or partial nucleotide sequence data, or remain unassigned where fewer than 3 examples of a new subtype have been described. In conclusion, these proposals provide the framework by which the HCV databases store and provide access to data on HCV, which will internationally coordinate the assignment of new genotypes and subtypes in the future.

Cochrane A, Hughes GJ, Seaton RA, Simmonds P. 2005. First evidence of HIV infection of CD8 lymphocytes expressing CD4 during primary HIV-1 infection. AIDS, 19 (11), pp. 1237-1239. | Read more

Jones MS, Kapoor A, Lukashov VV, Simmonds P, Hecht F, Delwart E. 2005. New DNA viruses identified in patients with acute viral infection syndrome. J Virol, 79 (13), pp. 8230-8236. | Show Abstract | Read more

A sequence-independent PCR amplification method was used to identify viral nucleic acids in the plasma samples of 25 individuals presenting with symptoms of acute viral infection following high-risk behavior for human immunodeficiency virus type 1 transmission. GB virus C/hepatitis G virus was identified in three individuals and hepatitis B virus in one individual. Three previously undescribed DNA viruses were also detected, a parvovirus and two viruses related to TT virus (TTV). Nucleic acids in human plasma that were distantly related to bacterial sequences or with no detectable similarities to known sequences were also found. Nearly complete viral genome sequencing and phylogenetic analysis confirmed the presence of a new parvovirus distinct from known human and animal parvoviruses and of two related TTV-like viruses highly divergent from both the TTV and TTV-like minivirus groups. The detection of two previously undescribed viral species in a small group of individuals presenting acute viral syndrome with unknown etiology indicates that a rich yield of new human viruses may be readily identifiable using simple methods of sequence-independent nucleic acid amplification and limited sequencing.

Anthony IC, Ramage SN, Carnie FW, Simmonds P, Bell JE. 2005. Does drug abuse alter microglial phenotype and cell turnover in the context of advancing HIV infection? Neuropathol Appl Neurobiol, 31 (3), pp. 325-338. | Show Abstract | Read more

The aim of this study was to test the effects of drug abuse, in particular opiate abuse, on the phenotype and turnover of microglial cells within the brain in the context of advancing HIV infection. Basal ganglia and hippocampus sections were studied in 51 cases divided into six groups: HIV-negative normal controls, HIV-negative drug abusers, AIDS nondrug abusers, AIDS drug abusers, HIV encephalitis (HIVE) nondrug abusers and HIVE drug abusers. None of the cases studied had received highly active anti-retroviral therapy (HAART). Microglial phenotypes were defined using CD14, CD16, CD68 and major histocompatibility class II (MHC II). Microglial turnover was assessed using terminal deoxynucleotidyl transferase mediated dUTP nick end labelling (TUNEL) (DNA damage), BAX (proapoptotic marker), Fas (CD95) (proapoptotic), proliferating cell nuclear antigen (PCNA) (proliferation and DNA repair), Ki-67 (cell proliferation) and BCL-2 (antiapoptosis). We find increased expression of MHC II and CD16 in response to drug abuse. We also noted increased levels of TUNEL positivity in drug abusers compared to nondrug abusers, although conversely we found lower levels of BAX in those who had abused drugs. We find no evidence of microglial proliferation in any of our study groups, including HIVE, although HIV infection leads to increased expression of CD16, CD68 and MHC II. CD14 expression was restricted to perivascular microglia in all groups (including normal controls) apart from the two HIVE groups where some but not all cases also showed parenchymal expression of CD14. In contrast, CD16 was found in parenchymal microglia in all groups. Using high-pressure antigen retrieval and tyramide signal amplification, we find moderately high levels of CD16 expression in the parenchyma of normal brains which is not normally observed using standard avidin/biotin complex (ABC) techniques. This suggests that a low basal expression of CD16 occurs in many resident microglial cells which may potentially be upregulated in HIV-infected individuals. From these data, we suggest that not all the CD16+ parenchymal cells detected in AIDS brains (using ABC) represent influx of monocyte lineage cells from the blood. Finally the increased expression of MHC II and CD68 detected in drug abusers with HIVE compared to nondrug abusers with HIVE suggests that the combination of drug abuse and HIV infection has a greater deleterious effect on the brain than either individual insult on its own.

Anthony IC, Ramage SN, Carnie FW, Simmonds P, Bell JE. 2005. Influence of HAART on HIV-related CNS disease and neuroinflammation. J Neuropathol Exp Neurol, 64 (6), pp. 529-536. | Show Abstract | Read more

Neuroinflammation has an established link with AIDS-related dementia but has not been investigated in the post-highly active anti-retroviral therapy (HAART) era. In this autopsy study we examined post-HAART cases in Edinburgh for the presence of HIV-related pathology and in well-treated cases for evidence of neuroinflammation. We focused on basal ganglia and the hippocampus, 2 key areas of the brain for cognitive functioning and compared pre- and post-HAART cases for neuroinflammatory status. We find evidence, post-HAART, that there is a high level of microglial/macrophage activation that is comparable with the levels seen, pre-HAART, in HIV encephalitis (HIVE) and AIDS cases. This result was maximal in the hippocampus where microglial/macrophage upregulation in the HAART-treated group exceeded that seen in HIVE. In the basal ganglia, HAART-treated cases showed significantly higher levels of CD68-positive microglia/macrophages than in control brains (p = 0.004), and in the hippocampus levels were significantly higher than those seen in control cases, pre-HAART AIDS, and presymptomatic brains (p = 0.01). However, lymphocyte levels in the areas examined were low in HAART-treated cases. We conclude that there is a surprising degree of ongoing neuroinflammation in HAART-treated patients, particularly in the hippocampus. This may pose a threat for the future health of individuals maintained long-term on HAART therapy.

Peters PJ, Bhattacharya J, Hibbitts S, Dittmar MT, Simmons G, Bell J, Simmonds P, Clapham PR. 2005. Biological analysis of human immunodeficiency virus type 1 R5 envelopes amplified from brain and lymph node tissues of AIDS patients with neuropathology reveals two distinct tropism phenotypes and identifies envelopes in the brain that confer an enhanced tropism and fusigenicity for macrophages (vol 78, pg 6915, 2004) JOURNAL OF VIROLOGY, 79 (5), pp. 3227-3227. | Read more

Sall AA, Starkman S, Reynes JM, Lay S, Nhim T, Hunt M, Marx N, Simmonds P. 2005. Frequent infection of Hylobates pileatus (pileated gibbon) with species-associated variants of hepatitis B virus in Cambodia. J Gen Virol, 86 (Pt 2), pp. 333-337. | Show Abstract | Read more

As well as being distributed widely in human populations, hepatitis B virus (HBV) infections occur frequently in chimpanzee, gibbon and other ape populations in sub-Saharan Africa and South-East Asia. To investigate the frequency and genetic relationships of HBV infecting gibbons in Cambodia, pileated gibbons (Hylobates pileatus) that were originally wild-caught were screened for surface antigen. Twelve of 26 (46 %) were positive, of which 11 were positive for HBV DNA. Phylogenetic analysis of complete genome sequences revealed two distinct genetic groups in the gibbon/orangutan clade. Three were similar to previously described variants infecting H. pileatus in Thailand and eight formed a distinct clade, potentially representing distinct strains of HBV circulating in geographically separated populations in South-East Asia. Because of the ability of HBV to cross species barriers, large reservoirs of infection in gibbons may hamper ongoing attempts at permanent eradication of HBV infection from human populations in South-East Asia through immunization.

Bertuzis R, Hardie A, Hottentraeger B, Izopet J, Jilg W, Kaesdorf B, Leckie G, Leete J, Perrin L, Qiu C et al. 2005. Clinical performance of the LCx HCV RNA quantitative assay. J Virol Methods, 123 (2), pp. 171-178. | Show Abstract | Read more

This study was conducted to assess the performance of the Abbott laboratories LCx HCV RNA Quantitative Assay (LCx assay) in the clinical setting. Four clinical laboratories measured LCx assay precision, specificity, and linearity. In addition, a method comparison was conducted between the LCx assay and the Roche HCV Amplicor Monitor, version 2.0 (Roche Monitor 2.0) and the Bayer VERSANT HCV RNA 3.0 Assay (Bayer bDNA 3.0) quantitative assays. For precision, the observed LCx assay intra-assay standard deviation (S.D.) was 0.060-0.117 log IU/ml, the inter-assay S.D. was 0.083-0.133 log IU/ml, the inter-lot S.D. was 0.105-0.177 log IU/ml, the inter-site S.D. was 0.099-0.190 log IU/ml, and the total S.D. was 0.113-0.190 log IU/ml. The specificity of the LCx assay was 99.4% (542/545; 95% CI, 98.4-99.9%). For linearity, the mean pooled LCx assay results were linear (r=0.994) over the range of the panel (2.54-5.15 log IU/ml). A method comparison demonstrated a correlation coefficient of 0.881 between the LCx assay and Roche Monitor 2.0, 0.872 between the LCx assay and Bayer bDNA 3.0, and 0.870 between Roche Monitor 2.0 and Bayer bDNA 3.0. The mean LCx assay result was 0.04 log IU/ml (95% CI, -0.08, 0.01) lower than the mean Roche Monitor 2.0 result, but 0.57 log IU/ml (95% CI, 0.53, 0.61) higher than the mean Bayer bDNA 3.0 result. The mean Roche Monitor 2.0 result was 0.60 log IU/ml (95% CI, 0.56, 0.65) higher than the mean Bayer bDNA 3.0 result. The LCx assay quantitated genotypes 1-4 with statistical equivalency. The vast majority (98.9%, 278/281) of paired LCx assay-Roche Monitor 2.0 specimen results were within 1 log IU/ml. Similarly, 86.6% (240/277) of paired LCx assay and Bayer bDNA 3.0 specimen results were within 1 log, as were 85.6% (237/277) of paired Roche Monitor 2.0 and Bayer specimen results. These data demonstrate that the LCx assay may be used for quantitation of HCV RNA in HCV-infected individuals.

Pybus OG, Cochrane A, Holmes EC, Simmonds P. 2005. The hepatitis C virus epidemic among injecting drug users. Infect Genet Evol, 5 (2), pp. 131-139. | Show Abstract | Read more

Given the economic and health costs of hepatitis C virus (HCV) infection, and the ongoing transmission within the injecting drug user (IDU) population, there is a need for improved understanding of HCV epidemiology within this risk group. We employed a recently developed method based on phylogenetic analysis to infer HCV epidemic history and to provide the first estimates of the rate of spread of subtypes 1a and 3a circulating within injecting drug user populations. The data indicates that HCV subtype 1a entered the IDU population on at least three separate occasions. Both subtypes demonstrate exponential population growth during the 20th century, with a doubling time of 7-8 years. The results provide a baseline for prediction of the future course of the HCV epidemic, and its likely response to transmission control policies.

Simmonds P. 2004. Genetic diversity and evolution of hepatitis C virus--15 years on. J Gen Virol, 85 (Pt 11), pp. 3173-3188. | Show Abstract | Read more

In the 15 years since the discovery of hepatitis C virus (HCV), much has been learned about its role as a major causative agent of human liver disease and its ability to persist in the face of host-cell defences and the immune system. This review describes what is known about the diversity of HCV, the current classification of HCV genotypes within the family Flaviviridae and how this genetic diversity contributes to its pathogenesis. On one hand, diversification of HCV has been constrained by its intimate adaptation to its host. Despite the >30 % nucleotide sequence divergence between genotypes, HCV variants nevertheless remain remarkably similar in their transmission dynamics, persistence and disease development. Nowhere is this more evident than in the evolutionary conservation of numerous evasion methods to counteract the cell's innate antiviral defence pathways; this series of highly complex virus-host interactions may represent key components in establishing its 'ecological niche' in the human liver. On the other hand, the mutability and large population size of HCV enables it to respond very rapidly to new selection pressures, manifested by immune-driven changes in T- and B-cell epitopes that are encountered on transmission between individuals with different antigen-recognition repertoires. If human immunodeficiency virus type 1 is a precedent, future therapies that target virus protease or polymerase enzymes may also select very rapidly for antiviral-resistant mutants. These contrasting aspects of conservatism and adaptability provide a fascinating paradigm in which to explore the complex selection pressures that underlie the evolution of HCV and other persistent viruses.

Macfarlane C, Simmonds P. 2004. Allelic variation of HERV-K(HML-2) endogenous retroviral elements in human populations. J Mol Evol, 59 (5), pp. 642-656. | Show Abstract | Read more

Human endogenous retroviruses (HERVs) are the remnants of ancient germ cell infection by exogenous retroviruses and occupy up to 8% of the human genome. It has been suggested that HERV sequences have contributed to primate evolution by regulating the expression of cellular genes and mediating chromosome rearrangements. After integration approximately 28 million years ago, members of the HERV-K (HML-2) family have continued to amplify and recombine. To investigate the utility of HML-2 polymorphisms as markers for the study of more recent human evolution, we compiled a list of the structure and integration sites of sequences that are unique to humans and screened each insertion for polymorphism within the human genome databases. Of the total of 74 HML-2 sequences, 18 corresponded to complete or near-complete proviruses, 49 were solitary long terminal repeats (LTRs), 6 were incomplete LTRs, and 1 was a SVA retrotransposon. A number of different allelic configurations were identified including the alternation of a provirus and solitary LTR. We developed polymerase chain reaction-based assays for seven HML-2 loci and screened 109 human DNA samples from Africa, Europe, Asia, and Southeast Asia. Our results indicate that the diversity of HML-2 elements is higher in African than non-African populations, with population differentiation values ranging from 0.6 to 9.8%. These findings denote a recent expansion from Africa. We compare the phylogenetic relationships of HML-2 sequences that are unique to humans and consider whether these elements have played a role in the remodeling of the hominid genome.

Tuplin A, Evans DJ, Simmonds P. 2004. Detailed mapping of RNA secondary structures in core and NS5B-encoding region sequences of hepatitis C virus by RNase cleavage and novel bioinformatic prediction methods. J Gen Virol, 85 (Pt 10), pp. 3037-3047. | Show Abstract | Read more

There is accumulating evidence from bioinformatic studies that hepatitis C virus (HCV) possesses extensive RNA secondary structure in the core and NS5B-encoding regions of the genome. Recent functional studies have defined one such stem-loop structure in the NS5B region as an essential cis-acting replication element (CRE). A program was developed (STRUCTUR_DIST) that analyses multiple rna-folding patterns predicted by mfold to determine the evolutionary conservation of predicted stem-loop structures and, by a new method, to analyse frequencies of covariant sites in predicted RNA folding between HCV genotypes. These novel bioinformatic methods have been combined with enzymic mapping of RNA transcripts from the core and NS5B regions to precisely delineate the RNA structures that are present in these genomic regions. Together, these methods predict the existence of multiple, often juxtaposed stem-loops that are found in all HCV genotypes throughout both regions, as well as several strikingly conserved single-stranded regions, one of which coincides with a region of the genome to which ribosomal access is required for translation initiation. Despite the existence of marked sequence conservation between genotypes in the HCV CRE and single-stranded regions, there was no evidence for comparable suppression of variability at either synonymous or non-synonymous sites in the other predicted stem-loop structures. The configuration and genetic variability of many of these other NS5B and core structures is perhaps more consistent with their involvement in genome-scale ordered RNA structure, a structural configuration of the genomes of many positive-stranded RNA viruses that is associated with host persistence.

Simmonds P, Tuplin A, Evans DJ. 2004. Detection of genome-scale ordered RNA structure (GORS) in genomes of positive-stranded RNA viruses: Implications for virus evolution and host persistence. RNA, 10 (9), pp. 1337-1351. | Show Abstract | Read more

Discrete RNA secondary and higher-order structures, typically local in extent, play a fundamental role in RNA virus replication. Using new bioinformatics analysis methods, we have identified genome-scale ordered RNA structure (GORS) in many genera and families of positive-strand animal and plant RNA viruses. There was remarkably variability between genera that possess this characteristic; for example, hepaciviruses in the family Flaviviridae show evidence for extensive internal base-pairing throughout their coding sequences that was absent in both the related pestivirus and flavivirus genera. Similar genus-associated variability was observed in the Picornaviridae, the Caliciviridae, and many plant virus families. The similarity in replication strategies between genera in each of these families rules out a role for GORS in a fundamentally conserved aspect of this aspect of the virus life cycle. However, in the Picornaviridae, Flaviviridae, and Caliciviridae, the existence of GORS correlated strongly with the ability of each genus to persist in their natural hosts. This raises the intriguing possibility of a role for GORS in the modulation of innate intracellular defense mechanisms (and secondarily, the acquired immune system) triggered by double-stranded RNA, analogous in function to the expression of structured RNA transcripts by large DNA viruses. Irrespective of function, the observed evolutionary conservation of GORS in many viruses imposes a considerable constraint on genome plasticity and the consequent narrowing of sequence space in which neutral drift can occur. These findings potentially reconcile the rapid evolution of RNA viruses over short periods with the documented examples of extreme conservatism evident from their intimate coevolution with their hosts.

Cochrane A, Imlach S, Leen C, Scott G, Kennedy D, Simmonds P. 2004. High levels of human immunodeficiency virus infection of CD8 lymphocytes expressing CD4 in vivo. J Virol, 78 (18), pp. 9862-9871. | Show Abstract | Read more

Human immunodeficiency virus (HIV)-infected CD8 lymphocytes have been reported in vivo, but the mechanism of infection remains unclear. Experiments using the thy/hu mouse model support export of intrathymically infected CD8 precursors, while recent in vitro data suggest that mature CD8 lymphocytes upregulate CD4 upon activation (generating a CD8bright CD4dim phenotype) and are susceptible to HIV infection. To determine whether these mechanisms operate in vivo and to assess their relative importance in the generation of circulating HIV-infected CD8 lymphocytes, we quantified HIV long terminal repeat (LTR) DNA in CD8+ CD4- and CD8bright CD4dim lymphocytes isolated from HIV-infected individuals by fluorescence-activated cell sorting. HIV infection of CD8 lymphocytes was demonstrated in 17 of 19 subjects, with a significant inverse relationship between level of infection and CD4 lymphocyte count (R = -0.73; P < 0.001). The level of HIV infection of CD8bright CD4dim lymphocytes was significantly higher (median, 1,730 HIV LTR copies/10(6) cells; n = 9) than that of CD8+ CD4- lymphocytes (undetectable in seven of nine individuals; P < 0.01) and approached that of CD4 lymphocytes from the same individuals (median, 3,660 HIV LTR copies/10(6) cells). CD8bright CD4dim lymphocytes represented 0.8 to 3.3% of total CD8 lymphocytes and were most prevalent in the memory subset. Thus, HIV-infected CD8 lymphocytes commonly circulate in HIV-infected individuals and are generated through infection of activated CD8 lymphocytes rather than through export of intrathymically infected precursors. The high level of infection of CD8bright CD4dim lymphocytes could have a direct role in the decline in CD8 lymphocyte function that accompanies HIV disease progression.

Peters PJ, Bhattacharya J, Hibbitts S, Dittmar MT, Simmons G, Bell J, Simmonds P, Clapham PR. 2004. Biological analysis of human immunodeficiency virus type 1 R5 envelopes amplified from brain and lymph node tissues of AIDS patients with neuropathology reveals two distinct tropism phenotypes and identifies envelopes in the brain that confer an enhanced tropism and fusigenicity for macrophages. J Virol, 78 (13), pp. 6915-6926. | Show Abstract | Read more

Complete envelope genes were amplified from autopsy brain tissue of five individuals who had died of AIDS and had neurological complications. Lymph node samples were included for two of the patients. Nineteen different envelope clones from the five patients had distinct V1V2 sequences. Thirteen of the envelopes were functional and conferred fusigenicity and infectivity for CD4(+) CCR5(+) cells. Infectivity and cell-cell fusion assays showed that most envelopes used both CCR5 and CCR3. One brain-derived envelope used a broad range of coreceptors, while three other brain envelopes from one individual were restricted to CCR5. However, there was no correlation between tissue of origin and coreceptor use. Envelopes showed two very distinct phenotypes depending on their capacity to infect macrophages and to exploit low levels of CD4 and/or CCR5 for infection. Envelopes that were highly fusigenic and tropic for macrophages were identified in brain tissue from four of the five patients. The enhanced macrophage tropism correlated with reduced sensitivity to inhibition by Q4120, a CD4-specific antibody, but not with sensitivity to the CCR5 inhibitor, TAK779. The highly macrophage-tropic envelopes were able to infect cells expressing low levels of CD4 and/or CCR5. Comparison with several well-characterized macrophage-tropic envelopes showed that the four identified patient envelopes were at the top limit of macrophage tropism. In contrast, all four lymph node-derived envelopes exhibited a non-macrophage-tropic phenotype and required high levels of CD4 for infection. Our data support the presence of envelopes that are highly fusigenic and tropic for macrophages in the brains of patients with neurological complications. These envelopes are able to infect cells that express low levels of CD4 and/or CCR5 and may have adapted for replication in brain macrophages and microglia, which are known to express limited amounts of CD4.

van Asten L, Verhaest I, Lamzira S, Hernandez-Aguado I, Zangerle R, Boufassa F, Rezza G, Broers B, Robertson JR, Brettle RP et al. 2004. Spread of hepatitis C virus among European injection drug users infected with HIV: a phylogenetic analysis. J Infect Dis, 189 (2), pp. 292-302. | Show Abstract | Read more

To describe the spread of hepatitis C virus (HCV) among HCV/human immunodeficiency virus (HIV)-coinfected injection drug users (IDUs), the molecular epidemiology of HCV was studied among 108 IDUs from 7 European countries. Phylogenetic analysis based on the NS5B region showed great sequence variation of HCV within each country and no clear phylogenetic clustering by geographic region. The most prevalent subtypes were 1a and 3a, but the percentage of genotype 4 was also relatively high, ranging from 7% in northern Europe to 24% in southern Europe. Genotype 4 consisted mainly of subtype 4d and has entered the majority of the IDU populations studied. The significantly lower evolutionary distances within subtype 4d suggest that this subtype may have entered the European IDU population relatively recently. In conclusion, HCV exchange between European IDU populations has occurred on a large scale, and, overall, country-specific clustering for HCV was less than that shown for HIV.

Pedersen JS, Meyer IM, Forsberg R, Simmonds P, Hein J. 2004. A comparative method for finding and folding RNA secondary structures within protein-coding regions. Nucleic Acids Res, 32 (16), pp. 4925-4936. | Show Abstract | Read more

Existing computational methods for RNA secondary-structure prediction tacitly assume RNA to only encode functional RNA structures. However, experimental studies have revealed that some RNA sequences, e.g. compact viral genomes, can simultaneously encode functional RNA structures as well as proteins, and evidence is accumulating that this phenomenon may also be found in Eukaryotes. We here present the first comparative method, called RNA-DECODER, which explicitly takes the known protein-coding context of an RNA-sequence alignment into account in order to predict evolutionarily conserved secondary-structure elements, which may span both coding and non-coding regions. RNA-DECODER employs a stochastic context-free grammar together with a set of carefully devised phylogenetic substitution-models, which can disentangle and evaluate the different kinds of overlapping evolutionary constraints which arise. We show that RNA-DECODER's parameters can be automatically trained to successfully fold known secondary structures within the HCV genome. We scan the genomes of HCV and polio virus for conserved secondary-structure elements, and analyze performance as a function of available evolutionary information. On known secondary structures, RNA-DECODER shows a sensitivity similar to the programs MFOLD, PFOLD and RNAALIFOLD. When scanning the entire genomes of HCV and polio virus for structure elements, RNA-DECODER's results indicate a markedly higher specificity than MFOLD, PFOLD and RNAALIFOLD.

Arango JC, Simmonds P, Brettle RP, Bell JE. 2004. Does drug abuse influence the microglial response in AIDS and HIV encephalitis? AIDS, 18 Suppl 1 (Supplement 3), pp. S69-S74. | Show Abstract | Read more

OBJECTIVES: To investigate the pathological evidence for a possible interaction between drugs of abuse and HIV infection in terms of microglial responses in early and late HIV/AIDS, and to discuss the possible long-term consequences of microglial activation in chronic HIV infection. DESIGN: This brain pathology study compared age and sex-matched control patients with HIV-negative intravenous drug users, and with HIV-positive drug users both in the presymptomatic stage and with AIDS. A further group of non-drug-using AIDS patients was included. All the AIDS patients had HIV encephalitis (HIVE) but no other significant HIV-associated brain pathology. METHODS: Microglia/macrophages were identified in the grey and white matter of the frontal and temporal lobes and the thalamus, using antibodies to CD68 and MHCII. Objective quantitation was used to compare subjects in the different groups. RESULTS: AIDS patients showed a significant increase in activated microglia/macrophages in both the grey and white matter of all areas compared with non-AIDS patients. Drug users with HIVE tended to have more activated microglia than non-drug-using comparison groups, but this difference was not found in all brain areas studied. CONCLUSION: Drug misuse appears to enhance the microglial activation resulting from HIV infection in some individuals. Other factors such as the severity of HIVE, or systemic immune factors, are also likely to affect the degree of microglial activation. The implications for drug-using patients who survive long term with HIV/AIDS are discussed, particularly in relation to premature neurodegeneration.

Sentjens R, Basaras M, Simmonds P, Vrielink H, Reesink H. 2003. HGV/GB virus C transmission by blood components in patients undergoing open-heart surgery. Transfusion, 43 (11), pp. 1558-1562. | Show Abstract | Read more

BACKGROUND: To establish the rate of HGV/GB virus C (GBV-C) transmission by blood components in open-heart surgery patients. STUDY DESIGN AND METHODS: From 55 patients receiving blood components, sera were collected before and 2, 4, 6, 8, 10, 12, 16, 20, 26, and 32 weeks after heart surgery. Serum samples from patients and implicated blood donations were tested for HGV/GBV-C RNA by PCR. Recipients of RNA-positive blood components were also tested for the presence of E2 antibodies (E2Ab) by ELISA. RESULTS: Of 55 recipients, 18 received RNA-positive blood components. Of 14 recipients of RNA-positive blood components, who were negative for RNA or E2Ab before transfusion, 8 became RNA positive and one developed E2Ab after transfusion. Three recipients of RNA-positive blood components had E2Ab before transfusion, and none of these became RNA positive after transfusion. One of 18 recipients was RNA positive before and after transfusion. Of 55 recipients, 37 received RNA-negative blood components: 34 were RNA negative before and after transfusion. Of 37 recipients, 3 were RNA positive before and after transfusion. CONCLUSION: Of susceptible patients, 64 percent became infected with HGV/GVC-C when transfused with RNA-positive blood components. E2Ab-positive patients were protected against HGV/GBV-C infection.

Welch J, Maclaran K, Jordan T, Simmonds P. 2003. Frequency, viral loads, and serotype identification of enterovirus infections in Scottish blood donors. Transfusion, 43 (8), pp. 1060-1066. | Show Abstract | Read more

BACKGROUND: Enteroviruses are non-enveloped, frequently pathogenic RNA viruses infecting humans. Infection is potentially transmissible through blood or blood component transfusion from donor in the viremic phase before seroconversion for antibody. To investigate the threat to blood safety from enteroviruses, a large-scale survey of frequency and levels of viremia in blood donors was conducted. STUDY DESIGNS AND METHODS: Blood donations from Scotland over a period of 22 calendar months were screened for enterovirus RNA sequences by PCR. Positive samples were quantified, and serotypes were identified by nucleotide sequencing of VP1. RESULTS: From a total of 3658 pools of 95 donations tested, 73 samples that were enterovirus-positive were identified (corrected annual frequency 0.024% or 1 in 4000). The highest rates of viremia were in late summer months (e.g., 0.055%, 1 in 1800 in July) and lowest from January to May (0.009 and 0.012%). Viral loads ranged from 500 (the lower cutoff of the assay) to greater than 100,000 amplifiable enterovirus template copies per mL. Coxsackievirus A16, echoviruses 11 and 30, and enterovirus 71 were most often identified. CONCLUSIONS: The detection of enterovirus-positive blood units indicates the potential for enteroviral transmission by blood components. Although the infrastructure established for PCR-based screening for HCV RNA would allow parallel screening for enteroviruses, any decision concerning donor testing would require further information on the outcome of transfusion-acquired enterovirus infections.

Anderson CE, Tomlinson GS, Pauly B, Brannan FW, Chiswick A, Brack-Werner R, Simmonds P, Bell JE. 2003. Relationship of Nef-positive and GFAP-reactive astrocytes to drug use in early and late HIV infection. Neuropathol Appl Neurobiol, 29 (4), pp. 378-388. | Show Abstract | Read more

Reactive astrocytosis is a well-documented feature of HIV encephalitis (HIVE), but it is unclear whether restricted infection of astrocytes contributes to this phenomenon. In addition, the part played by reactive and/or infected astrocytes in AIDS-related dementia is not fully understood. In this study of patients at different stages of the human immunodeficiency virus (HIV) infection, who had been treated at most with one antiretroviral drug, reactive astrocytes were identified by immunopositivity for glial fibrillary acidic protein (GFAP) and infected astrocytes by positivity for HIV Nef protein. Results were compared for drug-using AIDS patients with (n=9) and without (n=7) HIVE, for presymptomatic HIV-positive drug users (n=12) and for control HIV-negative subjects (n=20), including a group who used drugs (n=10). GFAP-reactive astrocytes in both grey and white matter were significantly more numerous in HIVE subjects than in each of the other groups but did not correlate with viral load. Nef-positive astrocytes were confined to HIVE cases and to white matter, but were numerous in only one subject who was treatment-naive. Nef-positive microglia were identified in all HIVE cases and in occasional AIDS and presymptomatic subjects who did not have HIVE. The results suggest that astrocytes may form an additional viral reservoir in late HIV infection and may contribute to HIVE. However, the number of GFAP-positive astrocytes was neither increased in pre-AIDS nor in drug abuse, in contrast with microglia which we have shown previously to be up-regulated in both states.

Roy KM, Bagg J, Kennedy C, Cameron S, Simmonds P, Lycett C, Hunter I, Taylor M. 2003. Prevalence of GBV-C infection among dental personnel. J Med Virol, 70 (1), pp. 150-155. | Show Abstract | Read more

Healthcare workers who carry out exposure-prone procedures are theoretically at increased risk of acquiring blood-borne virus infections. GB virus C (GBV-C) is a recently described blood-borne virus that is related distantly to hepatitis C virus. The occupational risk of GBV-C infection to healthcare workers is unknown. This study collected detailed occupational and personal risk data in parallel with a blood specimen, to establish the prevalence and determinants of GBV-C infection among dental healthcare workers. The presence of GBV-C antibodies was detected using commercially available ELISA; GBV-C RNA was detected by nested PCR using primers from the conserved 5' noncoding region. The overall prevalence of GBV-C antibodies among the study population was 11.1% (98/880, 95% confidence interval [CI], 9.1-13.4%) and 4.6% were positive for GBV-C RNA (46/879, 95% CI, 2.5-5.1%), resulting in a cumulative prevalence of 15.7%. These figures are similar to those described in other populations. There was no significant difference in lifetime exposure to GBV-C between dentists (17.7%) and dental nurses/hygienists (14.3%). Significantly more dental nurses/hygienists aged 16-30 years had been exposed to GBV-C compared to dentists of the same age (chi(2) = 13.75; P < 0.001). Conversely, significantly more dentists 46 years or older had evidence of exposure to GBV-C compared to dental nurses/hygienists (chi(2) = 6.79; P = 0.009). The high prevalence of GBV-C infection did not seem to be related to past parenteral exposure, and the data suggest that sexual transmission, rather than occupational transmission, was a more important route for GBV-C infection among this population.

Thom K, Morrison C, Lewis JC, Simmonds P. 2003. Distribution of TT virus (TTV), TTV-like minivirus, and related viruses in humans and nonhuman primates. Virology, 306 (2), pp. 324-333. | Show Abstract | Read more

TT virus (TTV) and TTV-like minivirus (TLMV) are small DNA viruses with single-stranded, closed circular, antisense genomes infecting man. Despite their extreme sequence heterogeneity (>50%), a highly conserved region in the untranslated region (UTR) allows both viruses to be amplified by polymerase chain reaction (PCR). TTV/TLMV infection was detected in 88 of 100 human plasma samples; amplified sequences were differentiated into TTV and TLMV by analysis of melting profiles, showing that both viruses were similarly prevalent. PCR with UTR primers also detected frequent infection with TTV/TLMV-related viruses in a wide range of apes (chimpanzees, gorillas, orangutans, gibbons) and African monkey species (mangabeys, drills, mandrills). These findings support the hypothesis for the co-evolution of TTV-like viruses with their hosts over the period of primate speciation, potentially analogous to the evolution of primate herpesviruses.

Imlach S, Leen C, Bell JE, Simmonds P. 2003. Phenotypic analysis of peripheral blood gammadelta T lymphocytes and their targeting by human immunodeficiency virus type 1 in vivo. Virology, 305 (2), pp. 415-427. | Show Abstract | Read more

There is increasing evidence that a wider range of lymphoid cell types other than CD4(+) T helper lymphocytes are infected with HIV-1 in vivo, including CD8 lymphocytes, natural killer cells, and reticulodendritic cells. Each potentially contributes to the reservoir of infected cells that resist antiviral treatment and to the impairment of immune responses in AIDS. By quantitative PCR for HIV proviral sequences we have now obtained evidence for substantial infection of gammadelta lymphocytes, contributing 3-45% of the proviral load in peripheral blood. A large proportion of gammadelta lymphocytes constitutively expressed the chemokine receptors CCR5 and CXCR4, with evidence for marked up-regulation of CD8 in samples from HIV-infected individuals, corresponding to an activated phenotype. That gammadelta lymphocytes might be susceptible to HIV infection was investigated using in vitro infectivity assays of recombinant HIV-expressing green fluorescent protein, followed by flow cytometry. gammadelta, CD4, and CD8 lymphocytes were each productively infected, with gammadelta lymphocytes showing the greatest susceptibility. For each cell type, blocking assays with an anti-CD4 monoclonal antibody indicated that entry was CD4-dependent.

Starkman SE, MacDonald DM, Lewis JC, Holmes EC, Simmonds P. 2003. Geographic and species association of hepatitis B virus genotypes in non-human primates. Virology, 314 (1), pp. 381-393. | Show Abstract | Read more

Infection with hepatitis B virus (HBV) has been detected in human populations throughout the world, as well as in a number of ape species (Pan troglodytes, Gorilla gorilla, gibbons [Nomascus and Hylobates species] and Pongo pygmaeus). To investigate the distribution of naturally occurring HBV infection in these species and other African Old World monkey species (Cercopithecidae), we screened 137 plasma samples from mainly wild caught animals by polymerase chain reaction (PCR) using several of highly conserved primers from the HB surface (HBs) gene, and for HBs antigen (HBsAg) by ELISA. None of the 93 Cercopithecidae screened (6 species) showed PCR or serology evidence for HBV infection; in contrast 2 from 8 chimpanzees and 5 from 22 gibbons were PCR-positive with each set of primers. Complete genome sequences from each of the positive apes were obtained and compared with all previously published complete and surface gene sequences. This extended phylogenetic analysis indicated that HBV variants from orangutans were interspersed by with HBV variants from southerly distributed gibbon species (H. agilis and H. moloch) occupying overlapping or adjacent habitat ranges with orangutans; in contrast, HBV variants from gibbon species in mainland Asia were phylogenetically distinct. A geographical rather than (sub)species association of HBV would account for the distribution of HBV variants in different subspecies of chimpanzees in Africa, and explain the inlier position of the previously described lowland gorilla sequence in the chimpanzee clade. These new findings have a number of implication for understanding the origins and epidemiology of HBV infection in non-human primates.

Simmonds P, Kurtz J, Tedder RS. 2002. Alternatives to nucleic acid testing in the blood transfusion service - Reply LANCET, 360 (9344), pp. 1519-1520. | Read more

Zeytinoğlu A, Erensoy S, Abacioğlu H, Sayiner AA, Ozacar T, Başçi A, Kaplan H, Simmonds P, Bilgiç A. 2002. Nosocomial hepatitis C virus infection in a renal transplantation center. Clin Microbiol Infect, 8 (11), pp. 741-744. | Show Abstract | Read more

Nosocomial hepatitis C virus (HCV) infections were recorded in the renal transplantation unit of the university hospital. There were cases of acute HCV infection with aggressive clinical courses diagnosed from a positive HCV RNA test in the early post-transplantation period and which remained anti-HCV negative. Their anti-HCV seronegativity was attributed to them having acquired HCV under intense immunosuppressive therapy and suggested that the aggressive clinical course could be due to the deficient immune response resulting in an inability to limit viral replication. There were also donors diagnosed as having acute HCV infection in the early post-operative period. Genotyping and sequence analysis for HCV were performed on the isolates of eight of these patients who were consecutively transplanted and of three donors whose recipients were infected with HCV prior to transplantation, and who acquired acute HCV infection after transplantation. Of the eight recipients in the first group three were genotype 1a, three were genotype 1b, one was genotype 3a, and the last one was genotype 4 according to Simmond's classification. Of the three donor-recipient couples both the HCV isolates from one couple were genotyped as 1b and the phylogenetic analysis indicated that the patients were infected with a common variant of HCV, but the genotypes of HCV isolates from the other couples were different. Recipients were genotype 1b and the donors were genotype 1a in these couples. Genotype results of the first group and donor-recipient couples, and sequence analysis of genotype 1b and 1a isolates, showed that the source of infection was not a unique strain and there were multiple breaks in universal precautions while managing these patients.

Cochrane A, Searle B, Hardie A, Robertson R, Delahooke T, Cameron S, Tedder RS, Dusheiko GM, De Lamballerie X, Simmonds P. 2002. A genetic analysis of hepatitis C virus transmission between injection drug users. J Infect Dis, 186 (9), pp. 1212-1221. | Show Abstract | Read more

Hepatitis C virus (HCV) genotype 1a and 3a partial NS5B gene segment sequences obtained from 154 HCV-infected injection drug users were studied to determine the extent to which HCV transmission occurs between injection drug user communities in London, Edinburgh, Glasgow (United Kingdom), Marseilles (France), and Melbourne. Phylogenetic relationships between sequences were analyzed by conventional methods and by a recently developed method that numerically scores the extent of sequence segregation between groups through calculation of association indices. The association indices revealed that none of the cities sampled support an HCV population that is completely isolated from that circulating in the other cities. Sequences from Melbourne were most isolated, whereas those from London were most dispersed. This suggests that HCV transmission between these cities occurs, with London playing a pivotal role. The degree of city-specific segregation of HCV subtype 1a sequences was linearly related to that of subtype 3a, indicating that these subtypes have spread through similar transmission networks.

Bell JE, Arango JC, Robertson R, Brettle RP, Leen C, Simmonds P. 2002. HIV and drug misuse in the Edinburgh cohort. J Acquir Immune Defic Syndr, 31 Suppl 2 (SUPPL. 2), pp. S35-S42. | Show Abstract | Read more

The Edinburgh cohort of intravenous drug users (IVDUs) became infected with HIV between 1983 and 1984. Before the era of effective therapy, many of these infected IVDUs displayed cognitive impairments on progressing to AIDS and were found to have HIV encephalitis (HIVE). Full autopsies were conducted on these patients, providing an opportunity to study the intersecting pathology of pure HIVE and drug use. High proviral load in the brain correlated well with the presence of giant cells and HIV p24 positivity. In presymptomatic HIV infection, IVDUs were found to have a lymphocytic infiltrate in the central nervous system (CNS). Apart from the expected microglial activation in the presence of HIV infection of the CNS, drug use in its own right was found to be associated with microglial activation. Examination of HIV-negative IVDUs revealed a number of neuropathologic features, including microglial activation, which may underpin HIV-related pathology in the CNS. HIV isolated from different regions of the brain was exclusively of R5-tropic type throughout the course of infection. Detailed studies of p17 and V3 sequences suggest that viral sequestration occurs in the CNS before the onset of AIDS and that increasing diversity of HIV variants within the brain is associated with increasing severity of HIVE. Because brain isolates have proved to be different from those in lymphoid tissue (and blood), it is likely that selective neuroadaptive pressures operate before HIVE supervenes. Drug abuse may be synergistic in this process through activation of microglia, breakdown of the blood-brain barrier, and direct neurotoxicity. Collections of clinically well-characterized HIV-infected tissues such as those in the Edinburgh Brain Bank are a vital resource to support ongoing studies of viral pathogenesis in the CNS and interactions with drug abuse.

Jarvis LM, Simmonds P. 2002. Scottish experience with NAT. Transfus Med, 12 (4), pp. 259-264. | Read more




Tuplin A, Wood J, Evans DJ, Patel AH, Simmonds P. 2002. Thermodynamic and phylogenetic prediction of RNA secondary structures in the coding region of hepatitis C virus RNA-A PUBLICATION OF THE RNA SOCIETY, 8 (6), pp. 824-841. | Read more

Tuplin A, Wood J, Evans DJ, Patel AH, Simmonds P. 2002. Thermodynamic and phylogenetic prediction of RNA secondary structures in the coding region of hepatitis C virus. RNA, 8 (6), pp. 824-841. | Show Abstract | Read more

The existence and functional importance of RNA secondary structure in the replication of positive-stranded RNA viruses is increasingly recognized. We applied several computational methods to detect RNA secondary structure in the coding region of hepatitis C virus (HCV), including thermodynamic prediction, calculation of free energy on folding, and a newly developed method to scan sequences for covariant sites and associated secondary structures using a parsimony-based algorithm. Each of the prediction methods provided evidence for complex RNA folding in the core- and NS5B-encoding regions of the genome. The positioning of covariant sites and associated predicted stem-loop structures coincided with thermodynamic predictions of RNA base pairing, and localized precisely in parts of the genome with marked suppression of variability at synonymous sites. Combined, there was evidence for a total of six evolutionarily conserved stem-loop structures in the NS5B-encoding region and two in the core gene. The virus most closely related to HCV, GB virus-B (GBV-B) also showed evidence for similar internal base pairing in its coding region, although predictions of secondary structures were limited by the absence of comparative sequence data for this virus. While the role(s) of stem-loops in the coding region of HCV and GBV-B are currently unknown, the structure predictions in this study could provide the starting point for functional investigations using recently developed self-replicating clones of HCV.

Simmonds P. 2002. TT virus infection: a novel virus-host relationship. J Med Microbiol, 51 (6), pp. 455-458. | Read more

Simmonds P, Kurtz J, Tedder RS. 2002. The UK blood transfusion service: over a (patent) barrel? Lancet, 359 (9319), pp. 1713-1714. | Read more

Sentiens R, Simmonds P, Basaras M, Vrielink H, Reesink H. 2002. HGV/GBV-C transmission by blood components in patients undergoing open-heart surgery JOURNAL OF HEPATOLOGY, 36 pp. 126-126.

Mokili JL, Rogers M, Carr JK, Simmonds P, Bopopi JM, Foley BT, Korber BT, Birx DL, McCutchan FE. 2002. Identification of a novel clade of human immunodeficiency virus type 1 in Democratic Republic of Congo. AIDS Res Hum Retroviruses, 18 (11), pp. 817-823. | Show Abstract | Read more

The high genetic heterogeneity of HIV-1 in the Democratic Republic of Congo (DRC) constitutes a real challenge for the development of vaccines to counter the spread of the HIV-1 epidemic. It is important to continue to monitor the epidemic by studying the circulating strains and their impact on the overall spread. As part of the ongoing effort to study the global distribution of HIV-1 subtypes and circulating recombinant forms (CRFs), here we describe a new phylogenetic clade of HIV-1 by the analysis of two full-length sequences (83CD003 and 90CD121E12) collected from two individuals at a 7-year interval (1983 and 1990, respectively). One of the two sequences (90CD121E12) was obtained from a vertically infected, 12-month-old baby in Kimpese, rural DRC, an area with low and stable seroprevalence of HIV-1 in women attending antenatal clinics. The two sequences are genetically similar by 95% of their full genome and topologically form a distinct cluster that is equidistant from the existing subtypes (A through K) by the analysis of both the full genome and subgenomic regions. Furthermore, they share several other genetic features, including an additional pair of cysteine residues, predictive of an extra disulfide bridge, in the V4 loop of gp120. This new clade is currently rare, spreading at a slower pace than the other subtypes found in the DRC region. Pending the identification of at least one partial length sequence of the same lineage from another patient who is epidemiologically unlinked to those described here, this clade is not yet named as a subtype as per the recommendation of the nomenclature committee.

Imlach S, McBreen S, Shirafuji T, Leen C, Bell JE, Simmonds P. 2001. Activated peripheral CD8 lymphocytes express CD4 in vivo and are targets for infection by human immunodeficiency virus type 1. J Virol, 75 (23), pp. 11555-11564. | Show Abstract | Read more

There is increasing evidence that CD8 lymphocytes may represent targets for infection by human immunodeficiency virus type 1 (HIV-1) in vivo whose destruction may contribute to the loss of immune function underlying AIDS. HIV-1 may infect thymic precursor cells destined to become CD4 and CD8 lymphocytes and contribute to the numerical decline in both subsets on disease progression. There is also evidence for the induction of CD4 expression and susceptibility to infection by HIV-1 of CD8 lymphocytes activated in vitro. To investigate the relationship between CD8 activation and infection by HIV-1 in vivo, activated subsets of CD8 lymphocytes in peripheral blood mononuclear cells (PBMCs) of HIV-seropositive individuals were investigated for CD4 expression and HIV infection. Activated CD8 lymphocytes were identified by expression of CD69, CD71, and the human leukocyte antigen (HLA) class II, the beta-chain of CD8, and the RO isoform of CD45. CD4(+) and CD4(-) CD8 lymphocytes, CD4 lymphocytes, other T cells, and non-T cells were purified using paramagnetic beads, and proviral sequences were quantified by PCR using primers from the long terminal repeat region. Frequencies of activated CD8 lymphocytes were higher in HIV-infected study subjects than in seronegative controls, and they frequently coexpressed CD4 (mean frequencies on CD69(+), CD71(+), and HLA class II(+) cells of 23, 37, and 8%, respectively, compared with 1 to 2% for nonactivated CD8 lymphocytes). The level of CD4 expression of the double-positive population approached that of mature CD4 lymphocytes. That CD4 expression renders CD8 cell susceptible to infection was indicated by their high frequency of infection in vivo; infected CD4(+) CD8 lymphocytes accounted for between 3 and 72% of the total proviral load in PBMCs from five of the eight study subjects investigated, despite these cells representing a small component of the PBMC population (<3%). Combined, these findings provide evidence that antigenic stimulation of CD8 lymphocytes in vivo induces CD4 expression that renders them susceptible to HIV infection and destruction. The specific targeting of responding CD8 lymphocytes may provide a functional explanation for the previously observed impairment of cytotoxic T-lymphocyte (CTL) function disproportionate to their numerical decline in AIDS and for the deletion of specific clones of CTLs responding to HIV antigens.

Wang TH, Donaldson YK, Brettle RP, Bell JE, Simmonds P. 2001. Identification of shared populations of human immunodeficiency virus type 1 infecting microglia and tissue macrophages outside the central nervous system. J Virol, 75 (23), pp. 11686-11699. | Show Abstract | Read more

Infection of microglia and other cells of the macrophage/monocyte lineage in the central nervous system (CNS) by human immunodeficiency virus type I (HIV-1) underlies the development of giant cell encephalitis (GCE). It is currently unknown whether GCE depends on the emergence of virus populations specifically adapted to replicate in cells of the monocyte/macrophage lineage and whether this also leads to the specific targeting of macrophages in other nonlymphoid tissues. Autopsy samples from lymph node, brain (frontal region), lung, and full-thickness colon sections were obtained from nine study subjects with GCE and from nine without. The two groups showed no significant differences in CD4 counts, disease progression, or treatment history before death. Genetic relatedness between variants recovered from lymph node and nonlymphoid tissues was assessed by sequence comparison of V3 and p17(gag) regions using a newly developed method that scores the sample composition at successive nodes in a neighbor-joining tree. The association index enabled objective, numerical comparisons on the degree of tissue compartmentalization to be made. High proviral loads and p24 antigen expression in the brain were confined to the nine individuals with GCE. GCE was also associated with significantly higher proviral loads in colon samples (median of the GCE(+) group: 1,010 copies/10(6) cells; median of GCE(-) group, 10/10(6) cells; P = 0.006). In contrast, there were no significant differences in proviral load between the GCE(+) and GCE(-) groups in lymph node or lung samples, where HIV infection was manifested predominantly by infiltrates of lymphoid cells. V3 sequences from brain samples of individuals with GCE showed the greatest compartmentalization from those of lymph node, although samples from other tissues, particularly the colon, frequently contained variants phylogenetically related to those found in brain. The existence of shared, distinct populations of HIV specifically distributed in cells of the monocyte/macrophage lineage was further indicated by immunocytochemical detection of CD68(+), multinucleated giant cells expressing p24 antigen in samples of lung and colon in two individuals with GCE. This study provides the basis for future investigation of possible phenotypic similarities that underline the shared distributions of HIV variants infecting microglia and tissue macrophages outside the CNS.

Simmonds P. 2001. Reconstructing the origins of human hepatitis viruses. Philos Trans R Soc Lond B Biol Sci, 356 (1411), pp. 1013-1026. | Show Abstract | Read more

Infections with hepatitis B and C viruses (HBV, HCV) are widespread in human populations throughout the world, and are major causes of chronic liver disease and liver cancer. HBV, HCV and the related hepatitis G virus or GB virus C (referred to here as HGV/GBV-C) are capable of establishing persistent, frequently lifelong infections characterized by high levels of continuous replication. All three viruses show substantial genetic heterogeneity, which has allowed each to be classified into a number of distinct genotypes that have different geographical distributions and associations with different risk groups for infection. Information on their past transmission and epidemiology might be obtained by estimation of the time of divergence of the different genotypes of HCV, HBV and HGV/GBV-C using knowledge of their rates of sequence change. While information on the latter is limited to short observation periods and is therefore subject to considerable error and uncertainty, the relatively recent times of origin for genotype of each virus predicted by this method (HCV, 500-2000 years; HBV, 3000 years; HGV/GBV-C, 200 years) are quite incompatible with their epidemiological distributions in human populations. They also cannot easily be reconciled with the recent evidence for species-associated variants of HBV and HGV/GBV-C in a range of non-human primates. The apparent conservatism of viruses over long periods implied by their epidemiological distributions instead suggests that nucleotide sequence change may be subject to constraints peculiar to viruses with single-stranded genomes, or with overlapping reading frames that defy attempts to reconstruct evolution according to the principles of the 'molecular clock'. Large population sizes and intense selection pressures that optimize fitness may be additional factors that set virus evolution apart from that of their hosts.

McBreen S, Imlach S, Shirafuji T, Scott GR, Leen C, Bell JE, Simmonds P. 2001. Infection of the CD45RA+ (naive) subset of peripheral CD8+ lymphocytes by human immunodeficiency virus type 1 in vivo. J Virol, 75 (9), pp. 4091-4102. | Show Abstract | Read more

To investigate the mechanism and functional significance of infection of CD8+ lymphocytes by human immunodeficiency virus type 1 (HIV-1) in vivo, we determined frequencies of infection, proviral conformation, and genetic relationships between HIV-1 variants infecting naive (CD45RA+) and memory (CD45RO+) peripheral blood CD4+ and CD8+ lymphocytes. Infection of CD3+ CD8+ CD45RA+ cells was detected in 9 of 16 study subjects at frequencies ranging from 30 to 1,400 proviral copies/10(6) cells, more frequently than CD3+ CD8+ lymphocytes expressing the RO isoform of CD45 (n = 2, 70 and 260 copies /10(6) cells). In agreement with previous studies, there was no evidence for a similar preferential infection of CD4+ naive lymphocytes. Proviral sequences in both CD4+ and CD8+ lymphocyte subsets were complete, as assessed by quantitation using primers from the long terminal repeat region spanning the tRNA primer binding site. In six of the seven study subjects investigated, variants infecting CD8+ lymphocytes were partially or completely genetically distinct in the V3 region from those recovered from CD4+ lymphocytes and showed a greater degree of compartmentalization than observed between naive and memory subsets of CD4+ lymphocytes. In two study subjects, arginine substitutions at position 306, associated with use of the chemokine coreceptor CXCR4, were preferentially found in CD4 lymphocytes. These population differences may have originated through different times of infection rather than necessarily indicating a difference in their biological properties. The preferential distribution of HIV-1 in naive CD8+ lymphocytes indeed suggests that infection occurred early in T-lymphocyte ontogeny, such as during maturation in the thymus. Destruction of cells destined to become CD8+ lymphocytes may be a major factor in the decline in CD8+ lymphocyte frequencies and function on disease progression and may contribute directly to the observed immunodeficiency in AIDS.

Welch JB, McGowan K, Searle B, Gillon J, Jarvis LM, Simmonds P. 2001. Detection of enterovirus viraemia in blood donors. Vox Sang, 80 (4), pp. 211-215. | Show Abstract | Read more

BACKGROUND AND OBJECTIVES: The infrastructure established for screening blood donations for hepatitis C virus has enabled large-scale population testing for other viruses which are potentially transmissible by transfusion of blood components and plasma-derived blood products. We have measured the frequency of viraemia of enteroviruses and parechoviruses in 83 600 Scottish blood donors to allow an initial assessment of their risk to blood safety. MATERIALS AND METHODS: Plasma samples collected from blood donors over 7 calendar months were tested anonymously in mini-pools of 95 donations, by polymerase chain reaction (PCR) for human enterovirus and parechovirus sequences. RESULTS: A total of 19 mini-pools, from the 880 that were tested, were PCR-positive for enterovirus RNA, predicting a donor prevalence of 0.023%. Enterovirus sequences were not detected in factor VIII or IX clotting factor concentrates. None of the 230 mini-pools or concentrates contained detectable parechovirus RNA. CONCLUSIONS: The prevalence of enterovirus viraemia detected in this study predicts that at least 1000 enterovirus-contaminated blood components are transfused per year in the UK. The frequency of transmission and clinical outcome after exposure to enterovirus-contaminated blood components in recipients is unknown.

Cuceanu NM, Tuplin A, Simmonds P. 2001. Evolutionarily conserved RNA secondary structures in coding and non-coding sequences at the 3′ end of the hepatitis G virus/GB-virus C genome Journal of General Virology, 82 (4), pp. 713-722. | Show Abstract

Hepatitis G virus (HGV)/GB virus C (GBV-C) causes persistent, non-pathogenic infection in a large proportion of the human population. Epidemiological and genetic evidence indicates a long-term association between HGV/GBV-C and related viruses and a range of primate species, and the cospeciation of these viruses with their hosts during primate evolution. Using a combination of covariance scanning and analysis of variability at synonymous sites, we previously demonstrated that the coding regions of HGV/GBV-C may contain extensive secondary structure of undefined function (Simmonds & Smith, Journal of Virology 73, 5787-5794, 1999). In this study we have carried out a detailed comparison of the structure of the 3′untranslated region (3′UTR) of HGV/GBV-C with that of the upstream NS5B coding sequence. By investigation of free energies on folding, secondary structure predictive algorithms and analysis of covariance between HGV/GBV-C genotypes 1-4 and the more distantly related HGV/GBV-C chimpanzee variant, we obtained evidence for extensive RNA secondary structure formation in both regions. In particular, the NS5B region contained long stem-loop structures of up to 38 internally paired nucleotides which were evolutionarily conserved between human and chimpanzee HGV/GBV-C variants. The prediction of similar structures in the same region of hepatitis C virus may allow the functions of these structures to be determined with a more tractable experimental model.

Simmonds P. 2001. The origin and evolution of hepatitis viruses in humans. J Gen Virol, 82 (Pt 4), pp. 693-712. | Show Abstract | Read more

The spread and origins of hepatitis C virus (HCV) in human populations have been the subject of extensive investigations, not least because of the importance this information would provide in predicting clinical outcomes and controlling spread of HCV in the future. However, in the absence of historical and archaeological records of infection, the evolution of HCV and other human hepatitis viruses can only be inferred indirectly from their epidemiology and by genetic analysis of contemporary virus populations. Some information on the history of the latter may be obtained by dating the time of divergence of various genotypes of HCV, hepatitis B virus (HBV) and the non-pathogenic hepatitis G virus (HGV)/GB virus-C (GBV-C). However, the relatively recent times predicted for the origin of these viruses fit poorly with their epidemiological distributions and the recent evidence for species-associated variants of HBV and HGV/GBV-C in a wide range of non-human primates. The apparent conservatism of viruses over long periods implied by these latter observations may be the result of constraints on sequence change peculiar to viruses with single-stranded genomes, or with overlapping reading frames. Large population sizes and intense selection pressures that optimize fitness may be the factors that set virus evolution apart from that of their hosts.

Hewson TJ, Logie JJ, Simmonds P, Howie SE. 2001. A CCR5-dependent novel mechanism for type 1 HIV gp120 induced loss of macrophage cell surface CD4. J Immunol, 166 (8), pp. 4835-4842. | Show Abstract

Type 1 HIV gp120 is especially effective in disrupting immune cell function because it is able to cause dysregulation of both infected and uninfected cells. We report a novel CCR5-dependent mechanism of gp120-induced CD4 loss from macrophages. An M-tropic gp120, using CCR5, is able to induce 70% loss of cell surface CD4 from macrophages within an hour. This cell surface CD4 loss is more substantial and rapid than the 20% loss observed with T-tropic gp120(IIIB) by 3 h. The rapid and substantial CD4 loss induced by M-tropic gp120 is not observed on macrophages homozygous for the ccr5Delta32 mutation, which fail to express cell surface CCR5. We have used confocal imaging to show that gp120 and CD4 are internalized together by a process resembling receptor-mediated endocytosis, and that both proteins enter HLA-DR containing compartments of the macrophage. We have also shown by semiquantitative RT-PCR that, in response to CD4 loss from the cell surface, mRNA for CD4 is up-regulated and the intracellular pool of CD4 increases. CCR5 mRNA levels are also increased. It is proposed that internalization of self and viral protein and increased pools of intracellular CD4 could modulate Ag presentation efficiencies and have implications for the induction and maintenance of both productive immune responses and self-tolerance.

Cuceanu NM, Tuplin A, Simmonds P. 2001. Evolutionarily conserved RNA secondary structures in coding and non-coding sequences at the 3' end of the hepatitis G virus/GB-virus C genome. J Gen Virol, 82 (Pt 4), pp. 713-722. | Show Abstract | Read more

Hepatitis G virus (HGV)/GB virus C (GBV-C) causes persistent, non-pathogenic infection in a large proportion of the human population. Epidemiological and genetic evidence indicates a long-term association between HGV/GBV-C and related viruses and a range of primate species, and the co-speciation of these viruses with their hosts during primate evolution. Using a combination of covariance scanning and analysis of variability at synonymous sites, we previously demonstrated that the coding regions of HGV/GBV-C may contain extensive secondary structure of undefined function (Simmonds & Smith, Journal of Virology 73, 5787-5794, 1999 ). In this study we have carried out a detailed comparison of the structure of the 3'untranslated region (3'UTR) of HGV/GBV-C with that of the upstream NS5B coding sequence. By investigation of free energies on folding, secondary structure predictive algorithms and analysis of covariance between HGV/GBV-C genotypes 1-4 and the more distantly related HGV/GBV-C chimpanzee variant, we obtained evidence for extensive RNA secondary structure formation in both regions. In particular, the NS5B region contained long stem-loop structures of up to 38 internally paired nucleotides which were evolutionarily conserved between human and chimpanzee HGV/GBV-C variants. The prediction of similar structures in the same region of hepatitis C virus may allow the functions of these structures to be determined with a more tractable experimental model.

Liew K, Lui HF, Sutherland S, Simmonds P, Finlayson NDC, Hayes PC. 2001. Precore HBV mutant infection is more common than expected among HBV carriers in Scotland GUT, 48 pp. A107-A108.

Connor MD, Lammie GA, Bell JE, Warlow CP, Simmonds P, Brettle RD. 2001. HIV-associated stroke - Response STROKE, 32 (2), pp. 583-583.

Cleland A, Davis C, Adams N, Lycett C, Jarvis LM, Holmes H, Simmonds P. 2001. Development of multiplexed nucleic acid testing for human immunodeficiency virus type 1 and hepatitis C virus. Vox Sang, 81 (2), pp. 93-101. | Show Abstract | Read more

BACKGROUND AND OBJECTIVES: In most Western countries, blood donations are routinely screened for hepatitis C virus (HCV) RNA by polymerase chain reaction (PCR) or other nucleic acid tests. We describe the development of a multiplexed assay for human immunodeficiency virus type 1 (HIV-1) and HCV in an internally controlled PCR suitable for large-scale blood donor screening. MATERIALS AND METHODS: The HIV/HCV multiplexed PCR used primers from highly conserved regions in the long terminal repeat region. The National Institute for Biological Standards and Controls (NIBSC) International HIV-1 RNA standard, run control and HIV-1 subtype panel were used for assay evaluation. RESULTS: The HIV-1 PCR showed a sensitivity of 24 IU/ml for HIV-1 RNA (a dilution where 95% of replicate reactions were positive), which was at least five times more sensitive than the Roche Monitor version 1.5 (using the ultrasensitive extraction protocol) and Organon NASBA assays. The assay was capable of detecting all subtypes of HIV-1 (A to H), as well as the more divergent group N and O variants. The sensitivity of the PCR was unaffected by multiplexing with HCV primers and by the presence of a bovine viral diarrhoea virus (BVDV) internal control. CONCLUSION: We have developed a highly sensitive multiplexed PCR for HIV-1 and HCV RNA screening that can be introduced into current PCR-based blood donor screening at minimal cost and without significant operational changes.

Simmonds P. 2000. 3 Hepatitis C virus genotypes Biomedical Research Reports, 2 (C), pp. 53-70. | Read more

Sentjens RE, Simmonds P, Vrielink H, Basaras M, Reesink HW. 2000. HGV/GBV-C transmission by blood components during heart surgery. HEPATOLOGY, 32 (4), pp. 592A-592A.

Sentjens RE, Simmonds P, Vrielink H, Basaras M, Reesink HW. 2000. HGV/GBV-C transmission by blood components during heart surgery TRANSFUSION, 40 (10), pp. 82S-82S.

Connor MD, Lammie GA, Bell JE, Warlow CP, Simmonds P, Brettle RD. 2000. Cerebral infarction in adult AIDS patients: observations from the Edinburgh HIV Autopsy Cohort. Stroke, 31 (9), pp. 2117-2126. | Show Abstract | Read more

BACKGROUND AND PURPOSE: Autopsy series of patients with AIDS have found a 4% to 29% prevalence of cerebral infarction. Little is known of the prevalence of cerebral infarction when not associated with non-HIV central nervous system (CNS) infection, lymphoma, or cardioembolic sources. Clinical correlation has seldom been available. We describe the pathological and clinical features of patients from the Edinburgh HIV Cohort Study found to have had cerebral infarcts without evidence of non-HIV CNS infection, CNS lymphoma, or cardioembolic sources at autopsy. METHODS: From 183 autopsy cases, 26 without evidence of opportunistic cerebral infection or lymphoma were selected. These 26 cases went through a second selection process in which the presence of cerebral infarction, in the absence of the conditions mentioned, was verified. Histology and clinical records for the remaining patients were reviewed. RESULTS: Ten (5.5%) cases fulfilled the inclusion criteria and demonstrated similar hypoxic-ischemic lesions. Small-vessel thickening was seen in all cases, and perivascular space dilatation, rarefaction, and pigment deposition, with vessel wall mineralization and perivascular inflammatory cell infiltrates, were seen in some cases. Vasculitis was not found. One patient had had a transient ischemic attack, and no patient had had a stroke. CONCLUSIONS: Cerebral infarcts in HIV-infected patients are not common in the absence of cerebral non-HIV infection, lymphoma, or embolic sources. We found an HIV-associated vasculopathy with similar features in all risk groups. In AIDS patients presenting with stroke or transient ischemic attack, potentially treatable causes, such as cerebral coinfection or tumor, should be sought.

Bell J, Arango JC, Nailon WI, Brannan F, Simmonds P. 2000. Drug abuse and HIV infection - Dual threat to the nervous system BRAIN PATHOLOGY, 10 (4), pp. 758-758.

Abacioglu YH, Bacaksiz F, Bahar IH, Simmonds P. 2000. Molecular evidence of nosocomial transmission of hepatitis C virus in a haemodialysis unit. Eur J Clin Microbiol Infect Dis, 19 (3), pp. 182-186. | Show Abstract | Read more

The molecular epidemiology of type 2a hepatitis C virus (HCV) infections in patients undergoing haemodialysis in the same unit in a Turkish hospital was investigated. Of nine HCV-infected patients four were infected with type 2a, four with type 1b and one with type 1a viruses. Since type 2 HCV infections in the Turkish population are rare, the possibility of nosocomial infection was investigated by means of phylogenetic analysis of viral sequences amplified by the polymerase chain reaction in the NS5b region. One of the samples failed to show amplification and therefore could not be sequenced. The sequences of the remaining three virus samples were grouped closely in a cluster within the type 2a group. The results thus showed that three patients were infected with the same HCV type 2a strain. Seroconversion and clinical data suggested that these patients may have been infected on different occasions, there being possibly more than one mode of transmission. Breaches in infection control procedures and lack of environmental decontamination between two haemodialysis sessions were probably the causes of HCV infections in these patients.

Jarvis L, Cleland A, Simmonds P, Dow B, Munro M, Jordan A, Prowse C, Yap PL. 2000. Screening blood donations for hepatitis C virus by polymerase chain reaction. Vox Sang, 78 (1), pp. 57-58. | Read more

Azim T, Islam MN, Bogaerts J, Mian MA, Sarker MS, Fattah KR, Simmonds P, Jenkins C, Choudhury MR, Mathan VI. 2000. Prevalence of HIV and syphilis among high-risk groups in Bangladesh. AIDS, 14 (2), pp. 210-211. | Read more




Smith DB, Basaras M, Frost S, Haydon D, Cuceanu N, Prescott L, Kamenka C, Millband D, Sathar MA, Simmonds P. 2000. Phylogenetic analysis of GBV-C/hepatitis G virus Journal of General Virology, 81 (3), pp. 769-780. | Show Abstract

Comparison of 33 epidemiologically distinct GBV-C/hepatitis G virus complete genome sequences suggests the existence of four major phylogenetic groupings that are equally divergent from the chimpanzee isolate GBV-C(tro) and have distinct geographical distributions. These four groupings are not consistently reproduced by analysis of the virus 5'-noncoding region (5'-NCR), or of individual genes or subgenomic fragments with the exception of the E2 gene as a whole or of 200-600 nucleotide fragments from its 3' half. This region is upstream of a proposed anti-sense reading frame and contains conserved potential RNA secondary structures that may be capable of directing the internal initiation of translation. Phylogenetic analysis of this region from certain South African isolates is consistent with previous analysis of the 5'-NCR suggesting that these belong to a fifth group. The geographical distribution of virus variants is consistent with a long evolutionary history that may parallel that of pre-historic human migrations, implying that the long-term evolution of this RNA virus is extremely slow.

Smith DB, Basaras M, Frost S, Haydon D, Cuceanu N, Prescott L, Kamenka C, Millband D, Sathar MA, Simmonds P. 2000. Phylogenetic analysis of GBV-C/hepatitis G virus. J Gen Virol, 81 (Pt 3), pp. 769-780. | Show Abstract | Read more

Comparison of 33 epidemiologically distinct GBV-C/hepatitis G virus complete genome sequences suggests the existence of four major phylogenetic groupings that are equally divergent from the chimpanzee isolate GBV-C(tro) and have distinct geographical distributions. These four groupings are not consistently reproduced by analysis of the virus 5'-noncoding region (5'-NCR), or of individual genes or subgenomic fragments with the exception of the E2 gene as a whole or of 200-600 nucleotide fragments from its 3' half. This region is upstream of a proposed anti-sense reading frame and contains conserved potential RNA secondary structures that may be capable of directing the internal initiation of translation. Phylogenetic analysis of this region from certain South African isolates is consistent with previous analysis of the 5'-NCR suggesting that these belong to a fifth group. The geographical distribution of virus variants is consistent with a long evolutionary history that may parallel that of pre-historic human migrations, implying that the long-term evolution of this RNA virus is extremely slow.

MacDonald DM, Holmes EC, Lewis JC, Simmonds P. 2000. Detection of hepatitis B virus infection in wild-born chimpanzees (Pan troglodytes verus): phylogenetic relationships with human and other primate genotypes. J Virol, 74 (9), pp. 4253-4257. | Show Abstract | Read more

Infection with hepatitis B virus (HBV) was detected by serological testing for HBV surface antigen and by PCR assay for HBV DNA in serum samples from two common chimpanzees (Pan troglodytes subsp. verus) born in West Africa. The complete genome sequences obtained by nucleotide sequencing of overlapping DNA fragments amplified by PCR were compared with HBV variants recovered from other primates and with human genotypes A to F. Both chimpanzee sequences were 3, 182 nucleotides in length, and the surface gene sequence predicted the existence of a, d, and w serological determinants. Neither sequence contained stop codons in the precore region. On phylogenetic analysis, the HBV variants infecting the chimpanzees clustered together with a third chimpanzee HBV isolate independently obtained from an infected captive animal (A. J. Zuckerman, A. Thornton, C. R. Howard, K. N. Tsiquaye, D. M. Jones, and M. R. Brambell, Lancet ii:652-654, 1978), with an overall sequence similarity of >94%. This provides strong evidence for a chimpanzee-specific genotype of HBV which circulates in nature. These findings add to the recent evidence for infection in the wild of other Old and New World primates (gibbon, orangutan, and woolly monkey) with species-specific variants of HBV. There is no evidence for close phylogenetic clustering of variants found so far in primates with any of the established HBV genotypes from humans. With the new evidence for the widespread distribution of HBV in primates, hypotheses for the origins of human infection are reviewed.

Jarvis L, Cleland A, Simmonds P, Dow B, Munro M, Jordan A, Prowse C, Yap PL. 2000. Screening blood donations for hepatitis C virus by polymerase chain reaction VOX SANGUINIS, 78 (1), pp. 57-58. | Read more

Simmonds P, Prescott LE, Logue C, Davidson F, Thomas AE, Ludlam CA. 1999. TT virus--part of the normal human flora? J Infect Dis, 180 (5), pp. 1748-1750. | Read more

Sathar MA, Soni PN, Pegoraro R, Simmonds P, Smith DB, Dhillon AP, Dusheiko GM. 1999. A new variant of GB virus C/hepatitis G virus (GBV-C/HGV) from South Africa. Virus Res, 64 (2), pp. 151-160. | Show Abstract | Read more

Phylogenetic analysis of the 5' non-coding region (5'NCR) sequences has demonstrated that GB virus C/hepatitis G virus (GBV-C/HGV) can be separated into three major groups that correlate with the geographic origin of the isolate. Sequence analysis of the 5'NCR of 54 GBV-C/HGV isolates from 31 blood donors, 11 haemodialysis patients and 12 patients with chronic liver disease suggests the presence of a new variant of GBV-C/HGV in the province of KwaZulu Natal, South Africa. Eleven isolates grouped as group 1 variants (bootstrap support, 90%) found predominantly in West and Central Africa, a further six isolates grouped as group 2 variants (bootstrap support, 58%) found in Europe and North America; five of which grouped as 2a (bootstrap support, 91%) and one as 2b (bootstrap support, 87%), the latter also includes isolates from Japan, East Africa and Pakistan. Although the remaining 37 GBV-C/HGV isolates were more closely related to group 1 variants (bootstrap support, 90%), they formed a cluster, which was distinct from all other known GBV-C/HGV sequences. None of the South African isolates grouped with group 3 variants described from Southeast Asia. Three variants of GBV-C/HGV exist in KwaZulu Natal: groups 1, 2 and a new variant, which is distinct from other African isolates.




Irish DN, Blake C, Christophers J, Craske JE, Burnapp L, Abbs IC, MacMahon EME, Muir P, Banatvala JE, Simmonds P. 1999. Identification of hepatitis C virus seroconversion resulting from nosocomial transmission on a haemodialysis unit: Implications for infection control and laboratory screening JOURNAL OF MEDICAL VIROLOGY, 59 (2), pp. 135-140. | Read more

Morris A, Marsden M, Halcrow K, Hughes ES, Brettle RP, Bell JE, Simmonds P. 1999. Mosaic structure of the human immunodeficiency virus type 1 genome infecting lymphoid cells and the brain: evidence for frequent in vivo recombination events in the evolution of regional populations. J Virol, 73 (10), pp. 8720-8731. | Show Abstract

In addition to immunodeficiency, human immunodeficiency virus type 1 (HIV-1) can cause cognitive impairment and dementia through direct infection of the brain. To investigate the adaptive process and timing of HIV-1 entry into the central nervous system, we carried out an extensive genetic characterization of variants amplified from different regions of the brain and determined their relatedness to those in lymphoid tissue. HIV-1 genomes infecting different regions of the brain of one study subject with HIV encephalitis (HIVE) had a mosaic structure, being assembled from different combinations of evolutionarily distinct lineages in p17(gag), pol, individual hypervariable regions of gp120 (V1/V2, V3, V4, and V5), and gp41/nef. Similar discordant phylogenetic relationships were observed between p17(gag) and V3 sequences of brain and lymphoid tissue from three other individuals with HIVE. The observation that different parts of the genome of HIV infecting a particular tissue can have different evolutionary histories necessarily limits the conclusions that can be drawn from previous studies of the compartmentalization of distinct HIV populations in different tissues, as these have been generally restricted to sequence comparisons of single subgenomic regions. The complexity of viral populations in the brain produced by recombination could provide a powerful adaptive mechanism for the spread of virus with new phenotypes, such as antiviral resistance or escape from cytotoxic T-cell recognition into existing tissue-adapted virus populations.

Tomlinson GS, Simmonds P, Busuttil A, Chiswick A, Bell JE. 1999. Upregulation of microglia in drug users with and without pre-symptomatic HIV infection. Neuropathol Appl Neurobiol, 25 (5), pp. 369-379. | Show Abstract | Read more

It is generally thought that infection of the central nervous system (CNS) by HIV-1 can occur early, even around the time of seroconversion, and evidence from animal studies supports this. However, the mode and timing of viral entry remain poorly understood since there have been comparatively few studies of the early neuropathology of HIV infection. In this study, samples of frontal and temporal lobes, and basal ganglia, were selected from 12 HIV-positive drug users who had been infected for 4-130 months before death, 10 HIV-negative drug users and 10 non-drug using controls, all age and sex matched. Routine and immunocytochemical staining showed that leptomeningeal and perivascular lymphocytic infiltrate was upregulated in HIV-infected cases compared with the two control groups, and choroid plexitis was confined to the HIV-positive subjects, suggesting an association with viral infection. In contrast, CD68-positive microglia were enhanced in both HIV- positive and HIV-negative drug users, considerably above the baseline seen in normal controls. However, there was no statistical difference between the three groups in relation to astrocytes. Screening and competitive polymerase chain reaction (PCR) undertaken on multiple samples including brain tissue, choroid plexus and leptomeninges from four of the HIV-positive subjects and one control case showed that the pro-viral burden was never more than 13 copies/microg DNA and was negative in multiple samples from one HIV-positive case and one control case. All the basal ganglia samples were PCR-negative. This study has not revealed any t spots' of viral load in brain tissue, choroid plexus or meninges, either early or late in the course of pre-symptomatic HIV infection. Drug use alone is associated with significant upregulation of microglia and this may predispose to HIV infection of the nervous system in drug users.

Irish DN, Blake C, Christophers J, Craske JE, Burnapp L, Abbs IC, MacMahon EM, Muir P, Banatvala JE, Simmonds P. 1999. Identification of hepatitis C virus seroconversion resulting from nosocomial transmission on a haemodialysis unit: implications for infection control and laboratory screening. J Med Virol, 59 (2), pp. 135-140. | Show Abstract | Read more

Hepatitis C virus (HCV) seroconversion was detected by routine screening in a haemodialysis patient, Patient 1. Serological investigations were undertaken over the following 3 months to determine if further transmission to other patients on the unit had occurred. No additional cases were identified. Twenty-two haemodialysis patients known to have HCV infection were investigated using molecular epidemiological methods to determine if transmission between patients had occurred. HCV viraemia was demonstrated by polymerase chain reaction in 19 of 22 patients (86%). Genotyping showed that eight patients were infected with genotype 1, three with genotype 3 and eight, including Patient 1, with genotype 2. Phylogenetic analysis of viral sequences from the eight patients with genotype 2 revealed three, including Patient 1,with a novel subtype of HCV type 2, and revealed close similarity between viral sequences from patient 1 and those from one other patient, suggesting transmission. This was consistent with haemodialysis histories. Among other patients with genotype 2, there were two with subtype 2a and three others with three separate novel subtypes, as yet undesignated. With the exception of patient 1, all patients infected with novel subtypes were of Afro-Caribbean origin. The HCV prevalence among patients on the haemodialysis unit was high (14%), which may reflect the ethnicity of our haemodialysis population. This case emphasises the risk of nosocomial transmission and the importance of infection control procedures on haemodialysis units, and highlights the usefulness of molecular epidemiological techniques for the investigation of outbreaks of HCV infection.

Mellor J, Hawkins A, Simmonds P. 1999. Genotype dependence of hepatitis C virus load measurement in commercially available quantitative assays. J Clin Microbiol, 37 (8), pp. 2525-2532. | Show Abstract

Standardization and genotype independence of methods used to quantify hepatitis C virus (HCV) RNA in clinical specimens are necessary for accurate assessment of the role of HCV quantitation as a prognostic marker for HCV infection and monitoring of the response to antiviral treatment. Commercially available methods used to measure HCV loads include PCR-based (Roche Monitor) and hybridization-based (Quantiplex bDNA-2) methods. Recently, a new version of the Roche Monitor assay (version 2.0) has become available; it has been modified to achieve more equal quantitation of different HCV genotypes. Consistent with previous reports, Roche Monitor version 1.0 substantially underestimated concentrations of RNA transcripts of types 2b, 3a, 4a, 5a, and 6a and virus loads in individuals infected with genotypes 2 to 6 relative to reference tests. However, version 2.0 achieved equivalent quantitation of each genotype over a narrow quantitative range (10(3) to 5 x 10(5) copies of RNA/ml) but significantly underestimated RNA concentrations above this range. The assay showed an equivalent inability to quantify high levels of HCV RNA in plasma samples, and this was responsible for the falsely narrow range of virus loads detected in HCV-infected individuals. In contrast, the Chiron bDNA-2 assay could only measure RNA concentrations in the upper quantitative range (2 x 10(5) to 5 x 10(7) copies of RNA/ml) but showed equivalent sensitivity for genotypes 1 to 5; however, concentrations of type 6a RNA transcripts and virus loads in clinical specimens from individuals infected with type 6a were underestimated by a factor of 2 to 4. Differences were observed between PCR- and hybridization-based assays in their relative quantitation of HCV RNA transcripts and HCV genomic RNA, which may cause problems with the use of transcripts for interassay calibration.

Simmonds P, Smith DB. 1999. Structural constraints on RNA virus evolution. J Virol, 73 (7), pp. 5787-5794. | Show Abstract

The recently discovered hepatitis G virus (HGV) or GB virus C (GBV-C) is widely distributed in human populations, and homologues such as HGV/GBV-CCPZ and GBV-A are found in a variety of different primate species. Both epidemiological and phylogenetic analyses support the hypothesis that GB viruses coevolved with their primate hosts, although their degree of sequence similarity appears incompatible with the high rate of sequence change of HGV/GBV-C over short observation periods. Comparison of complete coding sequences (8,500 bases) of different genotypes of HGV/GBV-C showed an excess of invariant synonymous sites (at 23% of all codons) compared with the frequency expected by chance (10%). To investigate the hypothesis that RNA secondary-structure formation through internal base pairing limited sequence variability at these sites, an algorithm was developed to detect covariant sites among HGV/GBV-C sequences of different genotypes. At least 35 covariant sites that were spatially associated with potential stem-loop structures were detected, whose positions correlated with positions in the genome that showed reductions in synonymous variability. Although the functional roles of the predicted secondary structures remain unclear, the restriction of sequence change imposed by secondary-structure formation provides a mechanism for differences in net rate of accumulation of nucleotide substitutions at different sites. However, the resulting disparity between short- and long-term rates of sequence change of HGV/GBV-C violates the assumptions of the "molecular clock." This places a major restriction on the use of nucleotide or amino acid sequence comparisons to calculate times of divergence of other viruses evolving under the same structural constraints as GB viruses.

Prescott LE, MacDonald DM, Davidson F, Mokili J, Pritchard DI, Arnot DE, Riley EM, Greenwood BM, Hamid S, Saeed AA et al. 1999. Sequence diversity of TT virus in geographically dispersed human populations. J Gen Virol, 80 ( Pt 7) (7), pp. 1751-1758. | Show Abstract | Read more

TT virus (TTV) is a newly discovered DNA virus originally classified as a member of the Parvoviridae. TTV is transmitted by blood transfusion where it has been reported to be associated with mild post-transfusion hepatitis. TTV can cause persistent infection, and is widely distributed geographically; we recently reported extremely high prevalences of viraemia in individuals living in tropical countries (e.g. 74% in Papua New Guinea, 83% in Gambia; Prescott & Simmonds, New England Journal of Medicine 339, 776, 1998). In the current study we have compared nucleotide sequences from the N22 region of TTV (222 bases) detected in eight widely dispersed human populations. Some variants of TTV, previously classified as genotypes 1a, 1b and 2, were widely distributed throughout the world, while others, such as a novel subtype of type 1 in Papua New Guinea, were confined to a single geographical area. Five of the 122 sequences obtained in this study (from Gambia, Nigeria, Papua New Guinea, Brazil and Ecuador) could not be classified as types 1, 2 or 3, with the variant from Brazil displaying only 46-50% nucleotide (32-35% amino acid) sequence similarity to other variants. This study provides an indication of the extreme sequence diversity of TTV, a characteristic which is untypical of parvoviruses.

Bell JE, Arango JC, Strappe P, Simmonds P. 1999. Drug use and HIV cause cumulative DNA damage in the CNS. JOURNAL OF NEUROPATHOLOGY AND EXPERIMENTAL NEUROLOGY, 58 (5), pp. 527-527. | Read more

Davidson F, MacDonald D, Mokili JL, Prescott LE, Graham S, Simmonds P. 1999. Early acquisition of TT virus (TTV) in an area endemic for TTV infection. J Infect Dis, 179 (5), pp. 1070-1076. | Show Abstract | Read more

TT virus (TTV) is widely distributed, with high frequencies of viremia in South America, Central Africa, and Papua New Guinea. The incidence and timing of infection in children born in a rural area of the Democratic Republic of Congo was investigated. TTV viremia was detected in 61 (58%) of 105 women attending an antenatal clinic and in 36 (54%) of 68 infants. Most infants acquired the infection at >/=3 months postpartum. Surprisingly, TTV infection was detected in a large proportion of children with TTV-negative mothers (13 [43%] of 30). Nucleotide sequences of TTV-infected children were frequently epidemiologically unlinked to variants detected in the mother. These three aspects contrast with the maternal transmission of hepatitis G virus/GB virus C in this cohort and suggest an environmental source of TTV infection comparable to hepatitis A virus and other enterically transmitted infections.

Mokili JL, Wade CM, Burns SM, Cutting WA, Bopopi JM, Green SD, Peutherer JF, Simmonds P. 1999. Genetic heterogeneity of HIV type 1 subtypes in Kimpese, rural Democratic Republic of Congo. AIDS Res Hum Retroviruses, 15 (7), pp. 655-664. | Show Abstract | Read more

A relatively low and stable seroprevalence of HIV-1 was previously reported among pregnant women attending for antenatal care between 1988 and 1993 in Kimpese, a rural town in the Democratic Republic of Congo (DRC, formerly Zaire). To characterize the HIV-1 subtypes circulating in this area, we have examined a 330-bp fragment of the p17 region of the gag gene of HIV-1 strains obtained from 70 patients (55 mothers, 15 children), of whom 61 were epidemiologically unlinked. Phylogenetic analyses revealed the existence of at least seven HIV-1 subtypes within the Kimpese region. Among the 61 epidemiologically unlinked patients, subtype A was predominant and found in 29 (47.5%) individuals. Other subtypes cocirculating in this rural part of DRC include subtypes C (1.6%), D (9.8%), F (3.2%), G (6.5%), H (21.3%), and J (4.9%). Sequences from four patients did not cluster with any of the currently documented HIV-1 subtypes, in analyses of fragments of both the gag (247 to 330 bp, 197 bp, and 310 bp) and env (340 bp) genes. Overall, comparisons of the gag(p17) gene regions revealed high pairwise divergences (mean, 19.9%; range, 1 to 46%). This level of gag(p17) gene variation in the DRC is considerably greater than previously appreciated. These results are relevant for the molecular epidemiology of HIV-1 in Africa and for the design of a future vaccine against HIV-1 in this region.

Cleland A, Nettleton P, Jarvis L, Simmonds P. 1999. Use of bovine viral diarrhoea virus as an internal control for amplification of hepatitis C virus. Vox Sang, 76 (3), pp. 170-174. | Show Abstract | Read more

BACKGROUND AND OBJECTIVES: Screening for hepatitis C virus (HCV) by polymerase chain reaction (PCR) will be mandatory for screening blood and plasma donors in Europe and elsewhere. This study describes an internally controlled, highly sensitive PCR method designed for screening blood donations in pools. MATERIAL AND METHODS: RNA extracted from bovine viral diarrhoea virus (BVDV) was used as an internal control to monitor the efficiency of extraction, reverse transcription and amplification steps in HCV PCR. RESULTS: Sensitivity of PCR for single molecules of HCV in the presence of 33 genome equivalents of BVDV RNA was achieved by reducing the efficiency of BVDV amplification. BVDV could be recovered at high efficiency from large volume pools (2-5 ml) by ultracentrifugation and by the NucliSens extraction method. CONCLUSION: Detection of BVDV validates the extraction, reverse transcription and amplification methods used for HCV detection in plasma pools and provides valuable quality assurance for negative results.

MacDonald DM, Scott GR, Clutterbuck D, Simmonds P. 1999. Infrequent detection of TT virus infection in intravenous drug users, prostitutes, and homosexual men. J Infect Dis, 179 (3), pp. 686-689. | Show Abstract | Read more

TT virus (TTV), a recently discovered DNA virus, has been implicated as a cause of non-A to non-C posttransfusion hepatitis. The frequency of TTV in persons considered at high risk for sexual and parenteral infection was investigated (52 prostitutes, 81 homosexual men, 65 intravenous drug users) to assess its mode of transmission. TTV DNA was assayed by polymerase chain reaction using primers from conserved regions in the N22 clone. Viremia frequency was 4.5%-13.0% in study subjects, not significantly different from that in low-risk controls (2 [4.5%] of 44). The frequency of TTV viremia increased significantly with age (P=.018) but was not associated with human immunodeficiency virus coinfection. The low frequency of infection detected in both risk groups suggests that spread by sexual contact or by intravenous drug use is relatively inefficient and unlikely to account for the high prevalence of TTV observed worldwide.

Casino C, McAllister J, Davidson F, Power J, Lawlor E, Yap PL, Simmonds P, Smith DB. 1999. Variation of hepatitis C virus following serial transmission: multiple mechanisms of diversification of the hypervariable region and evidence for convergent genome evolution. J Gen Virol, 80 ( Pt 3) (3), pp. 717-725. | Show Abstract | Read more

We have studied the evolution of hepatitis C virus (HCV) from a common source following serial transmission from contaminated batches of anti-D immunoglobulin. Six secondary recipients were each infected with virus from identifiable primary recipients of HCV-contaminated anti-D immunoglobulin. Phylogenetic analysis of virus E1/E2 gene sequences [including the hypervariable region (HVR)] and part of NS5B confirmed their common origin, but failed to reproduce the known epidemiological relationships between pairs of viruses, probably because of the frequent occurrence of convergent substitutions at both synonymous and nonsynonymous sites. There was no evidence that the rate at which the HCV genome evolves is affected by transmission events. Three different mechanisms appear to have been involved in generating variation of the hypervariable region; nucleotide substitution, insertion/deletion of nucleotide triplets at the E1/E2 boundary and insertion of a duplicated segment replacing almost the entire HVR. These observations have important implications for the phylogenetic analysis of HCV sequences from epidemiologically linked isolates.

Bell H, Hellum K, Harthug S, Myrvang B, Ritland S, Maeland A, von der Lippe B, Bjøro K, Skaug K, Gutigard BG et al. 1999. Treatment with interferon-alpha2a alone or interferon-alpha2a plus ribavirin in patients with chronic hepatitis C previously treated with interferon-alpha2a. CONSTRUCT Group. Scand J Gastroenterol, 34 (2), pp. 194-198. | Show Abstract | Read more

BACKGROUND: Preliminary results from combination therapy with interferon-alpha and ribavirin (IFN/Rib) in patients with chronic hepatitis C have been promising, with up to 50% sustained hepatitis C virus (HCV) RNA response. The aim of this study was to investigate whether a sustained HCV RNA response could be obtained with combination therapy in patients who were non-responders or relapsers after IFN treatment. METHODS: In a multicenter study we randomized 53 HCV RNA-positive patients into 2 treatment groups. They all had biopsy-confirmed chronic hepatitis C, and all were recruited from a previous IFN study: 26 were previous non-responders and 27 responders with relapse. Group A received interferon-alpha2a, 4.5 MIU thrice weekly for 6 months, and group B received ribavirin, 1000-1200 mg/day, in combination with the same dose of interferon-alpha2a for 6 months. Median Knodell index was 5.0 in both groups. Genotype 1 was found in 24 (45%), type 2 in 3 (6%), and type 3 in 26 (49%). RESULTS: Sustained clearance of HCV viremia 6 months after interferon-alpha2a treatment stop was obtained in 12 of 53 patients (23%): 6 of 27 in the IFN group (22%) and 6 of 26 (23%) in the IFN/Rib group (NS). Nine of 27 (33%) former responders with relapse, compared with 3 of 26 (12%) non-responders, obtained a sustained HCV RNA response (P = 0.054). In previous relapse patients sustained loss of viremia was more frequent in genotype 3 (50%) than in genotype 1 (11%) patients (P = 0.022). CONCLUSIONS: In a group of previous IFN-alpha2a-treated chronic HCV patients we obtained a similar sustained clearance of viremia when retreated either with IFN-alpha2a alone or with a combination of IFN-alpha2a and ribavirin for 6 months. Previous relapse patients with HCV genotype 3 obtained sustained loss of viremia significantly more often (50%) than type-patients (11%). Previous IFN responders with relapse responded better than previous non-responders.

Shepherd EJ, Brettle RP, Liberski PP, Aguzzi A, Ironside JW, Simmonds P, Bell JE. 1999. Spinal cord pathology and viral burden in homosexuals and drug users with AIDS. Neuropathol Appl Neurobiol, 25 (1), pp. 2-10. | Show Abstract | Read more

Unless treated with effective antiretroviral therapy many AIDS patients develop a characteristic vacuolar myelopathy of the spinal cord associated with moderate clinical disability. Opinion is divided as to whether vacuolar myelopathy is causally linked to HIV myelitis. To investigate this further, spinal cord pathology was assessed in 41 drug users, 33 homosexual men and 16 other patients, all with AIDS. Previous work has shown that HIV encephalitis is more common in Edinburgh drug users than in homosexual men. In the present study HIV myelitis (10% overall) was more common in drug users (17%) than in homosexual men (3%) (P = 0.05), whereas the incidence of opportunistic infections (7% v. 9%) and lymphomas (2% v. 6%) was comparable in the two groups, but with a slight trend in the reverse direction, reflecting similar findings in the brain. However, moderate or severe vacuolar myelopathy was equally represented in both groups (20% of drug users and 21% of homosexual men). The HIV proviral load, assessed by polymerase chain reaction in frozen samples of thoracic spinal cord in 37 cases, correlated closely with the presence of giant cells and/or with immunocytochemical evidence of productive HIV infection. In 13 cases, the proviral load was measured in cervical, thoracic and lumbar samples and proved to be uniformly high or low in individual cases. This study provides no evidence for direct involvement of HIV, cytomegalovirus, papovavirus or human foamy virus in the pathogenesis of vacuolar myelopathy.

el-Zayadi A, Selim O, Haddad S, Simmonds P, Hamdy H, Badran HM, Shawky S. 1999. Combination treatment of interferon alpha-2b and ribavirin in comparison to interferon monotherapy in treatment of chronic hepatitis C genotype 4 patients. Ital J Gastroenterol Hepatol, 31 (6), pp. 472-475. | Show Abstract

BACKGROUND AND AIM: Treatment of chronic hepatitis C patients, infected with genotype 4 with interferon-alpha yielded a limited response. Our aim was to compare the efficacy of interferon-alpha alone and in combination with ribavirin in chronic hepatitis C patients infected with genotype 4. PATIENTS: Fifty-two chronic hepatitis C patients (all males) infected with genotype 4, who had not received interferon, were randomized into 2 equal comparable groups. METHODS: Group I received interferon alpha-2b "Schering Plough" 3 MU, tiw combined with ribavirin (1000 mg/day). Group II received interferon alpha-2b alone in the same dose. Both groups were evaluated monthly, at the end of 24 weeks of treatment and 24 weeks later. Two patients were dropped from group I and one patient from group II. RESULTS: Biochemical response: at the end of treatment, a return to normal of ALT was obtained in 16/24 (66.7%) patients on combination therapy vs 8/25 (32%) patients on interferon alone (p = 0.0152). At the end of follow-up, a sustained response was achieved in 10/24 (41.7%) patients on combination therapy vs 4/25 (16%) patients on interferon (p = 0.0468). Virologic response: at the end of treatment, the rates of virological response were higher in the patients on combination therapy 9/24 (37.5%) than in those on interferon 4/25 (16%) (p = 0.0380). At the end of follow-up, loss of serum HCV RNA was reported in 5/24 (20.8%) patients on combination therapy vs 2/25 (8%) patients on interferon (p = 0.1916). Histologic response: mild histologic improvement was shown by a decrease in the inflammatory score, which was highest in patients in the combination group. CONCLUSIONS: In chronic hepatitis C patients infected with genotype 4, combination therapy with interferon-alpha and ribavirin was more effective than treatment with interferon monotherapy. At the end of the follow-up, about 50% of patients in both groups were still viraemic though their ALT remained normal.

Lawlor E, Power J, Garson J, Yap P, Davidson F, Columb G, Smith D, Pomeroy L, O'Riordan J, Simmonds P, Tedder R. 1999. Transmission rates of hepatitis C virus by different batches of a contaminated anti-D immunoglobulin preparation. Vox Sang, 76 (3), pp. 138-143. | Show Abstract | Read more

BACKGROUND AND OBJECTIVES: The aim of this study was to determine the hepatitis C virus (HCV) infection rate of recipients of different batches of anti-D immunoglobulin associated with an outbreak of HCV infection which occurred in 1977 and its relationship to the polymerase chain reaction (PCR) status of the implicated batches. This study was undertaken to determine the predictive value of HCV genome detection and quantification for subsequent infection in recipients of an HCV-contaminated anti-D immunoglobulin product for intravenous use. MATERIALS AND METHODS: Sera from recipients of anti-D were tested by HCV enzyme immunoassay and if found positive were subsequently tested by recombinant immunoblot assay and HCV PCR in a national HCV anti-D screening programme set up in 1994. The HCV status of 1,342 known recipients of infectious or potentially infectious batches has been compared to the amount of HCV RNA in the anti-D batch they received so as to determine the value of PCR in the prediction of infectivity in immunoglobulin preparations. RESULTS: It has been demonstrated that HCV-infected plasma derived from batches of anti-D showing levels of viral genome in excess of 10(4) genomes per millilitre led to infection of up to 60% of recipients. In contrast, batches with undetectable levels of HCV genome very rarely transmitted infection. CONCLUSIONS: The presence of HCV RNA in intravenous immunoglobulin preparations which have not undergone a specific viral inactivation step is a predictor of HCV infection in recipients.

Prescott LE, Simmonds P. 1999. Serological Genotyping Using Synthetic Peptides Derived from the NS4 Region. Methods Mol Med, 19 pp. 199-205. | Show Abstract | Read more

The RNA genome of hepatitis C virus (HCV) displays extensive sequence variation, and consequently, the virus is classified into six major genotypes. The severity of disease and response to antiviral treatment are thought to be influenced by both viral and host-related factors, including age of aquisition, duration of infection, circulating virus load, and the genotype of infecting virus Many investigators have reported that infections with HCV type 1 are associated with an increase in the likelihood of progression to hepatocellular carcinoma (1-3), and with nonresponsiveness to interferon therapy when compared to genotypes 2 or 3 (reviewed in refs. 4,5) This discovery emphasizes the need for genotyping methods in current clinical practice, in providing a predictor of the outcome of treatment, and perhaps helping to target appropriate treatment to the most relevant patient groups.

Smith DB, Lawlor E, Power J, O'Riordan J, McAllister J, Lycett C, Davidson F, Pathirana S, Garson JA, Tedder RS et al. 1999. A second outbreak of hepatitis C virus infection from anti-D immunoglobulin in Ireland. Vox Sang, 76 (3), pp. 175-180. | Show Abstract | Read more

OBJECTIVE: To investigate the infectivity for hepatitis C virus (HCV) of intravenous anti-D immunoglobulin batches manufactured in Ireland between 1991 and 1994. METHODS: Women who had received anti-D manufactured between 1991 and 1994 were screened for serological markers of HCV infection and for the presence of HCV RNA by RT-PCR amplification and virus genotyping. RESULTS: 44 women exposed to anti-D manufactured between 1991 and 1994 were polymerase chain reaction positive for HCV RNA, 19 of whom were infected with genotype 3a virus shown by phylogenetic analysis of the NS5B gene to be closely related to that from the single implicated donor. CONCLUSIONS: Anti-D manufactured in 1991-1994 transmitted infection of HCV genotype 3a. The prevalence of HCV-specific antibody in anti-D recipients was relatively low (0.59%), consistent with the low level of virus RNA in these anti-D batches.

Simmonds P. 1999. Viral heterogeneity of the hepatitis C virus. J Hepatol, 31 Suppl 1 (1), pp. 54-60. | Show Abstract | Read more

This review summarises the classification of hepatitis C virus as a flavivirus, the identification and detection of HCV genotypes, and reviews the current information concerning the geographical and risk group associations of the common genotypes in Europe.

Davidson F, Simmonds P. 1999. Determination of HCV Genotypes by RFLP. Methods Mol Med, 19 pp. 175-181. | Show Abstract | Read more

Several different methods have been developed for the typing of HCV variants: direct sequence analysis, slot-blot hybridization analysis of reverse transcriptase-polymerase chain reaction (RT-PCR) products using cDNA probes specific to each HCV genotype and PCR amplification using type-specific primers that are designed to match only virus sequences of a defined HCV type and will fail to amplify sequences of other types. An alternative method is the nonselective amplification of virus cDNA using conserved primers followed by restriction fragment length polymorphisms (RFLP) The coding regions of the HCV genome are highly variable, so reliable amplification of different HCV genotypes with the same set of primers is difficult. For this reason, typing methods based on analysis of amplified DNA are more reliable if carried out in the highly conserved 5' noncoding region (5' NCR). Different typing assays are discussed in Chapters 14 , 16 , and 17 . The RFLP assay method described in the following section has been developed to enable the detection HCV genotypes 1-6 (1).The advantages that this particular method offers are that it is relatively easy to carry out, the apparatus required is generally available in most laboratories or is inexpensive to buy and a large number of samples can be genotyped at the same time as those being tested by PCR in the laboratory. The major problem associated with the technique is the possible misidentification of novel subtypes of HCV type 6 as type 1 variants (2).

Berg ES, Jarvis LM, Simmonds P, Bell H, Skaug K. 1998. Detection of GB virus C RNA by GBV-C LCx and two PCR assays with primers from the 5' non-coding and NS5B region. J Virol Methods, 76 (1-2), pp. 43-49. | Show Abstract | Read more

The objective of this study was to compare the sensitivity of three different reverse transcriptase-polymerase chain reaction (RT-PCR) based tests, for detection of GB virus C (GBV-C) RNA. One commercial and two 'in house' RT-PCR assays were employed in the testing of serum samples from 114 chronic hepatitis C infected individuals. A part of the 5' non-coding region (5'NCR) of the GBV-C genome was amplified by the GBV-C LCx assay (Abbott) and one of the 'in house' RT-PCR tests. In the other 'in house' RT-PCR a segment of the NS5B region was amplified. The 'in house' assays included the use of internal controls that were co-amplified with use of the same outer PCR primers as the virus targets. The GBV-C LCx from Abbott and 5'NCR 'in house' PCR tests detected 28 and 27 GBV-C positive individuals, respectively. The sample positive only in the LCx test was confirmed by the 'in house' 5'NCR RT PCR using an increased virus input. In comparison, the NS5B 'in house' PCR test detected 24 of the GBV-C positive samples. One sample showed no amplification of internal controls/virus target in the 5'NCR 'in house' PCR and another samples was amplification negative in the NS5B PCR. The PCR assays with primers from the 5'NCR of the virus genome e.g. the GBV-C LCx, were more sensitive compared with RT-PCR using primers from the NS5B region. The GBV-C LCx seemed to be the most sensitive and robust assay. Internal controls included in the 'in house' assays identified two samples with failure of the amplification.

Robertson B, Myers G, Howard C, Brettin T, Bukh J, Gaschen B, Gojobori T, Maertens G, Mizokami M, Nainan O et al. 1998. Classification, nomenclature, and database development for hepatitis C virus (HCV) and related viruses: proposals for standardization. International Committee on Virus Taxonomy. Arch Virol, 143 (12), pp. 2493-2503. | Show Abstract | Read more

This paper presents a summary of the recommendations that were formulated for the purposes of unifying the nomenclature for hepatitis C virus (HCV), based upon guidelines of the International Committee on Virus Taxonomy (ICTV), and provides guidelines for the incorporation of sequence data into an HCV database that will be available to researchers through the internet. Based upon the available data, the genus Hepacivirus should be regarded as comprising a single species with HCV-1 as the prototype. All currently known isolates of HCV can be divided into six phylogenetically distinct groups, and we recommend that these groups are described as clades 1 to 6. Whether or not these should be regarded as different species within the Hepacivirus genus requires additional clinical, virological, and immunological information. Clades 1, 2, 4, and 5 would correspond to genotype 1, 2, 4, and 5 while clade 3 would comprise genotype 3 and genotype 10, and clade 6 comprise genotypes 6, 7, 8, 9, and 11. We propose that existing subtype designations are reassigned within these clades based upon publication priority, the existence of a complete genome sequence and prevalence. The assignment of isolates to new clades and subtypes should be confined to isolates characterized from epidemiologically unlinked individuals. Comparisons should be based on nucleotide sequences of at least two coding regions and preferably of complete genome sequences, and should be based on phylogenetic analysis rather than percent identity. A forum for discussion and contributions to these recommendations will be made available at the international HCV database at

Blair CS, Davidson F, Lycett C, McDonald DM, Haydon GH, Yap PL, Hayes PC, Simmonds P, Gillon J. 1998. Prevalence, incidence, and clinical characteristics of hepatitis G virus/GB virus C infection in Scottish blood donors. J Infect Dis, 178 (6), pp. 1779-1782. | Show Abstract | Read more

The prevalence, incidence, clinical features, and natural history of hepatitis G virus (HGV) or GB virus C (GBV-C) were investigated in a non-remunerated blood donor population to determine its clinical significance and its impact on blood safety. Of 1020 regular blood donors, 23 (2.25%) were positive for plasma HGV/GBV-C RNA. Alanine aminotransferase levels were lower than in uninfected donors (median, 20 IU/mL; 32 IU/mL in controls; P=.015). Clinical examination produced no other evidence for hepatitis or for shared nonhepatic diseases. Fifteen of 17 donors excreted HGV/GBV-C in saliva (mean level, 8x103 copies of RNA/mL). Testing of previous donations indicated an incidence of 170-200 new infections with HGV/GBV-C per 100,000 donor-years. The absence of further clinicopathologic data and the limitations of current polymerase chain reaction-based methods for screening suggests that it is neither necessary nor practical to commence screening.

Bell JE, Brettle RP, Chiswick A, Simmonds P. 1998. HIV encephalitis, proviral load and dementia in drug users and homosexuals with AIDS. Effect of neocortical involvement. Brain, 121 ( Pt 11) (11), pp. 2043-2052. | Show Abstract | Read more

In this consecutive autopsy study, the pathological evidence of HIV encephalitis, which included the presence of giant cells and/or HIV p24 immunopositivity, was found more frequently in drug users (25 of 45; 56%) than in homosexual men (6 of 35; 17%) with AIDS (P < 0.01). Productive infection, as shown by HIV p24 positivity, was found in frontal lobe white matter in 29 of the 31 HIV encephalitis cases, but was also present in grey matter in 50% of the HIV encephalitis cases. Immunopositivity was confined to microglia, monocytes and most but not all giant cells. HIV-1 proviral load was determined by quantitative PCR in 65 of the 80 cases (separately in grey and white matter in 49 of these), and correlated well with the presence of HIV encephalitis (P < 0.001). Twenty-five patients with AIDS (13 male homosexuals, 12 drug users) showed no HIV encephalitis, opportunistic infection or cerebral lymphoma, while 18 (2 male homosexuals, 16 drug users) showed pure HIV encephalitis. Cognitive function had been assessed prospectively in this cohort and graded as normal or mildly, moderately or severely impaired. Because opportunistic infections and lymphomas of the brain may also lead to dementia, patients found to have these conditions at autopsy were excluded from the final analysis of the cases with dementia, so that the precise correlation between cognitive impairment and pure HIV encephalitis could be determined in this cohort without possible confounding variables. Fourteen of 18 patients with pure HIV encephalitis had shown cognitive impairment. Severe dementia correlated better with pure HIV encephalitis in cases in which grey matter involvement was present (7 out of 9) than in those in which only white matter was involved (2 out of 9) (P < 0.05), although milder degrees of cognitive impairment had been present in a further 5 HIV encephalitis cases. No correlation was found between zidovudine therapy and the degree of cognitive impairment. Systemic and cerebral opportunistic infections and lymphoma showed a negative association with HIV encephalitis, being more common in homosexuals than in drug users, despite comparable CD4 counts in the two groups. These findings suggest that neocortical productive HIV infection is a significant factor in AIDS-related dementia, although this may reflect merely a higher overall viral burden in the brain.

Simmonds P, Davidson F, Jarvis LM. 1998. Transfusion transmitted virus - Reply LANCET, 352 (9136), pp. 1310-1311. | Read more

Haydon GH, Jarvis LM, Simmonds P, Hayes PC. 1998. Hepatitis G virus in patients with hepatocellular carcinoma. HEPATOLOGY, 28 (4), pp. 219A-219A.

Prescott LE, Simmonds P. 1998. Global distribution of transfusion-transmitted virus. N Engl J Med, 339 (11), pp. 776-777. | Read more

Simmonds P. 1998. Transfusion virology: progress and challenges. Blood Rev, 12 (3), pp. 171-177. | Show Abstract | Read more

Discoveries of new human viruses and new technologies for their detection have made, and will continue to make, major contributions to the safety of blood transfusion. This article discusses the practical issues involved in the implementation of additional serological screening tests for viruses such as human T-lymphotropic virus, and reviews current information on the prevalence and pathogenicity of more recently discovered viruses, such as hepatitis G virus (HGV) or GB virus-C (GBV-C) and human herpes virus 8, a potential aetiological agent of Kaposi's sarcoma. Progress in the technology behind nucleic acid amplification techniques, such as the polymerase chain reaction (PCR), makes direct detection of viruses such as human immunodeficiency virus and hepatitis C virus possible. The use of such methods for screening will allow the earlier detection of acutely-infected individuals and the elimination of transmission from 'window' period donations before seroconversion for antibody. Establishing a framework for PCR-based screening would also enable the testing for others such as hepatitis A virus, parvovirus B19 and GBV-C/HGV for which serological detection methods cannot be or have not been developed.

Adams NJ, Prescott LE, Jarvis LM, Lewis JC, McClure MO, Smith DB, Simmonds P. 1998. Detection in chimpanzees of a novel flavivirus related to GB virus-C/hepatitis G virus. J Gen Virol, 79 ( Pt 8) (8), pp. 1871-1877. | Show Abstract | Read more

Infection with hepatitis G virus (HGV) or GB virus-C (GBV-C) is widely distributed in human populations. Viruses related to GBV-C/HGV have been recovered from several New World primate species, including tamarins, owl monkeys and marmosets. To understand more about the relationship between GB viruses and their hosts, we used primers from the 5' non-coding (5'NC), non-structural 3 (NS3) and NS5 regions in nested polymerase chain reactions to screen for related viruses infecting non-captive chimpanzees (Pan troglodytes, troglodytes and verus subspecies). Sequences from the 5'NCR and NS5 regions were amplified from samples taken from 3 of 39 chimpanzees, and from one chimpanzee in the NS3 region. Sequence comparisons of each region revealed that the GB virus infecting chimpanzees was distinct from both GBV-C/HGV and from any of the known GBV-A sequences, but was more closely related to human viruses. GB viruses recovered from different chimpanzees were more diverse than variants of GBV-C/HGV found in humans, with 25% sequence divergence in the 5'NCR and 20% (9.5% amino acid) sequence divergence in NS5 between variants recovered from the troglodytes and verus subspecies, compared with 7.4% and 10.4% (1.9% amino acid) divergence amongst GBV-C/HGV variants infecting humans. Finding GBV-C/HGV-related viruses in an Old World monkey species suggests that GB-like viruses may be widely distributed in simians, and suggests a close evolutionary relationship with their natural hosts.

Shattock RM, Patrizio C, Simmonds P, Sutherland S. 1998. Detection of Chlamydia trachomatis in genital swabs: comparison of commercial and in house amplification methods with culture. Sex Transm Infect, 74 (4), pp. 289-293. | Show Abstract | Read more

AIMS: To evaluate the sensitivity of the Roche Cobas, Roche Amplicor plate kit, ligase chain reaction (LCR), and an in house polymerase chain reaction (PCR) by titration of purified elementary bodies (EB) and also to test 245 urethral and endocervical specimens for Chlamydia trachomatis by the four assays as well as conventional culture. STUDY DESIGN: EB titrations were run in duplicate in each commercial assay and six times in the in house PCR. Clinical samples were aliquoted and tested by each assay and were considered positive if C trachomatis was detected by two or more separate tests or if the sample was either culture or immunofluorescence positive. Major outer membrane protein (MOMP) specific primers were used as a confirmatory assay for the in house PCR. RESULTS: The in house PCR, Roche Cobas Amplicor, LCR, and Amplicor plate kit gave detection limits of approximately 1, 1-2, 2, and 2-4 EBs respectively. By the criteria described above for definition of a C trachomatis positive result in clinical samples we identified 23 true positives among the 245 clinical specimens. The in house PCR detected all 23 giving a sensitivity of 100% and a specificity of 98%. The Roche Cobas Amplicor, Roche Amplicor plate kit, and LCR detected 21, 19, and 19 of these respectively giving sensitivities of 87.5%, 82%, and 82% respectively and specificities of 99.5%, 99%, and 100% respectively. The culture gave a sensitivity of 78% and specificity of 100%. CONCLUSION: All four amplification assays had a greater sensitivity than the culture used routinely in this laboratory. The in house plasmid PCR had the greatest sensitivity and when combined with confirmation by immunofluorescence detected the greatest number of positives. This increased sensitivity is likely to have been achieved by the use of a DNA purification step and of nested primers in the amplification stage and their combined use in routine diagnostic assays for chlamydia might increase the frequency of C trachomatis detections. However, this assay is much less user friendly than the two semiautomated commercial assays investigated in this study.

Simmonds P, Davidson F, Lycett C, Prescott LE, MacDonald DM, Ellender J, Yap PL, Ludlam CA, Haydon GH, Gillon J, Jarvis LM. 1998. Detection of a novel DNA virus (TTV) in blood donors and blood products. Lancet, 352 (9123), pp. 191-195. | Show Abstract | Read more

BACKGROUND: A newly discovered DNA virus, transfusion-transmitted virus (TTV), has been implicated as a cause of post-transfusion hepatitis. We investigated the frequency of TTV viraemia in UK blood donors, and the extent to which TTV contaminates blood products such as factor VIII and IX clotting factors. We also investigated the possible aetiological role of TTV in cryptogenic fulminant hepatic failure (FHF). METHODS: We extracted DNA from plasma of blood donors and patients with FHF, and from blood products (factor VIII and IX clotting-factor concentrates, immunoglobulin preparations). We detected TTV by PCR using primers from a conserved region in the TTV genome. FINDINGS: TTV viraemia was detected in 19 (1.9%) of 1000 non-remunerated regular blood donors. Infection occurred more frequently in older donors (mean age 53 years), compared with the age prolife of donors infected with hepatitis C virus and other parenterally-transmitted viruses. TTV contamination was found in ten (56%) of 18 batches of factor VIII and IX concentrate manufactured from such non-remunerated donors, and in seven (44%) of 16 batches of commercially available products. Whereas solvent or detergent treatment had little effect on the detection of TTV in factor VIII and IX by PCR, this virucidal step seemed to inactivate TTV infectivity. TTV infection was detected in four (19%) of 21 patients with FHF; in three cases, infection was detected at the onset of disease and could thus not be excluded from its aetiology. INTERPRETATION: TTV viraemia is frequent in the blood-donor population, and transmission of TTV through transfusion of blood components may have occurred extensively. Clinical assessment of infected donors and recipients of blood and blood products, and assessment of TTV's aetiological role in hepatic and extra-hepatic disease, are urgently needed.

Scallan MF, Clutterbuck D, Jarvis LM, Scott GR, Simmonds P. 1998. Sexual transmission of GB virus C/hepatitis G virus. J Med Virol, 55 (3), pp. 203-208. | Show Abstract | Read more

Although it is established that infection with GB virus C (GBV-C) or hepatitis G virus (HGV) can be transmitted parenterally, the prevalence of GBV-C/HGV viremia in the general population (2-5%) is relatively high compared with other parenterally borne viruses such as hepatitis C virus. To investigate the possibility of sexual transmission of GBV-C/HGV, we determined the frequency of viremia by the polymerase chain reaction and serological reactivity to the E2 protein by ELISA in samples collected from individuals at risk for sexually transmitted diseases attending a city genitourinary medicine clinic. GBV-C/HGV viremia was detected in 27 of 87 male homosexuals (31%) and 9 of 50 prostitutes (18%), frequencies significantly greater than those in matched controls (2/63) and local blood donors (2.3%). Among nonviremic individuals, a high frequency of serological reactivity to the E2 protein of GBV-C/HGV was also observed in the risk groups (male homosexuals: 14/60; prostitutes: 11/41), although these figures are likely to be underestimates of the frequency of past infection as detectable anti-E2 reactivity may attenuate rapidly over time following resolution of infection. Infection with GBV-C/HGV was more frequent among those coinfected with human immunodeficiency virus type 1. Among male homosexuals from whom retrospective samples were available, evidence for de novo infection was found in 9 of 22 individuals over a mean sampling time of 2.9 years, predicting an annualized incidence of GBV-C/HGV infection of approximately 11% in this group. The high prevalence and incidence of GBV-C/HGV infection in these individuals and prostitutes provides strong evidence for its spread by sexual contact. Further studies are required to investigate the mechanism of its transmission and the clinical significance of acute and persistent infection in these risk groups.

McAllister J, Casino C, Davidson F, Power J, Lawlor E, Yap PL, Simmonds P, Smith DB. 1998. Long-term evolution of the hypervariable region of hepatitis C virus in a common-source-infected cohort. J Virol, 72 (6), pp. 4893-4905. | Show Abstract

The long-term evolution of the hepatitis C virus hypervariable region (HVR) and flanking regions of the E1 and E2 envelope proteins have been studied in a cohort of women infected from a common source of anti-D immunoglobulin. Whereas virus sequences in the infectious source were relatively homogeneous, distinct HVR variants were observed in each anti-D recipient, indicating that this region can evolve in multiple directions from the same point. Where HVR variants with dissimilar sequences were present in a single individual, the frequency of synonymous substitution in the flanking regions suggested that the lineages diverged more than a decade previously. Even where a single major HVR variant was present in an infected individual, this lineage was usually several years old. Multiple lineages can therefore coexist during long periods of chronic infection without replacement. The characteristics of amino acid substitution in the HVR were not consistent with the random accumulation of mutations and imply that amino acid replacement in the HVR was strongly constrained. Another variable region of E2 centered on codon 60 shows similar constraints, while HVR2 was relatively unconstrained. Several of these features are difficult to explain if a neutralizing immune response against the HVR is the only selective force operating on E2. The impact of PCR artifacts such as nucleotide misincorporation and the shuffling of dissimilar templates is discussed.

Mellor J, Haydon G, Blair C, Livingstone W, Simmonds P. 1998. Low level or absent in vivo replication of hepatitis C virus and hepatitis G virus/GB virus C in peripheral blood mononuclear cells. J Gen Virol, 79 ( Pt 4) (4), pp. 705-714. | Show Abstract | Read more

To investigate which subsets of peripheral blood mononuclear cells (PBMCs) are susceptible to infection with hepatitis C virus (HCV) and hepatitis G virus (HGV) or GB virus C (GBV-C), a PCR-based assay using tagged primers in the core region (HCV) and NS3 region (HGV/GBV-C) for the specific detection of negative strand (replicating) viral RNA sequences was developed. In liver biopsy samples both positive and negative strands of HCV RNA were detected, at levels ranging from 3 to 11 x 10(6) RNA copies per 10(6) cells and 3.7-4.2 x 10(3) copies per 10(6) cells respectively, while lower frequencies of positive strands of GBV-C/HGV RNA were detected (from 13 biopsies, the highest frequency was 7.3 x 10(3) per 10(6) cells). In no samples were negative RNA strands detected. To investigate extra-hepatic replication of HCV and GBV-C/HGV, CD4+, CD8+ and B lymphocytes, monocytes and putative dendritic cell populations were separated from PBMCs from ten study subjects. Detection of positive strand HCV RNA was largely confined to B lymphocytes (at levels of up to 5 x 10(3) copies per 10(6) cells), while detection of negative strands was confined to a single subset (dendritic cells) of one of the study individuals. Similarly, GBV-C/HGV was detected at low levels in only twelve of twenty PBMC samples, while negative strands were uniformly absent. The low levels of HCV and GBV-C/HGV RNA in PBMCs suggest that these cells are at most a minor reservoir for virus replication. The absence of detectable replication of GBV-C/HGV suggests that the actual site of GBV-C/HGV replication remains to be discovered.

Haydon GH, Jarvis LM, Blair CS, Simmonds P, Harrison DJ, Simpson KJ, Hayes PC. 1998. Clinical significance of intrahepatic hepatitis C virus levels in patients with chronic HCV infection. Gut, 42 (4), pp. 570-575. | Show Abstract | Read more

BACKGROUND: The clinical significance of a single assessment of circulating hepatitis C virus (HCV) RNA and its relation to the level of intrahepatic HCV RNA remains unclear. AIMS: To investigate the relation between intrahepatic HCV levels and clinicopathological characteristics of chronic HCV infection. PATIENTS: Ninety eight consecutive patients with chronic HCV infection were studied; none had received alpha interferon therapy. Of these, 12 patients were repeatedly negative for HCV RNA in serum by reverse transcriptase polymerase chain reaction (RT-PCR). METHODS: After diagnostic laparoscopy and liver biopsy, semiquantitative analysis of intrahepatic HCV RNA levels was carried out by limiting dilution of HCV cDNA. HCV genotypes were assessed in 96 patients by restriction fragment length polymorphism analysis of HCV cDNA. RESULTS: Ten out of 12 patients who were RT-PCR negative for HCV RNA in serum were RT-PCR positive in liver; however, this group had a significantly lower intrahepatic HCV level and serum aminotransferase level than the remaining 86 patients. Histological severity (cirrhosis: n = 10); histological activity index; HCV genotype (genotype 1: n = 41; genotype 2: n = 12; genotype 3: n = 36; genotype 4: n = 7); mode of infection (intravenous drug abuse: n = 58; post-transfusion: n = 10; haemophiliac: n = 4; sporadic: n = 26) and alcohol abuse did not affect the intrahepatic virus level. There was no correlation between patient age, duration of infection, and intrahepatic HCV level. CONCLUSIONS: Intrahepatic virus levels were not determined by host factors (age of patient, mode or duration of infection) or by virus factors (HCV genotype). Repeatedly negative RT-PCR for HCV RNA in serum does not indicate absence of HCV from the liver.

Jarvis LM, Bell H, Simmonds P, Hawkins A, Hellum K, Harthug S, Maeland A, Ritland S, Myrvang B, Von Der Lippe B et al. 1998. The effect of treatment with alpha-interferon on hepatitis G GBV-C viraemia SCANDINAVIAN JOURNAL OF GASTROENTEROLOGY, 33 (2), pp. 195-200. | Show Abstract | Read more

Background. Hepatitis G virus (HGV) or GBV-C is frequently detected in patients co-infected with hepatitis C virus (HCV). This study investigated host and virologic factors influencing the response to HGV/GBV-C to α-interferon treatment. Methods: HGV/GBV-C was detected and quantified by nested polymerase chain reaction. The influence of variables such as liver biopsy appearance, liver function abnormalities, and response of HCV to interferon treatment was monitored. Results: Fourteen of the 25 HGV/GBV-C-infected patients treated with interferon (3-6 MIU three times a week for 6 months) became non-viraemic during treatment, although all relapsed after treatment withdrawal at 6 months, with no net change in virus load between 0 and 12 months. Conclusions: Predictive factors for clearance of HGV/GBV-C viraemia by interferon were pre-treatment severity of liver disease (median Knodell score of 4, compared with 7 for non-responders; P = 0.030) and alanine aminotransferase levels (median, 114, 182 for non-responders; P = 0.039). Clearance was associated with the treatment response of HCV. Nine of 13 who cleared HGV/GBV-C also cleared HCV, compared with 3 of 11 HGV/GBV-C non-responders; P= 0.05). The shared susceptibility of HGV/GBV-C and HCV to interferon treatment suggests a link between the mechanism of clearance of the two viruses.

Hanley JP, Jarvis LM, Hayes PC, Lee AJ, Simmonds P, Ludlam CA. 1998. Patterns of hepatitis G viraemia and liver disease in haemophiliacs previously exposed to non-virus inactivated coagulation factor concentrates. Thromb Haemost, 79 (2), pp. 291-295. | Show Abstract

Hepatitis G virus (HGV), a novel flavivirus, has been implicated as a cause of posttransfusion hepatitis. We have performed a longitudinal study in a cohort of haemophiliacs (n = 68) who previously received non-virus inactivated coagulation factor concentrates to assess both patterns of HGV viraemia and any associated liver disease. Hepatitis C virus (HCV) RNA was present in 58/68 and co-infection with human immunodeficiency virus (HIV) was present in 15/68. HGV RNA was detected in 17/68 (25%) samples from the mid-1980s. There was no association between either HIV infection (p = 0.74) or co-infection with a particular HCV genotype (p = 0.62). However, there was a relationship between HGV viraemia and the severity of haemophilia (p = 0.0004) with HGV RNA detected in 5/19, 9/16 and 3/32 patients with mild, moderate and severe haemophilia respectively. A longitudinal study was performed in 15/17 haemophiliacs with HGV viraemia using stored serum samples from the 1980s and 1990s. HGV viraemia persisted in 8/15 and cleared in 7/15 over a variable period of time. A Weibull model was constructed to estimate the duration of HGV viraemia in the study group. The 75th and 90th percentiles for the duration of HGV were estimated to be 8.7 years (95%, confidence interval 4.8-15.7) and 23.6 years (95% confidence interval 11.8-47.1) respectively. Laparoscopic liver inspection/biopsy was performed in 25/68. There was no association between severity of liver disease and HGV viraemia (p = 0.43). This study demonstrates considerable variation in patterns of HGV viraemia in haemophiliacs. We found little evidence to implicate HGV as a major cause of chronic liver disease in haemophiliacs.

Strappe PM, Wang TH, McKenzie CA, Lowrie S, Simmonds P, Bell JE. 1998. In situ polymerase chain reaction amplification of HIV-1 DNA in brain tissue. J Virol Methods, 70 (2), pp. 119-127. | Show Abstract | Read more

A direct in situ polymerase chain reaction (IS-PCR) assay is described for the detection of HIV-1 proviral DNA in formalin fixed paraffin embedded brain tissue. Biotin-16-dUTP is incorporated during the PCR process and microwave pretreatment of tissue sections ensures that no non-specific incorporation into damaged or nicked genomic DNA occurs. Two methods are compared to detect the biotinylated amplified product, the use of an avidin-biotin-alkaline phosphatase complex (ABC) and the application of tyramide signal amplification (TSA) which allows both chromogenic and fluorescence detection. TSA detection enhances the sensitivity of IS-PCR, permitting fewer PCR cycles and preserving tissue morphology.

Jarvis LM, Bell H, Simmonds P, Hawkins A, Hellum K, Harthug S, Maeland A, Ritland S, Myrvang B, von der Lippe B et al. 1998. The effect of treatment with alpha-interferon on hepatitis G/GBV-C viraemia. The CONSTRUCT Group. Scand J Gastroenterol, 33 (2), pp. 195-200. | Show Abstract

BACKGROUND: Hepatitis G virus (HGV) or GBV-C is frequently detected in patients co-infected with hepatitis C virus (HCV). This study investigated host and virologic factors influencing the response to HGV/GBV-C to alpha-interferon treatment. METHODS: HGV/GBV-C was detected and quantified by nested polymerase chain reaction. The influence of variables such as liver biopsy appearance, liver function abnormalities, and response of HCV to interferon treatment was monitored. RESULTS: Fourteen of the 25 HGV/GBV-C-infected patients treated with interferon (3-6 MIU three times a week for 6 months) became non-viraemic during treatment, although all relapsed after treatment withdrawal at 6 months, with no net change in virus load between 0 and 12 months. CONCLUSIONS: Predictive factors for clearance of HGV/GBV-C viraemia by interferon were pre-treatment severity of liver disease (median Knodell score of 4, compared with 7 for non-responders; P = 0.030) and alanine aminotransferase levels (median, 114, 182 for non-responders; P = 0.039). Clearance was associated with the treatment response of HCV. Nine of 13 who cleared HGV/GBV-C also cleared HCV, compared with 3 of 11 HGV/GBV-C non-responders; P = 0.05). The shared susceptibility of HGV/GBV-C and HCV to interferon treatment suggests a link between the mechanism of clearance of the two viruses.

Beddows S, Louisirirotchanakul S, Cheingsong-Popov R, Easterbrook PJ, Simmonds P, Weber J. 1998. Neutralization of primary and T-cell line adapted isolates of human immunodeficiency virus type 1: role of V3-specific antibodies. J Gen Virol, 79 ( Pt 1) (1), pp. 77-82. | Show Abstract | Read more

The role of the third variable domain (V3) of gp120 in the neutralization of primary and T-cell line adapted (TCLA) strains of human immunodeficiency virus type 1 (HIV-1) by serum from HIV-1-infected individuals was investigated. A primary virus isolate, M2424/4, when adapted to H9 cells, was more sensitive to neutralization on MT2 cells than the same stock passaged in PBMC. Neutralization of the PBMC-passaged stock by V3-specific MAbs was abrogated by addition of V3 (MN) peptide. However, exogenous V3 (MN) peptide failed to reduce the neutralization of this isolate on PBMC, or MT2 cells, by high titre anti-HIV-1 polyclonal human sera in contrast to the extensive reduction of neutralization by the same sera on MT2 cells using the prototype MN strain (4- to > or = 24-fold) and the TCLA M2424/H9 isolate (2- to 8-fold). These results indicate that the neutralization of primary virus isolates by serum from HIV-1-infected individuals is not significantly mediated by V3-specific antibodies.

Poovorawan Y, Theamboonlers A, Chongsrisawat V, Seksarn P, Jarvis L, Simmonds P. 1998. High prevalence of hepatitis G virus infection in multiply transfused children with thalassaemia. J Gastroenterol Hepatol, 13 (3), pp. 253-256. | Show Abstract | Read more

We investigated the prevalence of hepatitis G virus (HGV) RNA in relation to the frequency of blood transfusions in thalassaemic children and in volunteer blood donors in Thailand. Furthermore, we studied the frequency of coinfection with hepatitis B virus (HBV) and/or hepatitis C virus (HCV) as well as a possible relationship to the alanine aminotransferase (ALT) status of the blood samples, taken at random from thalassaemic children who have received multiple blood transfusions and from volunteer blood donors. The results show detectable HGV-RNA in 32.6% of transfusion patients and in 5% of blood donors. The prevalence of HGV-RNA peaked between the 11th and 50th transfusion. The relationship between HGV infection and ALT status was not statistically relevant.

Gardiner N, Livingstone W, Simmonds P, Lawler M, McCann SR, Browne PV. 1998. Absence of HHV8/KSHV DNA in peripheral blood and bone marrow harvests from patients with multiple myeloma BRITISH JOURNAL OF CANCER, 78 pp. 24-24.

Simmonds P. 1998. Variability of the hepatitis C virus genome. Curr Stud Hematol Blood Transfus, 62 (62), pp. 38-63.

Nordøy I, Krarup HB, Bell H, Christensen PB, Elgjo K, von der Lippe B, De Muckadell OS, Maeland A, Ring-Larsen H, Samdal HH et al. 1997. Interferon-alpha 2b therapy in low-activity hepatitis C: a pilot study. Scand J Gastroenterol, 32 (12), pp. 1256-1260. | Show Abstract | Read more

BACKGROUND: Many patients with chronic hepatitis C have long periods of normal or near-normal liver enzyme levels, even though histologic alterations have been confirmed. The recommendation today is not to treat this patient group. METHODS: In a pilot study 23 hepatitis C virus (HCV) RNA-positive patients with alanine aminotransferase (ALAT) levels less than 1.5 times upper normal limits for at least 6 months on more than three occasions and with histologic liver abnormalities compatible with chronic hepatitis C were treated with 3 MU of interferon-alpha 2b three times a week for 6 months. RESULTS: Nine patients (39%) became HCV RNA-negative in serum during treatment, but only two (8.7%) remained so after 6 months' follow-up. Significantly more patients with genotype other than type 1 became HCV RNA-negative than patients with genotype 1 during treatment (P = 0.005). CONCLUSIONS: Patients with low-activity chronic hepatitis C have a response to interferon-alpha treatment similar to that of patients with increased ALAT levels. Genotype seems to influence the rate of response.

Neville JA, Prescott LE, Bhattacherjee V, Adams N, Pike I, Rodgers B, El-Zayadi A, Hamid S, Dusheiko GM, Saeed AA et al. 1997. Antigenic variation of core, NS3, and NS5 proteins among genotypes of hepatitis C virus. J Clin Microbiol, 35 (12), pp. 3062-3070. | Show Abstract

Assays that detect antibody to hepatitis C virus (HCV) are used to screen blood donors and patients with hepatitis. Current enzyme-linked immunosorbent assay (ELISA)-based methods are invariably based upon antigens from expressed recombinant proteins or oligopeptides from HCV type 1. Some HCV antigens used in screening assays are coded by regions of the HCV genome that show extensive variability; therefore, HCV type 1-based assays may be less effective for the detection of antibody elicited by infection with other genotypes. In this study, we have measured antibody reactivity of sera from 110 hepatitis C patients infected with type 1b, 3a, or 4a to genotype-specific and cross-reactive epitopes present in recombinant proteins from HCV genotypes 1b (core, NS3, and NS5), 3a (NS3, NS5), and 4a (core, NS3), corresponding to those used in current third-generation screening ELISAs. By comparing the serological reactivities of sera to type-homologous and type-heterologous antigens, we detected a significant type-specific component to the reactivity to NS3 (61 to 77% of the total reactivity) and NS5 (60% of the total reactivity). Furthermore, despite the similarities in the amino acid sequences of the core antigens of type 1b and type 4a, we also found significantly greater reactivity to type-homologous antigens, with approximately 25% of reactivity being type specific. These findings are consistent with previous findings of fivefold weaker reactivity of sera from HCV type 2- and HCV type 3-infected blood donors in the currently used third-generation ELISAs and suggest that these assays are suboptimal for screening populations in which the predominant genotype is not type 1.

Irish DN, Rice PS, Simmonds P, Abbs IC, Burnapp L, Craske J, Macmahon EME. 1997. Novel Hepatitis C sub-type in a renal unit Clinical Infectious Diseases, 25 (2), pp. 373. | Show Abstract

Seroconversion for Hepatitis C virus (HCV) was detected on routine serological screening in a 75 year old haemodialysis (HD) patient. Serum collected three months previously was both seronegative and PCR negative. The man, DP, of Greek Cypriot origin, had been receiving HD for nine months. As he had received a blood transfusion six months previously, the blood donors were recalled for repeat HCV testing and were negative. No other risk factors for HCV were identified. To determine whether other patients had been infected, all 140 hospital HD patients were screened serologically for HCV infection at monthly intervals and amino transferase (ALT) levels measured weekly. HCV PCR was performed in patients with elevated ALT. No new cases of HCV infection were identified after six months follow-up. To identify a possible source of Hepatitis C infection for DP, genotyping was performed using PCR and restriction fragment length polymorphism (RFLP) analysis on sera from DP and the eleven HD patients previously known to have HCV infection. DP and two others (FJ and RK), both of Afro-Caribbean origin were found to be infected with type 2a. However sequence analysis of NS5 showed that all three patients were infected not with type 2a, but with a novel subtype tentatively called 2k. FJ was consistently dialysed on the same shift as DP and was therefore considered to be the more likely source of infection. Further sequence analysis is underway to establish whether transmission can be conclusively demonstrated.




Prescott LE, Berger A, Pawlotsky JM, Conjeevaram P, Pike I, Simmonds P. 1997. Sequence analysis of hepatitis C virus variants producing discrepant results with two different genotyping assays JOURNAL OF MEDICAL VIROLOGY, 53 (3), pp. 237-244. | Read more

Prescott LE, Berger A, Pawlotsky JM, Conjeevaram P, Pike I, Simmonds P. 1997. Sequence analysis of hepatitis C virus variants producing discrepant results with two different genotyping assays. J Med Virol, 53 (3), pp. 237-244. | Show Abstract | Read more

Methods for identifying the genotype of hepatitis C virus (HCV) in clinical specimens are frequently based upon the direct characterisation of viral RNA sequences by polymerase chain reaction (PCR) amplification, or by serologically based methods, in which the infecting genotype is inferred from the pattern of antibody reactivity to type-specific peptides or recombinant proteins used as antigens in an Enzyme Linked Immunosorbent Assay (ELISA). Although genotyping by direct, PCR-based methods show generally highly concordant results with the genotype inferred from serological typing assays (> 95% agreement), there exist a small number of samples that produce discrepant results. To investigate the underlying reasons for the discrepancies, we obtained eleven samples from haemophiliacs and four samples from patients with chronic hepatitis C that produced discordant results between a PCR based assay (InnoLipa I and II) and a serotyping assay (Murex HC02). Nucleotide sequences in the 5'noncoding region (5'NCR), core, and NS4 region were used to identify the genotype of the circulating virus and to identify amino acid changes in NS4 that might alter antigenicity. In 14 samples, sequence analysis of all three regions was concordant with the results of the InnoLipa assay. There were few if any amino acid substitutions in NS4 that might have accounted for the discrepant serotyping results, which were found predominantly in samples from individuals with a history of multiple exposure to HCV. It remains unclear whether the detection of antibody in such discrepant samples corresponds to previous expression of a different genotype than detected by PCR, or whether the virus population in plasma is more restricted in genotype diversity than the population in the liver or at other sites of viral replication.

Hughes ES, Bell JE, Simmonds P. 1997. Investigation of population diversity of human immunodeficiency virus type 1 in vivo by nucleotide sequencing and length polymorphism analysis of the V1/V2 hypervariable region of env. J Gen Virol, 78 ( Pt 11) (11), pp. 2871-2882. | Show Abstract | Read more

In this study we have analysed variability in the V1 and V2 regions of human immunodeficiency virus type 1 (HIV-1) proviral sequences amplified from lymphoid tissue, brain and other non-lymphoid tissue collected at autopsy from three HIV-1-infected individuals with giant cell encephalitis. We found no evidence for any tissue-specific grouping of variants in the V1/V2 regions, in contrast to previous comparisons of sequences in the V3 region, but consistent with the existence of evolutionarily distinct lineages previously observed in these study subjects by sequence comparisons of the p17gag gene. Examination of inferred amino acid sequences from V1 and V2 revealed no correlations between tissue origin with overall charge, length or number of glycosylation sites. Length polymorphism analysis is a rapid method to compare whole populations of HIV-1 variants within a sample, and provides information on the length and diversity of the V1 and V2 hypervariable regions. Based upon a comparison of 42 individuals with CD4 counts ranging from 802 to < 1 at time of death, we found no evidence for changes in the length of V2 with development of AIDS. Using the number of length variants in the V1 and V2 hypervariable region as a marker of the overall degree of variability within HIV populations, we found no evidence for an increase or a decrease in diversity between those with and without AIDS defining illness.

Willson RA, Yap PL, Fischer SH, Ochs HD, Simmonds P. 1997. Long-term (7 yrs) interferon alfa maintenance therapy, with biochemical and virologic response, failed to prevent progression of chronic hepatitis C infection acquired through contaminated intravenous gamma globulin in a patient with common variable immune deficiency HEPATOLOGY, 26 (4), pp. 1181-1181.

Bouali MR, Khediri MF, Hmida J, Hila A, Davidson F, Simmonds P. 1997. Genotypes of hepatitis C virus: A new subtype 4. HEPATOLOGY, 26 (4), pp. 332-332.

Haydon GH, Jarvis LM, Blair CS, Simmonds P, Harrison DJ, Hayes PC. 1997. Clinicopathological characteristics of subpopulations serum RT-PCR negative and serum RT-PCR positive for hepatitis C virus GUT, 41 pp. A126-A127.

Haydon GH, Jarvis LM, Blair CS, Simmonds P, Harrison DJ, Hayes PC. 1997. Clinicopathological characteristics of subpopulations serum RT-PCR negative and serum RT-PCR positive for hepatitis C virus (HCV) RNA. HEPATOLOGY, 26 (4), pp. 51-51.

Smith DB, Simmonds P. 1997. Characteristics of nucleotide substitution in the hepatitis C virus genome: constraints on sequence change in coding regions at both ends of the genome. J Mol Evol, 45 (3), pp. 238-246. | Show Abstract | Read more

Comparison of complete genome sequences for different variants of hepatitis C virus (HCV) reveals several different constraints on sequence change. Synonymous changes are suppressed in coding regions at both 5' and 3' ends of the genome. No evidence was found for the existence of alternative reading frames or for a lower mutation frequency in these regions. Instead, suppression may be due to constraints imposed by RNA secondary structures identified within the core and NS5b genes. Nonsynonymous substitutions are less frequent than synonymous ones except in the hypervariable region of E2 and, to a lesser extent, in E1, NS2, and NS5b. Transitions are more frequent than transversions, particularly at the third position of codons where the bias is 16:1. In addition, nucleotide substitutions may not occur symmetrically since there is a bias toward G or C at the third position of codons, while T left and right arrow C transitions were twice as frequent as A left and right arrow G transitions. These different biases do not affect the phylogenetic analysis of HCV variants but need to be taken into account in interpreting sequence change in longitudinal studies.

Wyld R, Robertson JR, Brettle RP, Mellor J, Prescott L, Simmonds P. 1997. Absence of hepatitis C virus transmission but frequent transmission of HIV-1 from sexual contact with doubly-infected individuals. J Infect, 35 (2), pp. 163-166. | Show Abstract | Read more

Hepatitis C virus (HCV) is transmitted through infected blood and blood products, but evidence of other routes of transmission is less clearly understood. In a study designed to examine human immunodeficiency virus (HIV) transmission, the prevalence of HCV has also been measured. Sixty-one couples were analysed, 30 in which partners were at risk through sexual contact alone, of whom 12 (40%) became infected with HIV and none with HCV. Thirty-one partners were exposed sexually and additionally through intravenous drug use. Of these, 16 (52%) became infected with HIV and 25 (80%) contracted HCV infection. These findings support the evidence of others that HCV is only rarely transmitted by sexual intercourse in heterosexual relationships and that HIV is not a co-factor for HCV transmission.

Strappe PM, Wang TH, McKenzie CA, Lowrie S, Simmonds P, Bell JE. 1997. Enhancement of immunohistochemical detection of HIV-1 p24 antigen in brain by tyramide signal amplification. J Virol Methods, 67 (1), pp. 103-112. | Show Abstract | Read more

Human immunodeficiency virus type 1 (HIV-1) infection of the brain has been demonstrated in formalin fixed, paraffin embedded post-mortem brain tissue (PM) by chromogenic immunohistochemistry for the HIV p24 antigen. The sensitivity of antigen detection is increased significantly by tyramide signal amplification (TSA) compared to the conventional peroxidase labelled Avidin-Biotin complex (ABC) technique. The TSA method also permitted the use of a lower concentration of primary antibody than is conventionally used. Sensitivity was enhanced further by microwave irradiation of the paraffin embedded tissues in citrate buffer. HIV-1 p24 antigen was also detected in PM brain tissue by TSA enhanced immunofluorescence and demonstrated increased sensitivity compared to the conventional immunofluorescence technique with a greatly reduced autofluorescence background.

Chamberlain RW, Adams NJ, Taylor LA, Simmonds P, Elliott RM. 1997. The complete coding sequence of hepatitis C virus genotype 5a, the predominant genotype in South Africa. Biochem Biophys Res Commun, 236 (1), pp. 44-49. | Show Abstract | Read more

Hepatitis C virus (HCV) genotype 5a is the predominant genotype in southern Africa with a high prevalence amongst infected blood donors from areas in South Africa. We have determined the nucleotide sequence corresponding to the complete coding region of an HCV isolate, EUH1480, previously classified as genotype 5a, from an Edinburgh haemophiliac. The sequence contained a single open reading frame (ORF) coding for a polyprotein of 3014 amino acids. Comparison with the polyprotein sequences from other HCV genotypes, where the ORF varies from 3008 to 3037 amino acids, showed the observed variation in size was due to differences in lengths of the envelope 2 and the nonstructural 5A proteins. The sequence divergence of HCV genotype 5 ranged from 29.4% nucleotide differences (24.91% amino acid differences) compared with genotype 1c to 32.5% nucleotide differences (30.3% amino acid differences) compared with 2a. Phylogenetic analysis of the available full length nucleotide sequences showed EUH1480 to form a branch distinct from the other HCV types, confirming the classification of type 5a as a separate genotype.

Pawlotsky JM, Prescott L, Simmonds P, Pellet C, Laurent-Puig P, Labonne C, Darthuy F, Remire J, Duval J, Buffet C et al. 1997. Serological determination of hepatitis C virus genotype: comparison with a standardized genotyping assay. J Clin Microbiol, 35 (7), pp. 1734-1739. | Show Abstract

In patients with chronic hepatitis C, determination of hepatitis C virus (HCV) genotype could be routinely run in the future to tailor treatment schedules. The suitabilities of two versions of a serological, so-called serotyping assay (Murex HCV Serotyping Assay version 1-3 [SA1-3] and Murex HCV Serotyping Assay version 1-6 [SA1-6]; Murex Diagnostics Ltd.), based on the detection of genotype-specific antibodies directed to epitopes encoded by the NS4 region of the genome, for the routine determination of HCV genotypes were studied. The results were compared with those of a molecular biology-based genotyping method (HCV Line Probe Assay [INNO-LiPA HCV]; Innogenetics S.A.), based on hybridization of PCR products onto genotype-specific probes designed in the 5' noncoding region of the genome, obtained with pretreatment serum samples from 88 patients with chronic hepatitis C eligible for interferon therapy. Definitive genotyping was performed by sequence analysis of three regions of the viral genome in all samples with discrepant typing results found among at least two of the three assays studied. In all instances, sequence analysis confirmed the result of the INNO-LiPA HCV test. The sensitivity of SA1-3 was 75% relative to the results obtained by the genotyping assay. The results were concordant with those of genotyping for 92% of the samples typeable by SA1-3. The sensitivity of SA1-6 was 89% relative to the results obtained by the genotyping assay. The results were concordant with those of genotyping for 94% of the samples typeable by SA1-6. Overall, SA1-6 had increased sensitivity relative to SA1-3 but remained less sensitive than the genotyping assay on the basis of PCR amplification of HCV RNA. Cross-reactivities between different HCV genotypes could be responsible for the mistyping of 8 (SA1-3) and 6% (SA1-6) of the samples. Subtyping of 1a and 1b is still not possible with the existing peptides, but discriminating between subtypes may not be necessary for routine use.

Dittmar MT, Simmons G, Donaldson Y, Simmonds P, Clapham PR, Schulz TF, Weiss RA. 1997. Biological characterization of human immunodeficiency virus type 1 clones derived from different organs of an AIDS patient by long-range PCR. J Virol, 71 (7), pp. 5140-5147. | Show Abstract

In order to characterize the biological properties of human immunodeficiency virus type 1 (HIV-1) variants from different tissues (peripheral blood mononuclear cells [PBMC], lymph node, spleen, brain, and lung) of one patient, we have chosen long-range PCR to amplify virtually full-length HIV proviruses and to construct replication-competent viruses by adding a patient-specific 5' long terminal repeat. To avoid selection during propagation in CD4+ target cells, we transfected 293 cells and used the supernatants from these cells as challenge viruses for tropism studies after titration on human PBMC. Despite differences in the V3 loop of the major variants found in brain and lung compared to lymphoid tissues all recombinant HIV clones obtained showed identical cell tropism and replicative kinetics. After infection of human PBMC these viruses replicated with similar kinetics, with a slow/low-titer, non-syncytium-inducing phenotype. In contrast to the prediction of macrophage tropism, drawn from the V3 loop sequence, none of these viruses infected monocyte-derived macrophages. The challenge of blood dendritic cells by these recombinant viruses in the presence of tumor necrosis factor alpha, granulocyte-macrophage colony-stimulating factor, and interleukin-4 resulted in a productive infection only after adding stimulated CD4+ T lymphocytes. Therefore, the biological properties of the HIV-1 variants derived from nonlymphoid tissue of this patient did not differ from those of HIV-1 variants from lymphoid tissue with respect to tropism for primary cells such as PBMC, macrophages, and blood dendritic cells.

Smith DB, McAllister J, Casino C, Simmonds P. 1997. Virus 'quasispecies': making a mountain out of a molehill? J Gen Virol, 78 ( Pt 7) (7), pp. 1511-1519. | Read more

Smith DB, Cuceanu N, Davidson F, Jarvis LM, Mokili JL, Hamid S, Ludlam CA, Simmonds P. 1997. Discrimination of hepatitis G virus/GBV-C geographical variants by analysis of the 5' non-coding region. J Gen Virol, 78 ( Pt 7) (7), pp. 1533-1542. | Show Abstract | Read more

We have investigated the ability of different subgenomic fragments to reproduce the phylogenetic relationships observed between six complete genome sequences of GBV-C/hepatitis G virus (HGV). While similar relationships were observed following analysis of part of the 5' non-coding region (5'NCR), for the coding region they were not accurately reproduced for some large fragments or for the majority of fragments of 300 or 600 nucleotides. Analysis of 5'NCR sequences from a large number of isolates, including newly obtained sequences from Pakistan, Zaïre and Scotland, produced separate groupings of Asian, African and European/North American variants. These groupings are associated with specific polymorphisms in the 5'NCR, many of which were covariant and consistent with a proposed secondary structure for this region. The relatively low level of amino acid sequence variation observed between these geographically and phylogenetically defined groups of variants suggests that they are unlikely to display significant biological differences.

Smith DB, Simmonds P. 1997. Review: molecular epidemiology of hepatitis C virus. J Gastroenterol Hepatol, 12 (7), pp. 522-527. | Show Abstract | Read more

Molecular techniques have been used to investigate the epidemiology of hepatitis C virus (HCV) at several different levels. At a global level, the time of divergence of the diverse HCV genotypes isolated from different geographical regions has been estimated from the rate of divergence observed among a cohort of individuals infected from a common source. Estimates of more than 300 years for virus subtypes and more than 500-2000 years for virus types are consistent with their current geographical distributions. Analysis of virus sequences has also provided evidence for a common source of infection in several large-scale outbreaks of HCV infection, although where there is evidence that the implicated source contains more than one variant it may be difficult to distinguish individuals infected by different sources. Finally, sequence analysis has been used to investigate the vertical or horizontal transmission of HCV between pairs of individuals. The hypervariable region of the E2 gene is the most informative region to study if samples are available soon after the transmission event, but evidence for more distant events can still be obtained from analysis of genes such as NS5b and E1. Interpretation of some studies is complicated by the conservation of the gene region studied, or by the failure to make comparisons with sequences from epidemiologically unrelated viruses.

Chamberlain RW, Adams N, Saeed AA, Simmonds P, Elliott RM. 1997. Complete nucleotide sequence of a type 4 hepatitis C virus variant, the predominant genotype in the Middle East. J Gen Virol, 78 ( Pt 6) (6), pp. 1341-1347. | Show Abstract | Read more

Hepatitis C virus (HCV) type 4 is the predominant genotype found throughout the Middle East and parts of Africa, often in association with high population prevalence as in Egypt. To investigate more fully its evolutionary relationship with other genotypes of HCV, and to study its overall genome organization, we have determined the entire sequence encompassing the coding region of the genotype 4a isolate ED43, obtained from an HCV-infected individual from Egypt. The sequence of ED43 contained a single open reading frame encoding a polyprotein of 3008 amino acids (aa), smaller than that reported for other HCV genotypes which vary from 3010 aa to 3037 aa. The nucleotide and amino acid sequences were compared with the full-length sequences already reported for genotypes 1a, 1b, 1c, 2a, 2b, 2c, 3a, 3b and those of isolates JKO49 and JKO46 described as types 10a and 11a. The differences in length of the polyprotein originated in variable regions in the E2 and NS5A genes. The complete sequence of ED43 confirmed the classification of type 4 as a separate major genotype.

Hanley JP, Jarvis L, Hayes PC, Simmonds P, Ludlam CA. 1997. Hepatitis G virus infection in haemophiliacs: Patterns of viraemia and severity of liver disease THROMBOSIS AND HAEMOSTASIS, pp. P1843-P1843.

Adams NJ, Chamberlain RW, Taylor LA, Davidson F, Lin CK, Elliott RM, Simmonds P. 1997. Complete coding sequence of hepatitis C virus genotype 6a. Biochem Biophys Res Commun, 234 (2), pp. 393-396. | Show Abstract | Read more

Hepatitis C virus (HCV) genotype 6a is found in a restricted part of South East Asia, including Hong Kong, Macau and Vietnam. We determined the full length coding sequence of a type 6a isolate (EUHK2) obtained from a Hong Kong blood donor. The sequence of EUHK2 contained a single open reading frame coding for a polyprotein of 3018 amino acids, within the range of 3008 to 3037 for other HCV genotypes. The full length sequence of EUHK2 showed 30.3%-32.9% nucleotide (24.3%-29.4% amino acid) sequence divergence from genotypes 1-4, but only 27.7% (20.7% amino acid) divergence from JK046 ("type 11a"). These similarity values were intermediate between those of other HCV genotypes (minimum 28.4%) and between subtypes (maximum 25%). The close evolutionary relationship of EUHK2 with JK046 was further indicated by their grouping together by phylogenetic analysis.

Bell H, Hellum K, Harthug S, Maeland A, Ritland S, Myrvang B, von der Lippe B, Raknerud N, Skaug K, Gutigard BG et al. 1997. Genotype, viral load and age as independent predictors of treatment outcome of interferon-alpha 2a treatment in patients with chronic hepatitis C. Construct group. Scand J Infect Dis, 29 (1), pp. 17-22. | Show Abstract | Read more

Patients with chronic hepatitis C respond differently when treated with interferon. We randomized 116 patients with chronic hepatitis C in order to compare two dosage regimens of recombinant interferon alpha 2a:3 MIU x 3 per week for 6 months (arm A) or 6 MIU x 3 per week for 3 months and then 3 MIU x 3 per week for 3 months (arm B). There were no significant differences concerning outcome between the two dose regimens: sustained clearance of HCV viremia 6 months after the end of treatment was obtained in 12/59 (20%) in group A compared with 18/57 (32%) in group B (p = 0.24). In patients with genotype 1a, 4/31 (13%), in genotype 1b, none of 9 (0%), 9/15 (60%) in genotype 2, and 17/58 (29%) in genotype 3, showed sustained clearance of HCV viremia 6 months after the end of treatment (p = 0.002). In a stepwise logistic regression analysis, only pretreatment viral load (p = 0.0001), genotype (p = 0.001) and age (p = 0.04) were identified as independent predictors of sustained clearance of HCV viremia. Liver histology as assessed by Knodell index was significantly improved in patients with sustained HCV RNA response 6 months after the end of treatment (5.2 +/- 2.2 vs 2.6 +/- 2.2, p < 0.001), but not in responders with relapse or in non-responders. In conclusion, stepwise logistic regression analysis showed that viral load, HCV genotype and age were the only independent predictors for sustained HCV RNA response.

Bell H, Simmonds P, Hellum K, Harthug S, Maeland A, Ritland S, Myrvang B, vonderLippe B, Skaug K, Raknerud N et al. 1997. No sustained HGV RNA response in patients with hepatitis G virus GBV-C infection when treated with interferon alfa. GASTROENTEROLOGY, 112 (4), pp. A1224-A1224.

Simmonds P. 1997. Clinical relevance of hepatitis C virus genotypes. Gut, 40 (3), pp. 291-293. | Read more

Dittmar MT, McKnight A, Simmons G, Clapham PR, Weiss RA, Simmonds P. 1997. HIV-1 tropism and co-receptor use. Nature, 385 (6616), pp. 495-496. | Read more

McLaughlin KJ, Cameron SO, Good T, McCruden E, Ferguson JC, Davidson F, Simmonds P, Mactier RA, McMillan MA. 1997. Nosocomial transmission of hepatitis C virus within a British dialysis centre. Nephrol Dial Transplant, 12 (2), pp. 304-309. | Show Abstract | Read more

Patients on renal replacement therapy are recognized as a group at increased risk of infection with hepatitis C virus (HCV). While the risk has been reduced by the use of erythropoietin for treatment of anaemia and the introduction of HCV screening of blood products and potential renal transplant donors, new cases of HCV are still being documented, with patients on hospital haemodialysis appearing to be particularly at risk. The exact mode of transmission of HCV within dialysis units is unclear, although there is evidence to support nosocomial transmission between patients. Third generation HCV antibody testing was performed on all dialysis patients when a new case of HCV was identified within our unit. Stored monthly serum samples were then examined retrospectively to determine when patients became HCV RNA and HCV antibody positive. Viral typing was carried out to identify the HCV strains responsible for transmission. Four new cases of HCV infection are described within a single dialysis shift. Viral typing identified two distinct strains of HCV as being responsible for these infections, both of which had previously been identified in dialysis patients within the unit known to have HCV infection. This information, taken in conjunction with knowledge of the location of each patient for dialysis, suggests two separate episodes of nosocomial transmission of HCV between haemodialysis patients. While evidence of nosocomial transmission of HCV is accumulating, with modern dialytic procedures evidence of transmission through the dialysis machine or equipment used for dialysis is lacking. This stresses the importance of strict applications of universal precautions as the key to prevention of further transmission of HCV infection. This information is obviously applicable not only to dialysis units but all units that may potentially come in contact with HCV patients.

Hughes ES, Bell JE, Simmonds P. 1997. Investigation of the dynamics of the spread of human immunodeficiency virus to brain and other tissues by evolutionary analysis of sequences from the p17gag and env genes. J Virol, 71 (2), pp. 1272-1280. | Show Abstract

The time of spread of human immunodeficiency virus type 1 (HIV-1) from lymphoid to nonlymphoid tissues in the course of infection was investigated by sequence comparisons of variants infecting a range of lymphoid and nonlymphoid tissues from three individuals with AIDS in the pl7gag gene and regions flanking the V1/V2 hypervariable regions. Phylogenetic analysis in both regions revealed several lineages in each individual that contained sequences from both lymphoid and nonlymphoid tissues such as the brain. This observation contrasts strongly with the previously described organ-specific sequences in the V3 region in this study population and other investigations. Although individual pairwise comparisons of relatively short sequences such as p17gag are subject to considerable stochastic error, we found that the diversity of gag sequences in variants from lymphoid tissue was consistently lower than that found among variants amplified from the brain. By estimating mean synonymous pairwise distances in the p17gag region, we were able to make an approximate calculation of the ages of populations in different tissues. Those from lymphoid tissue ranged from 2.65 to 5.6 years in the three study subjects, compared with 4.1 to 6.2 years for variants in the brain. Indeed, variants infecting the brain were no more closely related to each other than they were to variants infecting other tissues in the body. In two of the three individuals, these times of divergence indicate that infection of the brain may have occurred as an early event in the progression to disease, preceding the onset of AIDS by several years. This study is the first in which it was possible to estimate times of diversification in different tissues in vivo and is of importance in understanding the dynamics of the spread of HIV-1 into nonlymphoid tissues and its possible adaptation for replication in different cell types.

Smith DB, Pathirana S, Davidson F, Lawlor E, Power J, Yap PL, Simmonds P. 1997. The origin of hepatitis C virus genotypes. J Gen Virol, 78 ( Pt 2) (2), pp. 321-328. | Show Abstract | Read more

For many RNA viruses, relatively recent times of origin of extant viruses are implied by the high rate of substitution observed in longitudinal studies. However, extrapolation of short-term rates of substitution can give misleading estimates of times of divergence. We show here that the common ancestor of different types of hepatitis C virus (HCV) is older than previously thought. The rate of HCV sequence change was measured amongst a cohort of individuals infected following administration of anti-D immunoglobulin. Virus sequences were obtained in the E1 and NS5B genes and compared with each other and with sequences from an infective batch. Taking account of the bias towards synonymous transition substitutions, the time of divergence of variants of subtype 1b is estimated to have occurred 70-80 years ago. The numerous subtypes of HCV are proposed to derive from more than 300 years of endemic infection in certain geographical regions, with recent spread of some subtypes to other parts of the world. Estimation of the time of origin of the major HCV genotypes (types 1-6) is problematic, but our data and analogy with other viruses suggest that divergence occurred at least 500-2000 years ago.

Shah HA, Jafri W, Malik I, Prescott L, Simmonds P. 1997. Hepatitis C virus (HCV) genotypes and chronic liver disease in Pakistan. J Gastroenterol Hepatol, 12 (11), pp. 758-761. | Show Abstract | Read more

UNLABELLED: Hepatitis C virus (HCV) is classified into different types depending on nucleotide sequence variability. Detailed information on the distribution of various HCV genotypes in some geographical areas is available but little is known about Pakistan. In this study, a 5' non-coding region (NCR)-based restriction fragment length polymorphism (RFLP) genotyping assay was used to investigate the genotype distribution in a large series of HCV-infected patients in Karachi, Pakistan. Serum samples from 74 hepatitis B surface antigen (HBsAg)-negative patients with a clinical diagnosis of chronic liver disease (60 patients) and hepatocellular carcinoma (HCC) (14 patients) were assayed for anti-HCV antibody by second generation enzyme immunoassay and 48 were confirmed anti-HCV-positive (33 males, 15 females). Other causes of chronic liver disease (e.g. haemochromatosis, Wilson's disease and immune-mediated injury) were ruled out. Liver biopsy was done in 27/48 anti-HCV-positive patients and in all HCC patients. Genotypes were determined for 45/48 anti-HCV-positive study patients; 39/45 (87%) were type 3; four (9%) were type 1; one was type 2; and one was type 5. Past blood transfusion was the main identifiable risk factor found in 10 patients, all type 3. Seven of the 14 HCC patients were anti-HCV positive, (six were type 3). Most patients with hepatitis C presented with established cirrhosis and complications of portal hypertension and liver failure. IN CONCLUSION: (i) genotype 3 is the most common isolate in HCV-associated chronic liver disease in Pakistan; (ii) a significant proportion of HBsAg-negative cirrhotics are non-B, non-C in aetiology; and (iii) half of the patients with HCC have serological evidence of HCV infection.

Hawkins A, Davidson F, Simmonds P. 1997. Comparison of plasma virus loads among individuals infected with hepatitis C virus (HCV) genotypes 1, 2, and 3 by quantiplex HCV RNA assay versions 1 and 2, Roche Monitor assay, and an in-house limiting dilution method. J Clin Microbiol, 35 (1), pp. 187-192. | Show Abstract

The accuracy of different methods for the quantitation of hepatitis C virus in plasma was measured with samples from individuals infected with different genotypes and by using RNA transcripts of predetermined concentrations. Highly reproducible results were observed upon repeat testing of samples by both the original version of the Chiron branched-DNA (bDNA) assay (Quantiplex RNA assay; bDNA-1) and the currently available version (Quantiplex HCV RNA 2.0 assay; bDNA-2). A greater variability was observed in the Roche Monitor assay (correlation coefficient of 0.537, compared with 0.942 and 0.964 for the bDNA-1 and bDNA-2 assays, respectively). Significant differences in the efficiency of detection of genotypes 1, 2, and 3 were observed for the bDNA-1 and Roche Monitor assays, whereas the bDNA-2 assay and nested PCR at limiting dilution were able to quantify genotypes with equal sensitivity. By quantifying RNA transcripts of different genotypes, the sensitivities of the Roche Monitor assay for sequences of the type 2 and type 3 transcripts were estimated to be 11 and 8% of those achieved for genotype 1. When correction factors based upon these results and those from quantitation of circulating viral RNA sequences in samples from blood donors were used, the genotype-specific differences in virus load in samples from blood donors were no longer observed, consistent with previous studies with corrected values from the bDNA-1 assay. These results suggest that many of the previous studies evaluating the effect of genotype and virus load on the response to interferon using methods such as the Roche Monitor assay and other competitive PCR methods require reinterpretation. Differences in efficiency of quantitation should be taken into account in future investigations of the relationship between genotype and virus load.

Haydon GH, Jarvis LM, Simpson KJ, Hayes PC, Simmonds P. 1997. The clinical significance of the detection of hepatitis GBV-C RNA in the serum of patients with fulminant, presumed viral, hepatitis. J Viral Hepat, 4 (1), pp. 45-49. | Show Abstract | Read more

In a significant number of cases of fulminant (presumed viral) hepatitis worldwide, no aetiological agent has been identified. Recently, it has been suggested that a newly described flavivirus, GBV-C, is responsible for some of these cases. This study aimed to assess the clinical significance of GBV-C RNA, demonstrated by reverse transcriptase-polymerase chain reaction (RT-PCR), in the serum of patients with fulminant non-A to E hepatitis. Twenty-three consecutive cases of non-A to E fulminant hepatitis were included in the study. GBV-C RNA was reverse transcribed and amplified using two RT-PCR based detection methods. Medical records were examined to assess clinical history, duration and mode of infection, transfusion history, liver histology and clinical outcome. Five (three female, two male; mean age 21.2 years) of 23 patients had GBV-C RNA detected in their serum by RT-PCR: all five patients were RT-PCR positive following amplification by primers specific for the 5' non-coding region (NCR), whilst four were positive by primers for the NS3 region. Prior to the onset of illness, two patients had risk factors for transmission of an infectious agent; however, all five patients had been transfused during their illness, prior to testing for GBV-C. Of these, two (of two in whom serum was available) were negative for GBV-C after the onset of fulminant hepatitis but before their first transfusion. This study does not support the hypothesis that the detection of hepatitis G virus (HGV)/GBV-C RNA in the serum of patients with fulminant hepatitis indicates a causal association. However, it does demonstrate that a careful transfusion history and screening of blood products is vital before the importance of GBV-C in the aetiology of fulminant hepatitis can be established.

Simmonds P, Smith DB. 1997. Investigation of the pattern of diversity of hepatitis C virus in relation to times of transmission. J Viral Hepat, 4 Suppl 1 (s1), pp. 69-74. | Show Abstract | Read more

The rate of sequence change of HCV in vivo was used to date the spread of HCV genotype 1b in European, USA and Japanese populations. Silent substitution rates of 0.0011 and 0.0017 substitutions per site per year were observed in the NS5 and E1 regions by sequence comparisons from a cohort of individuals infected from a common source of infection 17 years previously. Mean silent substitution frequencies of 0.169 and 0.224 in NS5 and E1, respectively, were observed amongst type 1b variants infecting epidemiologically unrelated individuals from several countries. This predicted a time of divergence from a common ancestor of 64-69 years. The absence of country-specific groupings by phylogenetic analysis of these sequences suggested that the spread of this genotype occurred on a worldwide basis at a similar time. Dates for the spread of other genotypes varied from around 80 years (type 2a) to 54 years (type 3a), suggesting that different variants spread into communities at different times this century. By extrapolating the silent substitution rate, the time of divergence of type 1a from 1b can be estimated at 200-300 years, but even this is likely to be an underestimate of the true time due to inequalities on the transition and transversion ratios, and the greater frequency of G <--> A transitions, compared with C <--> U, which are not considered in the current analysis. Thus, the diversity of the globally distributed genotypes such as type 1b and 3a suggests a relatively recent origin for HCV in Western countries and Japan, and contrast with the much greater diversity of specific genotypes in Central Africa (type 4) and South East Asia (type 6). These data may assist in understanding the global epidemiology of HCV and the mechanisms by which it has spread.

Haydon GH, Jarvis LM, Simmonds P, Harrison DJ, Garden OJ, Hayes PC. 1997. Association between chronic hepatitis C infection and hepatocellular carcinoma in a Scottish population. Gut, 40 (1), pp. 128-132. | Show Abstract | Read more

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most common cancers in the world. The geographical prevalence varies considerably in different countries and Scotland is regarded as an area of low risk for the disease. AIMS: To assess the association between chronic hepatitis C infection (HCV) and HCC in a population of patients presenting to a single hospital. PATIENTS: One hundred and fourteen cases of histologically confirmed liver cancer presenting to the Royal Infirmary of Edinburgh between 1985 and 1994 were examined. METHODS: Of 114 cases of HCC, 80 samples of stored sera were available. Samples positive for HCV Ab were genotyped by restriction fragment length polymorphism analysis of HCV c-DNA. A population of 29 cirrhotic patients (diagnosed between 1985 and 1994) with chronic HCV infection was also genotyped. RESULTS: Chronic HCV infection was a major risk factor (30% of tested HCC patients) identified. HCV genotype 1b was predominant (16 of 20 patients). The time from HCV transmission to development of cancer ranged from 10 to 50 years (median 30). In the cirrhotic patient population, a broader distribution of genotypes was present (genotype 1a: 7; genotype 1b: 8; genotype 2b: 3; genotype 3a: 8 and genotype 4: 2). However, this population was significantly younger. (Mean (SD) 52 (14.5) years) (p = 0.0002) and demonstrated a significantly shorter duration of infection: range 10-40 years (median: 19). CONCLUSION: There is a strong association between chronic HCV infection, cirrhosis, and hepatocarcinogenesis in this Scottish population. The study was unable to distinguish whether the high prevalence of genotype 1b in the HCC population reflected increased oncogenicity in itself, or whether 1b was simply the most prevalent genotype in Scotland when these patients were infected.

Jarvis LM, Davidson F, Hanley JP, Yap PL, Ludlam CA, Simmonds P. 1996. Infection with hepatitis G virus among recipients of plasma products. Lancet, 348 (9038), pp. 1352-1355. | Show Abstract | Read more

BACKGROUND: Hepatitis G virus (HGV or GBV-C) is a newly discovered human flavivirus distantly related to hepatitis C virus (HCV). Little information is available on its natural history or routes of transmission, although it can be transmitted parenterally. We investigated the prevalence of persistent infection of HGV and HCV in patients exposed to non-virus-inactivated pooled blood products associated with transmission of HCV. METHODS: RNA was extracted from the plasma of 112 patients with haemophilia and 57 with hypogammaglobulinaemia, as well as from 64 different batches of archived coagulation-factor concentrates and immunoglobulins. RNA was reverse transcribed and amplified with primers from the 5' non-coding region of HCV and HGV by a nested polymerase chain reaction (PCR). Viral RNA was quantified by titration of complementary DNA before amplification. FINDINGS: Among non-renumerated UK blood donors HGV infection (detected by PCR) was more common than HCV infection (four [3.2%] of 125 compared with 137 [0.076%] of 180658 in southeast Scotland). Testing of batches of factor VIII and factor IX concentrates prepared without viral inactivation procedures showed high frequencies of contamination with HGV (16 of 17 factor VIII batches positive; six of six factor IX batches positive), with no difference between renumerated and non-renumerated donors. However, among 95 haemophiliacs who had received non-virus-inactivated concentrates, 13 (14%) were positive for HGV compared with 79 (83%) who were positive for HCV. Two of 37 recipients of long-term immunoglobulin replacement therapy were positive for HGV. Virus inactivation of blood products substantially reduced or eliminated contamination by HGV RNA sequences. INTERPRETATION: Despite the extremely high level of HGV contamination of non-virus-inactivated blood products, their use was not associated with high rates of persistent infection in recipients. The infectivity of HGV in blood products may be lower than that of HCV, or the virus may be less able to establish persistent infection in humans. Whatever the case, the high prevalence of active HGV infection in the general population remains difficult to explain.

Ahmed MM, Mutimer DJ, Elias E, Linin J, Garrido M, Hubscher S, Jarvis L, Simmonds P, Wilde JT. 1996. A combined management protocol for patients with coagulation disorders infected with hepatitis C virus. Br J Haematol, 95 (2), pp. 383-388. | Show Abstract | Read more

The case notes of 394 adults with bleeding disorders registered at our centre together with those of the 72 patients who had died since 1971 were reviewed. 36/72 deceased patients had evidence of HCV infection. Liver decompensation was present at time of death in six. 274 (70%) of the currently registered patients had received factor concentrate or cryoprecipitate and 174 of these were screened for HCV infection. 76% of tested patients were RIBA positive. 87% of RIBA-positive patients were RT-PCR positive. 50 RIBA-positive patients, including nine who were HIV infected, have undergone percutaneous liver biopsy following appropriate factor infusion with no complication. The biopsy was assessed using a Histological Activity Index (HAI) ranging from 0 to 13. Patients with HAI > or = 6 were offered treatment with interferon. Patients with HAI < 6 were followed up with a view to re-biopsy in 2-3 years to assess progression. The median HAI was 4.5 (range 0-10) with HAI > or = 6 in 13 cases (27%). HAI was not correlated with duration of infection. haemophilia severity. RT-PCR status. HIV status or HCV genotype. Liver biopsy, a safe procedure in our hands, is an important investigation in HCV-infected patients to assess suitability for interferon therapy.




Zhou S, Terrault NA, Ferrell L, Hahn JA, Lau JYN, Simmonds P, Roberts JP, Lake JR, Ascher NL, Wright TL. 1996. Severity of liver disease in liver transplantation recipients with hepatitis C virus infection: Relationship to genotype and level of viremia Hepatology, 24 (5), pp. 1041-1046. | Show Abstract | Read more

Infection with hepatitis C virus (HCV) genotype 1b has been reported to be associated with more severe posttransplantation liver disease than infection with non-1b genotypes. To address this issue, we evaluated the outcome in 124 patients who underwent liver transplantation for chronic HCV infection. The HCV genotype and/or serotype responsible for infection was determined by four different methods. HCV RNA was detected in serum samples by polymerase chain reaction (PCR) amplification, and quantified by branched DNA assay. Disease severity was expressed as a histological score (which included grading of portal inflammation, lobular activity, fibrosis, and cytopathic changes). Median duration of histological follow-up was 25 months (range 1-75 months). Genotype was assignable in 112 (92.5%) patients. Genotypes responsible for infection were as follows: 1a = 32.2%, 1b = 27.3%, 2a = 7.4%, 2b = 8.3%, 3a = 14%, and mixed infection (more than one subtype) 3.3%. Level of viremia, alanine aminotransferase (ALT), aspartate aminotransferase (AST), bilirubin, and total histological score were not significantly different in patients infected with type 1b compared with patients infected with other genotypes. While duration of histological follow-up was greater in patients infected with type lb versus other types (P = .02), by univariate and multivariate analysis neither HCV genotype (1b versus others), level of viremia nor duration of histological follow-up were associated with disease severity. Moreover, there was no significant difference in the actuarial graft survival in patients infected with type lb compared with that of patients infected with non-1b types (82% and 87% at 3 years, respectively). Reanalysis using HCV genotype 1 showed no association with disease severity, graft survival, and patient survival. We conclude that HCV genotype 1 and subtype 1b are not associated with disease severity or graft survival in liver transplantation recipients.

Healey CJ, Chapel H, Simmonds P, Chapman RWG. 1996. Hepatitis G virus in intravenous immunoglobulin - Reply GASTROENTEROLOGY, 111 (5), pp. 1399-1400.

Zhou S, Terrault NA, Ferrell L, Hahn JA, Lau JY, Simmonds P, Roberts JP, Lake JR, Ascher NL, Wright TL. 1996. Severity of liver disease in liver transplantation recipients with hepatitis C virus infection: relationship to genotype and level of viremia. Hepatology, 24 (5), pp. 1041-1046. | Show Abstract | Read more

Infection with hepatitis C virus (HCV) genotype 1b has been reported to be associated with more severe posttransplantation liver disease than infection with non-1b genotypes. To address this issue, we evaluated the outcome in 124 patients who underwent liver transplantation for chronic HCV infection. The HCV genotype and/or serotype responsible for infection was determined by four different methods. HCV RNA was detected in serum samples by polymerase chain reaction (PCR) amplification, and quantified by branched DNA assay. Disease severity was expressed as a histological score (which included grading of portal inflammation, lobular activity, fibrosis, and cytopathic changes). Median duration of histological follow-up was 25 months (range 1-75 months). Genotype was assignable in 112 (92.5%) patients. Genotypes responsible for infection were as follows: 1a = 32.2%, 1b = 27.3%, 2a = 7.4%, 2b = 8.3%, 3a = 14%, and mixed infection (more than one subtype) = 3.3%. Level of viremia, alanine aminotransferase (ALT), aspartate aminotransferase (AST), bilirubin, and total histological score were not significantly different in patients infected with type 1b compared with patients infected with other genotypes. While duration of histological follow-up was greater in patients infected with type lb versus other types (P = .02), by univariate and multivariate analysis neither HCV genotype (lb versus others), level of viremia nor duration of histological follow-up were associated with disease severity. Moreover, there was no significant difference in the actuarial graft survival in patients infected with type lb compared with that of patients infected with non-lb types (82% and 87% at 3 years, respectively). Reanalysis using HCV genotype 1 showed no association with disease severity, graft survival, and patient survival. We conclude that HCV genotype 1 and subtype 1b are not associated with disease severity or graft survival in liver transplantation recipients.

Simpson GR, Schulz TF, Whitby D, Cook PM, Boshoff C, Rainbow L, Howard MR, Gao SJ, Bohenzky RA, Simmonds P et al. 1996. Prevalence of Kaposi's sarcoma associated herpesvirus infection measured by antibodies to recombinant capsid protein and latent immunofluorescence antigen. Lancet, 348 (9035), pp. 1133-1138. | Show Abstract | Read more

BACKGROUND: Kaposi's sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus 8, may be the infectious cause of KS. Its prevalence in the general population, on the basis of detection of the virus genome, is controversial. To investigate the seroprevalence, we measured antibodies to a recombinant capsid-related (lytic cycle) KSHV antigen and a latent antigen complex. METHODS: We selected potentially immunoreactive capsid-related proteins of KSHV by expressing them as recombinant proteins and testing them in western blot assays. We used a truncated recombinant protein encoded by KSHV open reading frame 65 (orf 65) to develop a diagnostic enzyme-linked immunosorbent assay (ELISA) and tested sera from HIV-infected individuals with KS, HIV-uninfected patients with "classic" KS, other HIV risk groups, and blood donors. We also compared the antibody response to this capsid-related protein to the response to latent antigen(s) in an immunofluorescence assay. FINDINGS: 77/92 (84%) sera from KS patients reacted with the KSHV orf 65 protein and 84/103 (81.5%) reacted with KSHV latent antigen(s). The dominant immunogenic region of orf 65 is within the carboxyterminal 80 aminoacids, a region with little sequence similarity to the related Epstein-Barr virus, suggesting that orf 65 is a KSHV specific antigen. Only three sera from patients with haemophilia (1/84) or from intravenous drug users (2/63) had KSHV specific antibodies in the orf 65 assay whereas none of these sera reacted with latent antigen. Antibodies to KSHV were also infrequently found in UK and US blood donors by either assay (UK, 3/174 with orf 65 and 4/150 with latent antigen; US, 6/117 with orf 65 and 0/117 with latent antigen). They were more common among HIV-infected gay men without KS (5/16 by orf 65 ELISA, 10/33 by IFA), HIV-uninfected STD clinic attenders (14/166 by IFA), and Ugandan HIV-uninfected controls (6/17 by orf 65 ELISA, 9/17 by IFA). Antibody reactivity to the orf 65 protein (ELISA) and to latent antigen(s) (IFA) was concordant in 89% of 462 sera tested but reactive blood donor sera were discordant in both assays. Four AIDS-KS sera were unreactive in both assays. INTERPRETATION: The distribution of antibodies to both a capsid-related recombinant protein and latent antigen(s) of KSHV strongly supports the view that infection with this virus is largely confined to individuals with, or at increased risk for, KS. However, infection with KSHV does occur, rarely, in the general UK and US population and is more common in Uganda. Antibodies to latent antigen(s) or to orf 65 encoded capsid protein will not detect all cases of KSHV infection, and a combination of several antigens will probably be required for accurate screening and confirmatory assays.

Bell H, Hellum K, Harthug S, Maeland A, Ritland S, Myrvang B, von der Lippe B, Raknerud N, Skaug K, Prescott L, Simmonds P. 1996. Prevalence of hepatitis C genotypes among patients with chronic hepatitis C in Norway. Construct Group. Scand J Infect Dis, 28 (4), pp. 357-359. | Show Abstract | Read more

Among 116 patients with biopsy-confirmed chronic hepatitis C (Riba 2 or Riba 3 positive) in a multicenter study in southern Norway on interferon, we determined hepatitis C virus genotype by restriction fragment length polymorphism (RFLP) of the 5' NCR. The RFLP method was supplemented by and compared with a serological typing method based on the detection of type-specific antibody to peptide from the NS-4 region. A total of 102/106 (96%) patient sera showed detectable type-specific antibody to NS-4 peptides and corresponded in all cases, except two, to the genotype detected by polymerase chain reaction. Combining the results from RFLP genotyping and serotyping, genotype 1 was found in 40 (35%) (27 with 1a and 10 with 1b, 3 subtypes not determined), genotype 2 in 15 (13%) (subtype 2b in 14 and 1 subtype not determined), and genotype 3 in 58 (50%) of patients. The low mean age of the patients (34 years), the low prevalence of cirrhosis (3.5%), the short duration of the disease, and a high prevalence of intravenous-drug abusers may account for the low prevalence of infection with genotype 1b (9%). The epidemiological features of hepatitis C patients are markedly different from patient groups described in southern Europe in terms of risk factors, age, and genotype distribution.

Forns X, Maluenda MD, López-Labrador FX, Ampurdanès S, Olmedo E, Costa J, Simmonds P, Sánchez-Tapias JM, Jimenez De Anta MT, Rodés J. 1996. Comparative study of three methods for genotyping hepatitis C virus strains in samples from Spanish patients. J Clin Microbiol, 34 (10), pp. 2516-2521. | Show Abstract

Hepatitis C virus (HCV) genotypes may be investigated by a variety of laboratory methods that target different parts of the HCV genome and have various degrees of technical difficulty. Since the choice of a particular method is difficult, we compared the performance of (i) a type-specific PCR with type-specific primers from the core region, (ii) molecular hybridization of the PCR-amplified 5' noncoding region to type-specific probes, and (iii) identification of type-specific antibodies against epitopes of nonstructural region 4 by enzyme-linked immunosorbent assay (ELISA). One hundred fifty-one patients with biopsy-proved chronic hepatitis and HCV RNA in serum were investigated. The HCV genotype was identified in 99%, 100%, and 85% of the cases by type-specific PCR, probe hybridization, and ELISA, respectively. The type-specific PCR disclosed infection with type 1a in 3%, type 1b in 74%, and type 3a in 4% of the cases and suggested infection with two or more HCV types, including 2a/2c and 2b, in the remaining 18%. Apparently mixed infections were more prevalent in patients with past intravenous drug use (P < 0.001), but cloning and sequencing of PCR products did not confirm a mixed infection in any of the four cases investigated. Concordant results were obtained by the three procedures with virtually all of the samples in which the type-specific PCR revealed a single HCV genotype. Type-specific hybridization and ELISA usually recognized the genotype producing the strongest DNA band in samples in which type-specific PCR suggested a mixed infection. In conclusion, the three procedures evaluated in this study are reliable for investigation of HCV genotype. Type-specific PCR provides information about HCV subtypes, but a mixed infection detected with this method should be interpreted with caution.

Ahmed MM, Mutimer DJ, Elias E, Jarvis L, Simmonds P, Martin B, Garrido M, Hubscher S, Linin J, Wilde JT. 1996. Histological severity and hepatitis C (HCV) viraemia and genotype in patients with bleeding disorders. HEPATOLOGY, 24 (4), pp. 1066-1066.

Hamid S, Jafri W, Khurshid M, Jarvis L, Shah H, Abbas Z, Sultana T, Siddiqui AA, Khan H, Simmonds P. 1996. Hepatitis G in the developing world: Impact of HGV on liver disease in Pakistan. HEPATOLOGY, 24 (4), pp. 1576-1576.

Prescott LE, Simmonds P, Lai CL, Chan NK, Pike I, Yap PL, Lin CK. 1996. Detection and clinical features of hepatitis C virus type 6 infections in blood donors from Hong Kong. J Med Virol, 50 (2), pp. 168-175. | Show Abstract | Read more

The genotype distribution of hepatitis C virus (HCV) was investigated in 212 viraemic blood donors from Hong Kong. A subset of the samples was investigated using three different genotyping assays to establish the accuracy of each in this population. These assays were restriction fragment length polymorphism (RFLP) of amplified 5' noncoding region (5'NCR) sequences, RFLP of the core region, and a serotyping assay using peptides from two antigenic regions of NS4. Genotypes detected in Hong Kong blood donors were 1a (6.2%), 1b (58.8%), 2a (1.4%), 2b (1.4%), 3a (1.9%), and 6a (27.0%). All genotyping assays produced concordant results. No evidence was obtained for the presence of type 6 group variants recently identified in Southeast Asia, other than type 6a. A serotyping assay based upon the detection of type-specific antibody to epitopes in NS4 produced similar results to the genotyping assays (98% concordance), but a reduced sensitivity (75%) compared with genotyping methods. Sequence variation in NS4 was not the cause of the reduced rate of detection of type 6 antibody in this population. Eighty-four percent donors infected with type 6a were male, compared to 75% donors infected with type 1b. The median alanine transaminase (ALT) level in type 6 infected donors was lower than in type 1b, (43.8 and 51.1 U/l, respectively) although these values were not statistically significant (P = 0.094). There was no significant difference between the ages of donors infected with types 1b and 6a. Risk factors for HCV infection in the blood donors included blood transfusion, intravenous drug abuse, and tattooing. A significantly greater number of donors infected with HCV-6a reported a history of drug abuse (66%) than donors infected with HCV-1b (7%).

Watson HG, Ludlam CA, Rebus S, Peutherer JF, Simmonds P. 1996. Hepatitis B serology and DNA detection in multitransfused haemophiliacs and factor VIII and IX concentrates. Haemophilia, 2 (4), pp. 229-234. | Show Abstract | Read more

To assess the effect of HIV infection and the introduction of virus-inactivated concentrates, we conducted a retrospective 20-year longitudinal study of hepatitis B virus (HBV) serology and look for HBV DNA in recent serum samples of 63 multiply transfused haemophiliacs. Of 63 haemophiliacs, 51 had evidence of previous HBV infection and 12 vaccinees had anti-HBs only. Of 40 HIV-negative, two had persistent HBsAg but all were HBV DNA negative. All 23 HIV-positive were HBsAg-negative. Loss of anti-HBc(46% vs. 17.5%) and anti-HBs (32% vs. 14%) was more commonly seen in HIV-infected compared with noninfected individuals. One HIV-positive individual had HBV DNA detectable by PCR. Restrospective testing demonstrated that re-emergence was associated with loss of anti-HBs and advanced HIV infection (CD4<50 × 10(6-1) L CDC II), although eight other with CDC IV disease were HBV DNA negative. Forty-three batches of concentrates produced between 1965 and 1992 from both commercial and volunteer donors and subjected to different donor screening and virus inactivation methods were negative for HBV DNA. Some of these may have been infectious for HBV and therefore being negative for HBV may not equate with noninfectivity. We conclude that both HIV-positive and -negative haemophiliacs have lost protective antibodies against HBV since 1984 and that virus replication may re-emerge at least in the HIV-positive group. These observations may have implications for the management of their chronic liver disease and the risk of infection of sexual partners and medical attendants.

Haydon GH, Jarvis LM, Simmonds P, Simpson KJ, Hayes PC. 1996. Clinical significance of the detection of GBV-C virus in the serum of patients with acute liver failure of unknown aetiology. HEPATOLOGY, 24 (4), pp. 664-664.

Jarvis LM, Davidson F, Hanley JP, Ludlam CA, Simmonds P. 1996. Low frequency of persistent infection with HGV or GBV-C in haemophiliacs and other plasma product users. HEPATOLOGY, 24 (4), pp. 649-649.

Bell JE, Lowrie S, Graham J, Simmonds P. 1996. The Edinburgh HIV Brain and Organ Bank NEUROPATHOLOGY AND APPLIED NEUROBIOLOGY, 22 (5), pp. 448-448.

Livingstone WJ, Moore M, Innes D, Bell JE, Simmonds P. 1996. Frequent infection of peripheral blood CD8-positive T-lymphocytes with HIV-1. Edinburgh Heterosexual Transmission Study Group. Lancet, 348 (9028), pp. 649-654. | Show Abstract

BACKGROUND: Although lymphocytes expressing the CD4 surface receptor for HIV-1 have been identified as the principal target of the virus, the extent to which infection of other cell types of the immune system contributes to immunodeficiency is unknown. We investigated the cell types in peripheral blood infected with HIV and the relation of viral load in different subsets to disease progression. METHODS: The study group consisted of 16 HIV-infected individuals, eight of whom had clinically defined AIDS with CD4 cell counts less than 200/microL blood. The main component subsets of peripheral blood mononuclear cells were purified by magnetic bead separation, and included CD4 and CD8 lymphocytes, B lymphocytes, monocytes, and dendritic cells. HIV proviral sequences within these separate populations were quantified by limiting-dilution nested polymerase chain reaction. FINDINGS: HIV-1 proviral sequences were detected in T-helper cells, cytotoxic T cells, dendritic cells, and monocytes. CD4 T lymphocytes constituted the main reservoir of HIV in all but one of the symptom-free individuals studied (those with CD4 count > 200/microL). However, in all the individuals with CD4 counts of less than 200/microL, most infected cells within the peripheral blood mononuclear cell fraction were either dendritic cells or CD8 lymphocytes. Infection of CD8 cells accounted for between 66% and 97% of total proviral load in five of the eight AIDS patients. A strong inverse relation between total CD8 count and the frequency of CD8 T-lymphocyte infection was found. INTERPRETATION: This study provides evidence for widespread infection of lymphocytes of the CD8 phenotype, indicating that HIV-1 has a broader tropism for different cell types in vivo than described for cultured virus. Infection of CD8 cells may contribute to the decline of this subset upon disease progression in HIV-infected individuals. Infection of CD8 cells may or may not occur by a non-CD4-dependent mechanism of virus entry.




Livingstone WJ, Moore M, Innes D, Bell JE, Simmonds P, Whitelaw J, Wyld R, Robertson JR, Brettle RP. 1996. Frequent infection of peripheral blood CD8-positive T-lymphocytes with HIV-1 LANCET, 348 (9028), pp. 649-654. | Show Abstract | Read more

Background Although lymphocytes expressing the CD4 surface receptor for HIV-1 have been identified as the principal target of the virus, the extent to which infection of other cell types of the immune system contributes to immunodeficiency is unknown. We investigated the cell types in peripheral blood infected with HIV and the relation of viral load in different subsets to disease progression. Methods The study group consisted of 16 HIV-infected individuals, eight of whom had clinically defined AIDS with CD4 cell counts less than 200/μL blood. The main component subsets of peripheral blood mononuclear cells were purified by magnetic bead separation, and included CD4 and CD8 lymphocytes, B lymphocytes, monocytes, and dendritic cells. HIV proviral sequences within these separate populations were quantified by limiting-dilution nested polymerase chain reaction. Findings HIV-1 proviral sequences were detected in T-helper cells, cytotoxic T cells, dendritic cells, and monocytes. CD4 T lymphocytes constituted the main reservoir of HIV in all but one of the symptom-free individuals studied (those with CD4 count 200/μL). However, in all the individuals with CD4 counts of less than 200/μL, most infected cells within the peripheral blood mononuclear cell fraction were either dendritic cells or CD8 lymphocytes. Infection of CD8 cells accounted for between 66% and 97% of total proviral load in five of the eight AIDS patients. A strong inverse relation between total CD8 count and the frequency of CD8 T-lymphocyte infection was found. Interpretation This study provides evidence for widespread infection of lymphocytes of the CD8 phenotype, indicating that HIV-1 has a broader tropism for different cell types in vivo than described for cultured virus. Infection of CD8 cells may contribute to the decline of this subset upon disease progression in HIV-infected individuals. Infection of CD8 cells may or may not occur by a non-CD4-dependent mechanism of virus entry.

el-Zayadi A, Simmonds P, Dabbous H, Prescott L, Selim O, Ahdy A. 1996. Response to interferon-alpha of Egyptian patients infected with hepatitis C virus genotype 4. J Viral Hepat, 3 (5), pp. 261-264. | Show Abstract | Read more

Hepatitis C virus (HCV) genotype 4 is the principal HCV genotype found in Egypt and the Middle East. Little is known concerning its propensity to cause disease and the frequency with which infected individuals respond to interferon-alpha (IFN-alpha). We have investigated the response to treatment in a cohort of 100 chronic hepatitis C patients infected with genotype 4. All patients had biopsy-proven chronic active liver disease. Each was treated with 3 million units (MU) IFN-alpha, thrice weekly. Response was monitored, in 92 patients who completed treatment, by alanine aminotransferase (ALT) measurements and by polymerase chain reaction (PCR) for HCV. ALT levels remained abnormal in 64 patients during treatment (69.6%). Of the 28 patients who showed a biochemical response during treatment (30.4%), 18 maintained this over the 6-month posttreatment period. Amongst the sustained biochemical responders, HCV RNA was cleared from serum in only four of the 18 (22.2%) in this period. Histological improvement was observed in 26/51 (50.9%) of the patients who had a second biopsy. Hence, patients infected with HCV genotype 4 show a poor response to IFN-alpha therapy compared with genotypes 2 and 3, but a similar response to IFN-alpha compared with those infected with type 1b HCV. These findings have major implications for treatment strategies in the Middle East, including Egypt, where HCV genotype 4 is widely distributed.

Hanley JP, Jarvis LM, Simmonds P, Ludlam CA. 1996. Development of anti-interferon antibodies and breakthrough hepatitis during treatment for HCV infection in haemophiliacs. Br J Haematol, 94 (3), pp. 551-556. | Show Abstract | Read more

The development of anti-interferon antibodies may lead to treatment failure during interferon therapy. We have studied the development of such antibodies in a group of 39 haemophiliacs receiving interferon-alpha 2a for chronic hepatitis C virus (HCV) infection. Anti-interferon antibodies developed in five (13%) patients and were associated with "breakthrough hepatitis' in three cases. There was an association between the development of anti-interferon antibodies and infection with HCV genotype 3a (P = 0.01). This study suggests that the development of anti-interferon antibodies may lead to treatment failure in a proportion of haemophiliacs with HCV infection. The association with genotype 3a has not previously been reported. Monitoring for the development of breakthrough hepatitis due to anti-interferon antibodies may provide the opportunity to develop strategies to overcome their effects.

Echevarría JM, León P, Domingo CJ, López JA, Elola C, Madurga M, Salmerón F, Yap PL, Daub J, Simmonds P. 1996. Laboratory diagnosis and molecular epidemiology of an outbreak of hepatitis C virus infection among recipients of human intravenous immunoglobulin in Spain. Transfusion, 36 (8), pp. 725-730. | Show Abstract | Read more

BACKGROUND: Passive transfer of antibody to hepatitis C virus (HCV) has been thought to occur after infusion of human intravenous immunoglobulin (IVIG), as anti-HCV and/or HCV RNA was commonly found in that product. Recently, however, HCV RNA was detected in the serum of recipients of IVIG. Establishment of a causal relationship between IVIG therapy and HCV infection in recipients was attempted. STUDY DESIGN AND METHODS: Anti-HCV and HCV RNA sequences were investigated in serum samples from 39 persons who received a human IVIG product in seven different hospitals in Spain. HCV RNA was also investigated in two batches of the IVIG shared by some recipients. All the viral RNA detected were characterized with a line probe assay, restriction fragment length polymorphism analysis of the 5'-noncoding and core regions, and sequencing of the nonstructural 5 region. RESULTS: On the basis of both clinical and laboratory data, a relationship could be established between the IVIG therapy and the acquisition of the HCV infection by the recipients. Several HCV strains were detected among the recipients, with most of the recipients coming from the same hospital presenting with closely related strains. Moreover, an HCV strain almost identical to the main strain detected among the recipients was found in one batch of the IVIG that probably was shared by most of them. Follow-up studies and evaluation of low-avidity anti-HCV IgG suggested that both acute primary infections and reinfections were produced. In one case, direct evidence of reinfection by a different HCV strain was obtained. CONCLUSION: The results did not exclude the possibility that a second HCV strain associated with a further, unidentified batch of the IVIG could have contributed to this outbreak.

Romeo R, Colombo M, Rumi M, Soffredini R, Del Ninno E, Donato MF, Russo A, Simmonds P. 1996. Lack of association between type of hepatitis C virus, serum load and severity of liver disease. J Viral Hepat, 3 (4), pp. 183-190. | Show Abstract | Read more

Chronic infection with the hepatitis C virus (HCV) may lead to a variety of hepatic lesions from benign inflammation to liver cancer, but the relationships between infection and development of liver disease are poorly understood. To assess whether virus type and load are of pathogenetic importance, 197 Italian carriers with various hepatic lesions were investigated consecutively. Of these, 187 (95%) patients had serum HCV RNA, by reverse transcription-polymerase chain reaction (RT-PCR) with a median level of 1003 x 10(3) genomic equivalents ml-1 according to the branched-DNA assay (b-DNA). One hundred and seven patients (54%) had serotype 1, 22 (11%) had serotype 2, 9 (5%) had serotype 3, 17 (9%) had mixed serotypes and 42 (21%) had no specified serotype. One hundred and thirty four patients were also tested for genotype. The genotype distribution was as follows: 17 (13%) had genotype 1a; 67 (50%) 1b; 29 (22%) 2a; 12 (9%) 3a; 3 (2%) had genotype 1 not classified (NC); 3 (2%) had genotype 2 NC; 2 (1.4%) had genotype 4 and 1 (1%) had mixed genotype 1a + 3a. No virus type was associated with any particular histological diagnosis and all were equally distributed between progressive and non-progressive liver disease groups. Serum HCV-RNA levels were similar in the liver diseased groups. By analogy to hepatitis B, there was no direct correlation between type and level of viraemia and the severity of the underlying liver damage.

Hanley JP, Jarvis LM, Andrews J, Dennis R, Lee R, Simmonds P, Piris J, Hayes P, Ludlam CA. 1996. Investigation of chronic hepatitis C infection in individuals with haemophilia: assessment of invasive and non-invasive methods. Br J Haematol, 94 (1), pp. 159-165. | Show Abstract | Read more

Hepatitis C virus (HCV) infection is the major cause of chronic liver disease in individuals with haemophilia. A wide spectrum of disease severity is found in this group, ranging from mild hepatitis to cirrhosis. We have studied a cohort of 87 anti-HCV positive haemophiliacs who have been infected with HCV for 10-25 years and assessed the relative value of invasive and non-invasive methods of evaluating liver disease. The severity of liver disease was assessed using ultrasound scan (n = 77), upper GI endoscopy (n = 50), laparoscopic liver inspection (n = 33) and liver biopsy (n = 22). Invasive investigations were performed without any significant bleeding complications. Evidence of severe liver disease was found in approximately 25% of patients. There was agreement between the severity of liver histology and the information derived from the laparoscopic liver inspection, endoscopy and ultrasound in 86%. Co-infection with HIV was significantly associated with more severe liver disease (P = 0.006). This study provides further evidence that liver disease is emerging as a major complication in haemophiliacs and severe liver disease is more common in those co-infected with HIV. We have shown the potential value of laparoscopic liver inspection, in combination with endoscopy and ultrasound, in staging the extent of liver disease, and suggest that most patients may be managed without resorting to liver biopsy.

Dow BC, Buchanan I, Munro H, Follett EA, Davidson F, Prescott LE, Yap PL, Simmonds P. 1996. Relevance of RIBA-3 supplementary test to HCV PCR positivity and genotypes for HCV confirmation of blood donors. J Med Virol, 49 (2), pp. 132-136. | Show Abstract | Read more

HCV antibody screening of 624,910 blood donations resulted in 3,832 samples being referred for confirmation. All were tested by RIBA-3 with 2,710 negative, 945 indeterminate and 177 positive results. HCV RNA was detected by PCR in an average of 69.5% of RIBA-3 positives (4 bands 84.1%; 3 bands 74.1%; 2 bands 34.1%) and only 0.53% of RIBA-3 indeterminates. Eighty-four percent of samples with a total RIBA-3 band intensity score (maximum 16) of > or = 8 were PCR positive compared with only 22% of those with a score of < 8. Total mean band intensities for HCV genotype 1 samples (n = 65) were 13.2, genotype 2 (n = 17) 11.4 and genotype 3 (n = 65) 11.2 with type 1 samples showing greater reactivity with c100 and c33 antibodies. No PCR positive type 1 samples were found with RIBA-3 total band scores less than 8, no PCR positive type 2 samples less than 6, whilst PCR positive type 3 samples were found with scores as low as 2. NS5 indeterminates were the most common (40.2%) single band pattern but yielded no PCR positive samples, followed by c33 (23.3%) with one PCR positive and c100 (20.2%) with one PCR positive whilst c22 indeterminates were least common (16.3%) but included three PCR positive donors. All five RIBA-3 indeterminate PCR positive donors were type 3.

Dow BC, Munro H, Buchanan I, Follett EA, Davidson F, Yap PL, Simmonds P. 1996. Third-generation recombinant immunoblot assay: comparison of reactivities according to hepatitis C virus genotype. Transfusion, 36 (6), pp. 547-551. | Show Abstract | Read more

BACKGROUND: Recombinant immunoblot assay (RIBA) is widely used as a supplemental test in hepatitis C virus (HCV) confirmatory algorithms. As this assay is based on HCV type 1, its performance was examined with the common European HCV genotypes (1, 2, and 3). STUDY DESIGN AND METHODS: A study was performed to retest in third-generation RIBA (RIBA-3) all 146 second-generation RIBA (RIBA-2)-positive polymerase chain reaction-positive samples detected by second-generation enzyme-linked immunosorbent assays and having known HCV genotypes (74 HCV type 1, 21 type 2, 51 type 3). RIBA band intensities were examined according to HCV genotype. An additional 90 RIBA-3-confirmed PCR-positive samples (47 HCV type 1, 5 type 2, 38 type 3) detected by third-generation enzyme-linked immunosorbent assays were also examined. RESULTS: In the first group of 146 samples, the RIBA-3 NS4 (c100p) band showed a marked improvement in sensitivity for the detection of HCV types 2 and 3 over that of the c100 antigen of RIBA-2, but the mean band intensities of HCV types 2 and 3 remained significantly lower than those of type 1. Improved sensitivity of the NS3 band of RIBA-3 to HCV type 3 was also apparent, but, again, the mean band intensity measured was lower for type 3 than for either type 1 or type 2. The c22 band of RIBA-2 and RIBA-3 exhibited equal sensitivity for all HCV genotypes. These differences were also apparent when RIBA-3 was used in conjunction with third-generation enzyme-linked immunosorbent assays. CONCLUSION: The current RIBA-3 lacks sensitivity to the NS4 antibody for HCV types 2 and 3. The incorporation of type-specific components to other genotypes for NS4 (and NS3) antigens should be considered by the manufacturers.

Hanley J, Jarvis L, Simmonds P, Parker A, Ludlam C. 1996. HCV and non-Hodgkin lymphoma. Lancet, 347 (9011), pp. 1339. | Read more




Lau JYN, Davis GL, Prescott LE, Maertens G, Lindsay KL, Qian KP, Mizokami M, Simmonds P, Perrillo RP, Schiff ER et al. 1996. Distribution of hepatitis C virus genotypes determined by line probe assay in patients with chronic hepatitis C seen at tertiary referral centers in the United States ANNALS OF INTERNAL MEDICINE, 124 (10), pp. 868-&. | Show Abstract

Objective: To 1) verify the validity of a new line probe assay for hepatitis C virus (HCV) genotyping and 2) determine the distribution of HCV genotypes and the association between HCV genotype and clinical variables in patients with chronic hepatitis C seen in tertiary referral centers in the United States. Design: Retrospective cross-sectional analysis. Patients: 438 patients with chronic hepatitis C from 10 tertiary referral centers. Measurements: The validity of the line probe assay was first verified against a panel of serum specimens that had previously been characterized by six different HCV genotyping methods. Specimens from all 438 patients were then genotyped using this line probe assay. The associations between HCV genotype and clinical variables were examined using analysis of variance. Pairwise testing was used when the F test showed a statistically significant difference. Nonparametric alternatives were used for variables for which normality could not be assumed. Results: The line probe assay was quick and reproducible, and it showed good concordance with other tests. In our sample, the proportions of patients with HCV types 1, 2, 3, and 4 were 71.5%, 13.5%, 5.5%, and 1.1%, respectively. Subtypes 1a and 1b were seen in approximately equal proportions of patients with HCV type 1. Mixed infection was detected in 3.7% of specimens, and 4.8% of specimens either had negative results on polymerase chain reaction or could not be typed. A higher proportion of patients with HCV type 1 than of patients with HCV-type 1 had acquired HCV through transfusion of blood products (50% compared with 25%; P < 0.001). Patients with HCV type 1 also had a longer estimated duration of infection compared with patients with HCV type 3 (P = 0.004) and type 4 (P = 0.049). Disease activity did not differ among patients infected with HCV types 1, 2, or 3. Levels of viremia were similar in patients with HCV types 1, 2, or 3, but patients with HCV type 4 had a lower level of viremia than did patients with HCV type 1 (P = 0.047). Conclusions: The line probe assay can be used in patients with chronic HCV infection in the United States. In patients with chronic hepatitis C referred to tertiary centers in the United States, type 1 is the most common HCV genotype. Disease activity and viremia levels do not differ among patients chronically infected with HCV types 1, 2, or 3.

Bell JE, Donaldson YK, Lowrie S, McKenzie CA, Elton RA, Chiswick A, Brettle RP, Ironside JW, Simmonds P. 1996. Influence of risk group and zidovudine therapy on the development of HIV encephalitis and cognitive impairment in AIDS patients. AIDS, 10 (5), pp. 493-499. | Show Abstract | Read more

OBJECTIVE: To determine the associations between HIV encephalitis and other central nervous system (CNS) pathology, viral burden, cognitive impairment, zidovudine therapy and risk group in AIDS patients. DESIGN: Planned autopsy study in AIDS patients evaluated prospectively for numerous clinical parameters. SETTING: Regional academic centre for clinical care and pathology examination of patients with HIV infection. PATIENTS: Edinburgh cohort of HIV-positive patients prospectively assessed for cognitive impairment, immunosuppression and clinical course. Unbiased series of consecutive autopsies in 27 homosexual men and 39 drug-using patients with AIDS. INTERVENTIONS: Zidovudine therapy monitored in all patients. MAIN OUTCOME MEASURES: Determination of CNS viral burden and pathology including immunocytochemically confirmed HIV encephalitis in injecting drug users (IDU) versus homosexual AIDS patients with known CD4 counts and cognitive function. RESULTS: HIV encephalitis was present in 59% of IDU and 15% of homosexuals: 88% of patients with encephalitis had displayed cognitive impairment. HIV encephalitis was strongly associated with a high viral load and HIV p24 immunopositivity. Opportunistic infections and lymphomas were more common in homosexuals (63%) than in IDU (31%) and were associated with the degree of immunosuppression before death. Within both groups, prolonged zidovudine treatment was associated with a lower incidence of HIV encephalitis. CONCLUSIONS: This study documents two separate CNS outcomes in AIDS patients in that HIV encephalitis occurs independently of opportunistic infections and lymphomas and shows different associations with risk group, immunosuppression and antiviral treatment before death.

Simmonds P, Mellor J, Craxi A, Sanchez-Tapias JM, Alberti A, Prieto J, Colombo M, Rumi MG, Lo Iacano O, Ampurdanes-Mingall S et al. 1996. Epidemiological, clinical and therapeutic associations of hepatitis C types in western European patients. J Hepatol, 24 (5), pp. 517-524. | Show Abstract | Read more

BACKGROUND/AIMS: Different variants of hepatitis C virus might show different susceptibility to interferon alpha treatment, but it is important to understand whether this difference in sensitivity reflects an association with other factors, such as cirrhosis or age. METHODS: We have used an enzyme-linked immunosorbent hepatitis C virus typing assay based upon the detection of antibody in patient's era to type-specific NS-4 antigens to investigate the effect of hepatitis C virus type in 610 patients with chronic hepatitis C virus infection. The influence of viral types and their interdependency with host factors were separately analyzed to establish which factors executed an independent effect on the probability of sustained response. RESULTS: There was a marked difference in the distribution of hepatitis C virus types with age: infection with type 3 was more common in younger patients. The distribution of hepatitis C virus type with age is accounted for by differences in risk-factors for infection in different age groups. The frequency of cirrhosis increased markedly with age. Even after standardization for age, center and the presence of cirrhosis, viral type was strongly related to the outcome of infection. CONCLUSIONS: Our data suggest that enzyme-linked immunosorbent hepatitis C virus typing could assist in patient selection for interferon treatment to improve sustained response rates. Together with measurement of viral load, hepatitis C virus typing may serve to indicate the probability of response in patients with chronic hepatitis C, and to elucidate antiviral mechanisms in the disease. The serotyping assay provides a relatively inexpensive screening method to determine the infecting hepatitis C virus type, which could facilitate therapeutic decisions and strategies in patients with chronic hepatitis C.

Abacioğlu YH, Davidson F, Simmonds P. 1996. Sequence analysis of the 5' non coding region of Turkish HCV isolates: implications for PCR diagnosis. Clin Diagn Virol, 5 (2-3), pp. 211-214. | Show Abstract | Read more

Hepatitis C virus (HCV) is a positive-strand RNA virus related to pestiviruses and flaviviruses. The 5' noncoding region (NCR) of the virus genome consists of 324-341 nucleotides and is generally highly conserved among different HCV isolates which has made this region the choice for primer selection in amplification of HCV sequences by polymerase chain reaction (PCR). In this study, we report the partial nucleotide sequences of the 5'-NCR from type 1a (n = 4), type 1b (n = 6) and type 4 (n = 1) Turkish HCV isolates. Sequence information was obtained by direct sequencing of RT-PCR product using biotinylated primers and single strands were sequenced using T7 DNA polymerase after binding to streptavidin coated magnetic beads. In comparison to prototype type 1a consensus sequence, all type 1b sequences had A-G substitution at position - 99. Nucleotid changes from the prototype 1a sequence were found in 12 of the 174 nucleotide positions. The most variable domain spans 51 nucleotides (positions - 167 to - 117) where nine polymorphic sites were identified. Although the nucleotide sequence of the 5'-noncoding region is highly conserved there are type-specific polymorphic sites within this region that has to be taken into consideration in the design of oligonucleotide primers for reliable amplification of sequences from different HCV genotypes.

Healey CJ, Sabharwal NK, Daub J, Davidson F, Yap PL, Fleming KA, Chapman RW, Simmonds P, Chapel H. 1996. Outbreak of acute hepatitis C following the use of anti-hepatitis C virus--screened intravenous immunoglobulin therapy. Gastroenterology, 110 (4), pp. 1120-1126. | Show Abstract | Read more

BACKGROUND & AIMS: Hepatitis C virus (HCV) infection has been associated with intravenous (IV) immunoglobulin (Ig), and plasma donations used to prepare IV Ig are now screened to prevent transmission. Thirty-six patients from the United Kingdom received infusions from a batch of anti-HCV antibody-screened intravenous Ig (Gammagard; Baxter Healthcare Ltd., Thetford, Norfolk, England) that was associated with reports of acute hepatitis C outbreak in Europe. The aim of this study was to document the epidemiology of this outbreak. METHODS: Forty-six patients from the United Kingdom treated with Gammagard (34 exposed and 12 unexposed to the batch) returned epidemiological questionnaires. RESULTS: Eighty-two percent of the exposed patients (28 of 34) became positive for HCV RNA. Eighteen percent of the patients (6 of 34) who had infusions with this batch tested negative for HCV RNA, but 2 of the patients had abnormal liver function and subsequently seroconverted to anti-HCV antibody positive. Twenty-seven percent of the patients (9 of 34) developed jaundice, and 79% (27 of 34) had abnormal liver transferase levels. Virus isolates (n=21), including an isolate from the implicated batch, were genotype 1a and virtually identical by sequence analysis of the NS5 region, consistent with transmission from a single source. CONCLUSIONS: Hepatitis C infection can be transmitted by anti-HCV-screened IV Ig. Careful documentation of IV Ig batch numbers and regular biochemical monitoring is recommended for all IV Ig recipients.

Jarvis LM, Ludlam CA, Ellender JA, Nemes L, Field SP, Song E, Chuansumrit A, Preston FE, Simmonds P. 1996. Investigation of the relative infectivity and pathogenicity of different hepatitis C virus genotypes in hemophiliacs. Blood, 87 (7), pp. 3007-3011. | Show Abstract

To assess the relative infectivity and pathogenicity of variants of hepatitis C virus (HCV) genotypes, the distribution of genotypes in hemophilic patients who had been treated with nonvirally inactivated factor concentrates or cryoprecipitates prepared from local blood donors was compared with those found in the respective blood donor populations. Genotype frequencies differed markedly in the four countries investigated (Scotland, Hungary, South Africa, and Thailand) but in each, the HCV genotype distributions in hemophiliacs and blood donors were similar. In addition, HCV genotypes in recipients of commercially manufactured concentrates were similar to those found in the US general population. These findings provide no evidence that HCV genotypes differ significantly from each other in replication rate, transmissibility, or infectivity.

Shah HA, Jafri SW, Malik IA, Prescott L, Simmonds P. 1996. The association of hepatitis C virus genotype 3 with severe chronic liver disease in Pakistan. GASTROENTEROLOGY, 110 (4), pp. A1320-A1320.

Hanley J, Jarvis L, Prescott L, Simmonds P, Parker A, Ludlam C. 1996. B-cell non-Hodgkins lymphoma and chronic HCV infection - No evidence of an association BRITISH JOURNAL OF HAEMATOLOGY, 93 pp. 272-272.




Smith DB, Davidson F, Yap PL, Brown H, Kolberg J, Detmer J, Urdea M, Simmonds P, Mellor J, Neville J et al. 1996. Levels of hepatitis C virus in blood donors infected with different viral genotypes JOURNAL OF INFECTIOUS DISEASES, 173 (3), pp. 727-730. | Show Abstract | Read more

The level of hepatitis C virus (HCV) in the serum of 337 blood donors infected with different viral genotypes was investigated by branched DNA assay. Viral genotype was deduced by restriction analysis of the virus 5'- noncoding region. Samples included genotypes 1a, 1b, 2a, 2b, 3, 4, 5, and 6. Multivariate analysis revealed that the ranges of HCV levels were similar for all viral genotypes and subtypes (P = .18), with the possible exception of genotype 4. Virus levels were significantly lower in female than male subjects (P < .001) but did not correlate with donor age (P = .06) or country of origin (P = .07). Alanine aminotransferase level was not correlated with virus level or genotype or with donor age, sex, or country. These results indicate a similar replicative capacity in vivo for different HCV genotypes and clarify the influence of host and virus factors on disease severity and responsiveness to interferon treatment.

Hanley JP, Jarvis LM, Andrew J, Dennis R, Hayes PC, Piris J, Lee R, Simmonds P, Ludlam CA. 1996. Interferon treatment for chronic hepatitis C infection in hemophiliacs--influence of virus load, genotype, and liver pathology on response. Blood, 87 (5), pp. 1704-1709. | Show Abstract

In this study, we assessed the effectiveness of interferon treatment in 31 hemophiliacs with chronic hepatitis C virus (HCV) infection. Interferon alfa-2a (3 MU three times weekly) was administered for 6 months. Response was assessed by both serial alanine transaminase (ALT) and HCV RNA levels measured by a sensitive semiquantitative polymerase chain reaction (PCR) method. HCV genotype was determined by restriction fragment length polymorphism (RFLP), and evidence of changing genotypes during interferon therapy was sought. Severity of liver disease was assessed by both noninvasive and invasive methods, including laparoscopic liver inspection and biopsy. Sustained normalization of ALT levels occurred in eight patients (28%), and seven (24%) became nonviremic as assessed by PCR (<80 HCV/mL). Responders universally cleared HCV RNA within 2 months of starting interferon. Genotype 3a was associated with a favorable response to interferon. No evidence was found for a change in circulating genotype in patients who failed to respond to interferon or who relapsed. This study confirms that response rates to interferon are low in hemophiliacs as compared with other groups with chronic HCV infection. We have also demonstrated that virus load measurement over the first 8 to 12 weeks of treatment is an extremely useful method to identify responders at an early stage.

Smith DB, Davidson F, Yap P, Brown H, Kolberg J, Detmer J, Urdea M, Simmonds P, International HCV Collaborati. 1996. Levels of hepatitis C virus in blood donors infected with different viral genotypes. International HCV Collaborative Study Group. J Infect Dis, 173 (3), pp. 727-730. | Show Abstract

The level of hepatitis C virus (HCV) in the serum of 337 blood donors infected with different viral genotypes was investigated by branched DNA assay. Viral genotype was deduced by restriction analysis of the virus 5'-noncoding region. Samples included genotypes 1a, 1b, 2a, 2b, 3,4,5, and 6. Multivariate analysis revealed that the ranges of HCV levels were similar for all viral genotypes and subtypes (P=.18), with the possible exception of genotype 4. Virus levels were significantly lower in female than in male subjects (P<.001) but did not correlate with donor age (P=.06) or genotype or with donor age, sex, or country. These results indicate a similar replicative capacity in vivo for different HCV genotypes and clarify the influence of host and virus factors on disease severity and responsiveness to interferon treatment.

Watson JP, Brind AM, Chapman CE, Bates CL, Gould FK, Johnson SJ, Burt AD, Ferguson J, Simmonds P, Bassendine MF. 1996. Hepatitis C virus: epidemiology and genotypes in the north east of England. Gut, 38 (2), pp. 269-276. | Show Abstract | Read more

The epidemiology of hepatitis C virus (HCV) infection was studied in an English teaching hospital over an 18 month period. A total of 104 HCV antibody positive patients were referred for further investigation. They were divided into those diagnosed through screening (blood donors and intravenous drug abusers) and those diagnosed for other reasons, and their mean ages, known risk factors for HCV transmission, genotypes, and liver biopsy histology were analysed. Screened patients were significantly younger than the others. No significant difference in age was found between genotypes. Most patients genotyped (69%) were genotype 1. Intravenous drug abusers had a higher proportion of subtype 1a, and patients who acquired HCV through blood transfusion had a higher proportion of subtype 1b. Liver biopsy specimens were scored using a histological activity index for liver inflammation and fibrosis. Patients with subtype 1b had significantly more severe liver disease than other genotypes when the histological activity index scores for fibrosis were analysed (p < 0.05). Liver disease worsened significantly with age according to all three histological activity index scores (portal activity: p < 0.01, acinar activity: p < 0.001, fibrosis: p < 0.0001). Liver disease worsened with increased duration of infection (p < 0.002), and patients who also abused alcohol presented at a significantly younger age (cirrhosis, p < 0.05, hepatocellular carcinoma, p < 0.02).

Mellor J, Walsh EA, Prescott LE, Jarvis LM, Davidson F, Yap PL, Simmonds P. 1996. Survey of type 6 group variants of hepatitis C virus in Southeast Asia by using a core-based genotyping assay. J Clin Microbiol, 34 (2), pp. 417-423. | Show Abstract

Previous surveys of the prevalences of genotypes of hepatitis C virus (HCV) in different populations have often used genotyping assays based upon analysis of amplified sequences from the 5' noncoding region (5'NCR), such as restriction fragment length polymorphism (RFLP) or hybridization with type-specific probes (e.g., InnoLipa). Although highly conserved, this region contains several type-specific nucleotide polymorphisms that allow major genotypes 1 to 6 to be reliably identified. Recently, however, novel HCV variants found in Vietnam and Thailand that are distantly related to the type 6a genotype (type 6 group) by phylogenetic analysis of coding regions of the genome often have sequences in the 5'NCR that are similar or identical to those of type 1 and could therefore not be identified by an assay of sequences in this region. We developed a new genotyping assay based upon RFLP of sequences amplified from the more variable core region to investigate their distribution elsewhere in southeast (SE) Asia. Among 108 samples from blood donors in seven areas that were identified as type 1 by RFLP in the 5'NCR, type 6 group variants were found in Thailand (7 from 28 samples originally identified as type 1) and Burma (Myanmar) (1 of 3) but were not found in Hong Kong (n = 43), Macau (n = 8), Taiwan (n = 6), Singapore (n = 2), or Malaysia (n = 18). Although this small survey suggests a relatively limited distribution for type 6 group variants in SE Asia, larger studies will be required to explore their distribution in other geographical regions and the extent to which their presence would limit the practical usefulness of 5'NCR-based genotyping assays for clinical or epidemiological purposes.

Dhaliwal SK, Prescott LE, Dow BC, Davidson F, Brown H, Yap PL, Follett EA, Simmonds P. 1996. Influence of viraemia and genotype upon serological reactivity in screening assays for antibody to hepatitis C virus. J Med Virol, 48 (2), pp. 184-190. | Show Abstract | Read more

Detection of antibody to recombinant proteins derived from hepatitis C virus (HCV) genotype 1 represents the principal method for diagnosis of HCV infection. A method was developed for quantifying antibody reactivity in two third-generation enzyme immunoassays (Ortho EIA 3.0 and Murex VK48), and the influence of viraemia, HCV genotype, and host factors such as age, gender, and risk group upon antibody levels were investigated in a consecutive series of 117 anti-HCV-positive volunteer blood donors. Viraemic donors (as assessed by the polymerase chain reaction; PCR) showed significantly higher levels of anti-HCV by the Ortho EIA than those who were nonviraemic (adjusted mean difference of 10.1 fold after multiple regression analysis). The only other factor to influence significantly antibody level was genotype, where it was found that donors infected with type 1 showed 4 to 4.5 times greater serological reactivity by the Ortho assay than those infected with type 2 or 3. Antibody levels by the Ortho assay correlated closely to those detected by the Murex VK48 assay, and similar differences between PCR-positive and negative donors and between those infected with different genotypes were found. Differences in serological reactivity between genotypes indicate that a large proportion of epitopes of the type 1a or 1b recombinant proteins used in current assays are genotype specific. Variation in sensitivity of screening assays for different genotypes is of potential concern when used in countries where non-type 1 genotypes predominate in the blood donor or patient population.

Simmonds P. 1996. Neurotropism of HIV type 1? AIDS RESEARCH AND HUMAN RETROVIRUSES, 12 (6), pp. 469-470.

Simmonds P, Mellor J, Sakuldamrongpanich T, Nuchaprayoon C, Tanprasert S, Holmes EC, Smith DB. 1996. Evolutionary analysis of variants of hepatitis C virus found in South-East Asia: comparison with classifications based upon sequence similarity. J Gen Virol, 77 ( Pt 12) (12), pp. 3013-3024. | Show Abstract | Read more

Variants of hepatitis C virus (HCV) have been classified by nucleotide sequence comparisons in different regions of the genome. Many investigators have defined the ranges of sequence similarity values or evolutionary distances corresponding to divisions of HCV into types, subtypes and isolates. Using these criteria, novel variants of HCV from Vietnam, Thailand and Indonesia have been classified as types 7, 8, 9, 10 and 11, many of which can be further subdivided into between two to four subtypes. In this study, this distance-based method of virus classification was compared with phylogenetic analysis and statistical measures to establish the confidence of the groupings. Using bootstrap resampling of phylogenetic trees in several subgenomic regions (core, E1, NS5) and with complete genomic sequences, we found that one set of novel HCV variants ('types 7, 8, 9 and 11') consistently grouped together into a single clade that also contained type 6a, while 'type 10a' grouped with type 3. In contrast, no robust higher-order groupings were observed between any of the other five previously described HCV genotypes (types 1-5). In each subgenomic region, the distribution of pairwise distances between members of the type 6 clade were consistently bi-modal and therefore provided no justification for classification of these variants into the three proposed categories (type, subtype, isolate). Based on these results, we propose that a more useful classification would regard all these variants as subtypes of type 6 or type 3, even though the level of sequence diversity within the clade was greater than observed for other genotypes. Classification by phylogenetic relatedness rules out simple sequence similarity measurements as a method for assigning HCV genotypes, but provides a more appropriate description of the evolutionary and epidemiological history of a virus.

Gerken G, Pontisso P, Roggendorf M, Grazia Rumi M, Simmonds P, Trepo C, Zeuzem S, Colucci G. 1996. Clinical evaluation of a single reaction, diagnostic polymerase chain reaction assay for the detection of hepatitis C virus RNA. J Hepatol, 24 (1), pp. 33-37. | Show Abstract | Read more

BACKGROUND/AIMS: In the past few years the detection of HCV-RNA by polymerase chain reaction has become a well-established diagnostic tool for patients with chronic hepatitis C. However, the lack of reproducible results between laboratories and the relatively high proportion of false-positive results, has indicated the need for a standardized and reliable polymerase chain reaction assay. In the present study we have analyzed the performance of a commercial, HCV-RNA polymerase chain reaction assay based on a single, combined reverse transcription and amplification reaction and on the use of Uracil-N-glycosilase to prevent carry-over contamination (Amplicor HCV, Roche Molecular Systems). METHODS: In this assay the amplification products are detected in microwell plates using biotinylated primer and the HRP avidin colorimetric system. Serum samples collected from 446 patients, including 181 with chronic active hepatitis C, 50 with autoimmune chronic hepatitis, 117 in hemodialysis, 30 asymptomatic carriers of anti-HCV and 68 with indeterminate serology (RIBA indeterminate results), as well as from 121 controls were tested with the commercial, single-step assay and with nested polymerase chain reaction. Both techniques use primers located within the 5' non-coding region of the HCV genome. RESULTS: In all cases a good concordance was observed between the commercial, single-step assay and nested polymerase chain reaction which, for patients with chronic active hepatitis, showed a sensitivity and specificity of 100% and 99.3% for the former and of 98.8% and 100% for the latter, when compared to clinical diagnosis taken as the gold standard. Most of the 11 discordant samples were seen in the group of RIBA-indeterminate cases and in patients with chronic active hepatitis C. Further analysis of these cases, based on repeat testing and clinical data showed that 64% and 36% of the discrepancies were due, respectively, to nested polymerase chain reaction and Amplicor inconsistent reactions. In hemodialyzed patients, patients with autoimmune hepatitis and asymptomatic carriers of anti-HCV, both assays produced results which were consistent with the clinical diagnosis. In the former group, polymerase chain reaction was able to identify the presence of active viral replication in some antibody negative samples. CONCLUSIONS: Taken together, these results indicate that the commercial, single-step polymerase chain reaction assay has the same clinical sensitivity and specificity as nested polymerase chain reaction and that, because of its simplified procedures and fast turn-around time, it may be a valuable test for routine diagnostic applications.

Romeo R, Colombo M, Rumi MG, Soffredini R, DelNinno E, Donato MF, Russo A, Simmonds P. 1996. Lack of association between type of hepatitis C virus, serum load and severity of liver disease HEPATOLOGY, 23 (1), pp. T55-T55.

ElZayadi A, Simmonds P, Dabbous H, Prescott L, Selim O, Ahdy A. 1996. Response of chronic hepatitis C type 4 Egyptian patients to alfa-interferon HEPATOLOGY, 23 (1), pp. T17-T17.

Simmonds P. 1996. Virology of hepatitis C virus. Clin Ther, 18 Suppl B (SUPPL. B), pp. 9-36. | Show Abstract | Read more

Hepatitis C virus (HCV) has been identified as the main causative agent of posttransfusion non-A, non-B hepatitis. Through recently developed diagnostic assays, routine serologic screening of blood donors has prevented most cases of posttransfusion hepatitis. The purpose of this paper is to comprehensively review current information regarding the virology of HCV. Recent findings on the genome organization, its relationship to other viruses, the replication of HCV ribonucleic acid, HCV translation, and HCV polyprotein expression and processing are discussed. Also reviewed are virus assembly and release, the variability of HCV and its classification into genotypes, the geographic distribution of HCV genotypes, and the biologic differences between HCV genotypes. The assays used in HCV genotyping are discussed in terms of reliability and consistency of results, and the molecular epidemiology of HCV infection is reviewed. These approaches to HCV epidemiology will prove valuable in documenting the spread of HCV in different risk groups, evaluating alternative (nonparenteral) routes of transmission, and in understanding more about the origins and evolution of HCV.

Jobanputra P, Davidson F, Graham S, O'Neill H, Simmonds P, Yap PL. 1995. High frequency of parvovirus B19 in patients tested for rheumatoid factor. BMJ, 311 (7019), pp. 1542. | Read more

Hanley JP, Jarvis LM, Andrews J, Hayes PC, Simmonds P, Ludlam CA. 1995. Treatment of hepatitis C infection in haemophiliacs: the Edinburgh experience. Haemophilia, 1 Suppl 4 (S4), pp. 36-38. | Read more

Jarvis LM, Ludlam CA, Simmonds P. 1995. Hepatitis C virus genotypes in multi-transfused individuals. Haemophilia, 1 Suppl 4 (S4), pp. 3-7. | Read more

Mellor J, Holmes EC, Jarvis LM, Yap PL, Simmonds P. 1995. Investigation of the pattern of hepatitis C virus sequence diversity in different geographical regions: implications for virus classification. The International HCV Collaborative Study Group. J Gen Virol, 76 ( Pt 10) (10), pp. 2493-2507. | Show Abstract | Read more

Genotypes of hepatitis C virus (HCV) present within 104 samples from HCV-infected individuals from Africa, the Middle East, the Indian subcontinent and South-East Asia were identified by sequence comparisons in the core and NS-5 regions. Relatively short sequences (such as the 222 bp fragment of NS-5) provided effective discrimination of types, subtypes and isolates, and produced equivalent relationships between genotypes as were found upon comparison of longer sequences of NS-5, of the core region, and by comparison of the limited number of complete genomic sequences currently available. Measurement of evolutionary distances in the core and NS-5 regions allowed 79 of the 104 samples to be identified as examples of known genotypes, while 17 of the remainder could be provisionally classified as new subtypes of types 1 (Nigeria), 2 (Gambia), 3 (India, Pakistan and Bangladesh) and 4 (Middle East) on the basis of sequence comparison in core and NS-5 (n = 9) or provisionally using core alone (n = 8). The remaining sequences from Thailand made up two groups showing no close similarity to any of the six major genotypes classified to date, although one corresponded to an as yet unclassified variant of HCV also found in Thailand. However, phylogenetic analysis of the core and NS-5 regions indicated a distant relationship between these sequences with variants found in Vietnam and with type 6a, and collectively they formed a diverse single phylogenetic group. The existence of great diversity within a single genotype was also found amongst type 3 sequences in the Indian subcontinent, amongst type 4 variants in Central Africa and the Middle East, and amongst type variants in Nigeria. These findings may provide clues for understanding the origins and mechanisms of transmission of HCV.

Kasper P, Simmonds P, Schneweis KE, Kaiser R, Matz B, Oldenburg J, Brackmann HH, Holmes EC. 1995. The genetic diversification of the HIV type 1 gag p17 gene in patients infected from a common source. AIDS Res Hum Retroviruses, 11 (10), pp. 1197-1201. | Show Abstract | Read more

An evolutionary analysis was undertaken of HIV-1 gag p17 sequences taken from a small cohort of hemophilia B patients infected from a common batch of clotting factor concentrate. The sequence population found at seroconversion was highly homogeneous, suggesting that the infecting batch also contained little sequence variation. Genetic diversification was found in follow-up sequences taken approximately 3 years later and was generally found to be complex. Greater rates of synonymous to nonsynonymous substitution were observed, especially when comparing distantly related isolates, and the rate of synonymous substitution was used to estimate times of divergence for a number of isolates of HIV-1 including the origin of the subtypes A to H. The p17 region is therefore proposed as a useful marker for future epidemiological studies.





Lau JY, Simmonds P, Urdea MS. 1995. Implications of variations of "conserved" regions of hepatitis C virus genome. Lancet, 346 (8972), pp. 425-426. | Read more

Power JP, Davidson F, O'Riordan J, Simmonds P, Yap PL, Lawlor E. 1995. Hepatitis C infection from anti-D immunoglobulin. Lancet, 346 (8971), pp. 372-373. | Read more

Smith DB, Mellor J, Jarvis LM, Davidson F, Kolberg J, Urdea M, Yap PL, Simmonds P. 1995. Variation of the hepatitis C virus 5' non-coding region: implications for secondary structure, virus detection and typing. The International HCV Collaborative Study Group. J Gen Virol, 76 ( Pt 7) pp. 1749-1761. | Show Abstract | Read more

Variation in the 5' non-coding region (5'NCR) of hepatitis C virus (HCV) was investigated in detail by comparing 314 5'NCR sequences of viruses of genotypes 1 to 6. Evidence was obtained for the existence of associations between particular 5'NCR sequence motifs and virus types and subtypes. No recombination was observed between the 5'NCR and coding regions of different genotypes, implying that the sequence of subgenomic regions such as the 5'NCR can be used to deduce virus genotype. The distribution of polymorphic sites within the 5'NCR is used to propose improved oligonucleotide primers for virus detection and quantification that would be equally efficient in detecting RNA of different virus genotypes. The accuracy of two different genotyping methods (RFLP and the line probe assay) based on analysis of sequence polymorphisms in the 5'NCR is predicted from the sequences surveyed to be 97% and 83% respectively for types 1 to 6, with higher accuracies for distinguishing between subtypes 1a/1b, 2a/2b or 3a/3b. Several sites of genotype-specific polymorphism were covariant and maintained the base pairings required for a secondary structure model of the 5'NCR. Other sites of variation suggest minor modifications to this model and have implications for the probable functions of the 5'NCR.

McClure MO, Bieniasz PD, Weber JN, Tedder RS, O'Shea S, Banatvala JE, Tudor-Williams G, Simmonds P, Holmes EC. 1995. HIV clearance in an infant? Nature, 375 (6533), pp. 637-638. | Read more



Power JP, Lawlor E, Davidson F, Holmes EC, Yap PL, Simmonds P. 1995. Molecular epidemiology of an outbreak of infection with hepatitis C virus in recipients of anti-D immunoglobulin. Lancet, 345 (8959), pp. 1211-1213. | Show Abstract | Read more

In a retrospective investigation of possible transmission of hepatitis C virus (HCV) by anti-rhesus D immunoglobulin (anti-D) in 1977, we compared variants infecting anti-D recipients in Ireland of one of the implicated batches with those of epidemiologically unrelated HCV-infected individuals. All 100 of the recipients of the batch investigated to date were infected with a single genotype (type 1), consistent with a single-source outbreak, whereas a wider range of genotypes (1, 2, and 3) were found in anti-HCV positive individuals from Ireland infected by different routes. Nucleotide sequences from a 222 base fragment from the NS-5 region of the genome amplified from stored aliquots of the implicated batch closely matched those detected in anti-D recipients 17 years after the transmission event. This study shows the value of molecular epidemiological techniques for identifying distant sources of infection, and for the epidemiological investigation of the current distribution and transmission of HCV in different populations.

Davidson F, Simmonds P, Ferguson JC, Jarvis LM, Dow BC, Follett EA, Seed CR, Krusius T, Lin C, Medgyesi GA. 1995. Survey of major genotypes and subtypes of hepatitis C virus using RFLP of sequences amplified from the 5' non-coding region. J Gen Virol, 76 ( Pt 5) (5), pp. 1197-1204. | Show Abstract | Read more

A method is described for identifying different genotypes of hepatitis C virus (HCV) by restriction endonuclease cleavage of sequences amplified by PCR from the 5' non-coding region. Using the enzymes HaeIII-RsaI and HinfI-MvaI, followed by cleavage with BstU1 or ScrFI, it was possible to identify and distinguish HCV genotypes 1a, 1b, 2a, 2b, 3a, 3b, 4, 5 and 6. The method was used to investigate the prevalence of these genotypes in 723 blood donors in 15 countries, the largest survey to date, and one which covered a wide range of geographical regions (Europe, America, Africa and Asia). These results, combined with a review of the existing literature, indicate the existence of several distinct regional patterns of HCV genotype distribution, and provide the framework for future detailed epidemiological investigations of HCV transmission.

Haydon GH, Jarvis LM, Simmonds P, Hayes PC. 1995. Association between chronic hepatitis C infection and hepatocellular carcinoma. Lancet, 345 (8954), pp. 928-929. | Read more


Kyriacou E, Simmonds P, Miller EK, Bouchier IA, Hayes PC, Harrison DJ. 1995. Liver biopsy findings in patients with alcoholic liver disease complicated by chronic hepatitis C virus infection. Eur J Gastroenterol Hepatol, 7 (4), pp. 331-334. | Show Abstract

OBJECTIVE: To identify the features of concurrent hepatitis C virus (HCV) infection in liver biopsies from patients thought to have alcoholic liver disease. PATIENTS: Fifty-five patients with a history of excess alcohol consumption were studied. METHODS: All patients underwent liver biopsy. RESULTS: Eight of the 55 patients studied were found to be HCV-positive. CONCLUSION: The histological features found to be most useful for identifying concurrent HCV infection were the presence of lymphoid aggregates in portal tracts (predictive value 100%), the presence of lymphocytes in the lobules (predictive value 83%), and the pattern of fibrosis, particularly periportal spurring rather than perivenular fibrosis (predictive value 75%).

Preston FE, Jarvis LM, Makris M, Philp L, Underwood JC, Ludlam CA, Simmonds P. 1995. Heterogeneity of hepatitis C virus genotypes in hemophilia: relationship with chronic liver disease. Blood, 85 (5), pp. 1259-1262. | Show Abstract

In this study we have determined the hepatitis C virus (HCV) serotype and genotype in a cohort of 96 HCV-infected hemophiliacs and have examined the relationship between HCV genotype and severity of chronic liver disease as determined by liver biopsy. HCV serotype was determined by specific enzyme-linked immunosorbent assays (ELISAs) and genotype by restriction fragment length polymorphism (RFLP) and HCV viral sequencing. The pattern of genotype distribution was quite unlike that of HCV-infected United Kingdom (UK) blood donors in that five of the six known HCV genotypes were represented, 50% were type 1, 13% type 2, and 18% type 3. An unexpected observation was the presence of HCV genotype 4 in four patients and type 5 in two patients. An additional feature was the presence of mixed infection, detected in 14% and 7% by serotype and genotype analysis, respectively. Liver biopsies were available from 51 patients. Cirrhosis was present in five of 27 (19%) of individuals with type 1, in 2 of 9 (22%) with type 2, and 5 of 8 (63%) of those with type 3. The heterogeneous pattern of HCV genotype distribution in this cohort of patients and the observed relationship between the severity of the related liver disease and specific HCV genotype may have important implications with respect to the natural history and treatment of HCV-related chronic liver disease in infected hemophiliacs worldwide.

Lau JY, Mizokami M, Kolberg JA, Davis GL, Prescott LE, Ohno T, Perrillo RP, Lindsay KL, Gish RG, Qian KP. 1995. Application of six hepatitis C virus genotyping systems to sera from chronic hepatitis C patients in the United States. J Infect Dis, 171 (2), pp. 281-289. | Show Abstract | Read more

Serum samples from 139 US patients with chronic hepatitis C virus (HCV) infection were studied using six different genotyping systems, including both molecular and serologic methods, to determine the applicability of these approaches and the prevalence of various HCV subtypes. The concordance of genotyping results based on the various systems (except for core polymerase chain reaction genotyping) was good (93.5%). Subtypes 1a and 1b were prevalent (37.4%). Subtypes 2a (2.2%), 2b (8.6%), and 3a (5.8%) were less common. HCV genotypes could not be determined in 3.4%-16.5% of samples depending on the method used. HCV type 2 was associated with greater histologic activity but lower serum HCV RNA levels (P < .05), whereas type 3 was associated with lower serum alanine aminotransferase levels (P < .05). These data demonstrate a high concordance between HCV genotyping systems and provide a foundation for comparison of genotyping data between studies using different systems. HCV types 1a and 1b are both prevalent in the United States.

Simmonds P. 1995. Variability of hepatitis C virus. Hepatology, 21 (2), pp. 570-583. | Read more

Pawlotsky JM, Roudot-Thoraval F, Simmonds P, Mellor J, Ben Yahia MB, André C, Voisin MC, Intrator L, Zafrani ES, Duval J, Dhumeaux D. 1995. Extrahepatic immunologic manifestations in chronic hepatitis C and hepatitis C virus serotypes. Ann Intern Med, 122 (3), pp. 169-173. | Show Abstract | Read more

OBJECTIVE: To determine, using a serotyping assay, whether the occurrence of extrahepatic immunologic disorders in patients with chronic hepatitis C is dependent on hepatitis C virus serotype. DESIGN: Prospective study. SETTING: Liver unit and virology laboratory of a university hospital. PATIENTS: 59 consecutive patients with chronic hepatitis C. MEASUREMENTS: Hepatitis C virus serotype was determined using a recently developed immunoenzymatic assay that detects antibodies directed to serotype-specific immunodominant epitopes. Cryoglobulin, rheumatoid factor, and numerous antitissue antibodies were sought. Biopsies of labial salivary glands were done in 49 of the 59 patients. RESULTS: Prevalence was 59% for serotype 1, 10% for serotype 2, 12% for serotype 3, and 3% for mixed infection. Fifteen percent of patients could not be serotyped. Cryoglobulinemia was found in 36% of patients and rheumatoid factor was found in the serum of 71%. At least one antitissue antibody was found in the serum of 41% of patients; salivary gland biopsy showed lymphocytic capillaritis in 49% of patients. These immunologic abnormalities were seen in patients infected with any of the three serotypes, and prevalences of the abnormalities did not differ significantly among patients infected with different serotypes. CONCLUSIONS: We confirm that the prevalence of extrahepatic immunologic abnormalities is high in patients with chronic hepatitis C. These abnormalities may occur in patients infected with any of the three major hepatitis C virus serotypes now present in developed countries.


An enzyme linked immunosorbent assay has been recently developed which detects and distinguishes between infections with the three major hepatitis C virus (HCV) genotypes prevalent in Europe. Using this assay we have investigated the sera of 30 Italian and 37 Spanish children with chronic hepatitis C. Infection with HCV type 1 was found in 43% of Italian and 46% of Spanish children. Of the Italian children 7% were infected with HCV of type 2 and 7% had a mixed type 1/type 2 serotype. Infection with HCV type 3 was found in 7% of Italian and 8% of Spanish children while 36% of Italian and 46% of Spanish children had non-reactive sera. Serotype 3 was significantly more frequent in children with anti-HCV positive mothers (often drug abusers) than in those with percutaneous exposure (25% vs. 2%, p < 0.05). Mean alanine aminotransferase values were significantly higher in children with HCV type 1 than in those with non-reactive sera (P < 0.05). These results indicate a similar distribution of HCV serotypes in Italian and Spanish children. Serotype 1 is prevalent and associated with a more severe liver damage. The relevant proportion of non-reactive sera could be related to the existence of genetic variants different from those explored by the test and with low pathogenicity, or to a poor antibody response to viral antigens of the NS4 region in a consistent subgroup of children. © 1995.




Smith DB, Mellor J, Jarvis LM, Davidson F, Kolberg J, Urdea M, Yap P-L, Simmonds P. 1995. Variation of the hepatitis C virus 5' non-coding region: implications for secondary structure, virus detection and typing Journal of General Virology, 76 (7), pp. 1749-1761. | Show Abstract | Read more

Variation in the 5' non-coding region (5'NCR) of hepatitis C virus (HCV) was investigated in detail by comparing 314 5'NCR sequences of viruses of genotypes 1 to 6. Evidence was obtained for the existence of associations between particular 5'NCR sequence motifs and virus types and subtypes. No recombination was observed between the 5'NCR and coding regions of different genotypes, implying that the sequence of subgenomic regions such as the 5'NCR can be used to deduce virus genotype. The distribution of polymorphic sites within the 5'NCR is used to propose improved oligonucleotide primers for virus detection and quantification that would be equally efficient in detecting RNA of different virus genotypes. The accuracy of two different genotyping methods (RFLP and the line probe assay) based on analysis of sequence polymorphisms in the 5'NCR is predicted from the sequences surveyed to be 97% and 83% respectively for types 1 to 6, with higher accuracies for distinguishing between subtypes 1a/1b, 2a/2b or 3a/3b. Several sites of genotype-specific polymorphism were covariant and maintained the base pairings required for a secondary structure model of the 5'NCR. Other sites of variation suggest minor modifications to this model and have implications for the probable functions of the 5'NCR.

Bhattacherjee V, Prescott LE, Pike I, Rodgers B, Bell H, El-Zayadi AR, Kew MC, Conradie J, Lin CK, Marsden H. 1995. Use of NS-4 peptides to identify type-specific antibody to hepatitis C virus genotypes 1, 2, 3, 4, 5 and 6. J Gen Virol, 76 ( Pt 7) (7), pp. 1737-1748. | Show Abstract | Read more

The 5' end of the NS-4 protein of different genotypes of hepatitis C virus (HCV) is highly variable in nucleotide and inferred amino acid sequence, with frequent predicted amino acid substitutions between all six of the major HCV genotypes described to date. This region has been shown to be antigenic by epitope mapping, and elicits antibody in HCV-infected individuals with a detectable type-specific component. We have used this sequence data to specify branched peptides for an indirect binding/competition assay to detect type-specific antibody to each major genotype. A total of 183 out of 210 samples (87%) from blood donors and patients with chronic hepatitis C infected with genotypes 1 to 6 showed detectable type-specific antibody to NS-4 peptides that in almost all cases (> 97 %) corresponded to the genotype detected by a PCR typing method. These findings demonstrate the existence of major antigenic differences between genotypes of HCV, and indicate how infection with different variants of HCV may be detected by a serological test.

Holmes EC, Zhang LQ, Robertson P, Cleland A, Harvey E, Simmonds P, Leigh Brown AJ. 1995. The molecular epidemiology of human immunodeficiency virus type 1 in Edinburgh. J Infect Dis, 171 (1), pp. 45-53. | Show Abstract | Read more

Human immunodeficiency virus (HIV) type 1 sequences obtained from HIV-infected persons in different risk groups in Edinburgh were studied to determine the number and origin of virus variants and patterns of virus transmission. Phylogenetic analysis revealed that 12 of 14 hemophiliac patients who had been exposed to a single common batch of factor VIII had closely related gag gene sequences. Sequences from intravenous drug users and patients infected through heterosexual contact formed another distinct group, and 2 other hemophiliacs formed a third group. However, epidemiologic relationships inferred from analysis of the V3 region of the env gene were less conclusive, especially when the V3 loop was taken in isolation. This appears to be due to the length of time since infection and the action of selection, which has favored the independent appearance of similar V3 loop variants.

Tisminetzky S, Gerotto M, Pontisso P, Chemello L, Prescott LE, Rose KA, Baralle F, Simmonds P, Alberti A. 1995. Comparison of genotyping and serotyping methods for the identification of hepatitis C virus types. J Virol Methods, 55 (3), pp. 303-307. | Show Abstract | Read more

The usefulness of identification of hepatitis C virus (HCV) genotype has recently been investigated for the clinical management of patients infected by HCV. In the present study, the HCV genotype infecting 127 patients was determined by two different methods: HCV genotyping using a dot-blot assay with type-specific probes derived from the 5'-UTR of HCV genome and HCV serotyping using an ELISA system in which type-specific antibodies against the NS4 region were detected. Overall, a good correlation of the two methods was observed, the main discrepancy being 4 patients with sequence-confirmed HCV-2 (2 cases) and HCV-3 (2 cases) genotypes recognized as HCV-1 by serotyping. Mixed infections were not detected by either method. In 19 PCR negative sera, in which the HCV genotype could not be evaluated, no particular serotype profile was observed. In conclusion, the molecular and serological techniques are almost equivalent in determining the viral type, although in individual cases, especially in PCR negative patients, the clinical meaning of the serotyping result remains to be determined.

Healey CJ, Smith DB, Walker JL, Holmes EC, Fleming KA, Chapman RW, Simmonds P. 1995. Acute hepatitis C infection after sexual exposure. Gut, 36 (1), pp. 148-150. | Show Abstract | Read more

A case is described of a woman with acute hepatitis C infection whose partner had chronic hepatitis C infection and where heterosexual contact was the only major risk factor. Infection of both partners was confirmed serologically and by the finding of virus RNA by reverse transcription and polymerase chain reaction amplification. Nucleotide sequence analysis of the NS5 region (RNA polymerase) was used to show that both partners were infected with virus of the same genotype (1a). The nucleotide sequence of virus RNA found in the female patient is closest to variants cocirculating in the male contact, consistent with transmission having occurred between the two.

Watson HG, Ludlam CA, McOmish F, Dennis R, Hart H, Simmonds P. 1995. Absence of hepatitis A virus transmission by high-purity solvent detergent treated coagulation factor concentrates in Scottish haemophiliacs. Br J Haematol, 89 (1), pp. 214-216. | Show Abstract | Read more

Recent reports of hepatitis A virus (HAV) infection in haemophiliacs receiving high-purity solvent detergent (HP.SD) treated factor VIII concentrates have brought into question the efficacy of this virucidal method for inactivating HAV. To assess whether HAV may have been transmitted by HP.SD concentrates, we compared seroprevalence in haemophiliacs with different disease severity, sought evidence of seroconversion to HAV since introduction of HP.SD products, and directly examined concentrates for HAV RNA by PCR. Our data suggest that Scottish haemophiliacs are not being infected with HAV by HP.SD concentrates produced initially by CRTS Lille and presently by PFC Edinburgh and supplied by the Scottish National Blood Transfusion Service (SNBTS).

Smith DB, Davidson F, Simmonds P. 1995. Hepatitis C virus variants and the role of genotyping. J Hepatol, 23 Suppl 2 (2), pp. 26-31. | Show Abstract

Hepatitis C virus demonstrates considerable divergence in nucleotide sequence. This variation may affect virus detection, disease outcome, and the effectiveness of interferon treatment. For example, infection with genotype 1 is associated with a lower response rate to interferon treatment. Hepatitis C virus can be classified into six distinct types, comprising at least 74 different subtypes. Both types and subtypes are subject to geographical differences in distribution, presumably reflecting the epidemiological history of the virus. However, because this history may be blurred by migration and by commerce in blood products between regions, screening and typing assays must recognize both the major indigenous and more exotic virus genotypes. Little information exists about the role of virus sequence variation in screening for hepatitis C virus antibodies, but there is some evidence that the reactivity of current serological screening assays for hepatitis C virus is genotype dependent. In the future, screening assays may need to include antigens specific for different virus types or may need to be designed with regard to the particular types found in a certain area. Antigenic variation may also mean that an effective vaccine needs to be multivalent to protect against all genotypes present in a given region. There are several polymerase chain reaction-based methods of distinguishing between hepatitis C virus variants. These methods must be continually updated, however, as new sequence variants are discovered. Alternative genotyping assays are based on the host's serological response to virus infection, but these cannot distinguish between virus subtypes and are unsuitable for immunocompromised patients. As more countries are sampled, it is likely that more genotypes will be identified and this may help elucidate the origins of hepatitis C virus. Detailed epidemiological studies may delineate past and current routes of transmission.