Expanded classification of hepatitis C virus into 7 genotypes and 67 subtypes: updated criteria and genotype assignment web resource.

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 (http://talk.ictvonline.org/links/hcv/hcv-classification.htm). 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.

Polyvalent vaccines for optimal coverage of potential T-cell epitopes in global HIV-1 variants.

HIV-1/AIDS vaccines must address the extreme diversity of HIV-1. We have designed new polyvalent vaccine antigens comprised of sets of 'mosaic' proteins, assembled from fragments of natural sequences via a computational optimization method. Mosaic proteins resemble natural proteins, and a mosaic set maximizes the coverage of potential T-cell epitopes (peptides of nine amino acids) for a viral population. We found that coverage of viral diversity using mosaics was greatly increased compared to coverage by natural-sequence vaccine candidates, for both variable and conserved proteins; for conserved HIV-1 proteins, global coverage may be feasible. For example, four mosaic proteins perfectly matched 74% of 9-amino-acid potential epitopes in global Gag sequences; 87% of potential epitopes matched at least 8 of 9 positions. In contrast, a single natural Gag protein covered only 37% (9 of 9) and 67% (8 of 9). Mosaics provide diversity coverage comparable to that afforded by thousands of separate peptides, but, because the fragments of natural proteins are compressed into a small number of native-like proteins, they are tractable for vaccines.

The LANL hemorrhagic fever virus database, a new platform for analyzing biothreat viruses.

Hemorrhagic fever viruses (HFVs) are a diverse set of over 80 viral species, found in 10 different genera comprising five different families: arena-, bunya-, flavi-, filo- and togaviridae. All these viruses are highly variable and evolve rapidly, making them elusive targets for the immune system and for vaccine and drug design. About 55,000 HFV sequences exist in the public domain today. A central website that provides annotated sequences and analysis tools will be helpful to HFV researchers worldwide. The HFV sequence database collects and stores sequence data and provides a user-friendly search interface and a large number of sequence analysis tools, following the model of the highly regarded and widely used Los Alamos HIV database [Kuiken, C., B. Korber, and R.W. Shafer, HIV sequence databases. AIDS Rev, 2003. 5: p. 52-61]. The database uses an algorithm that aligns each sequence to a species-wide reference sequence. The NCBI RefSeq database [Sayers et al. (2011) Database resources of the National Center for Biotechnology Information. Nucleic Acids Res., 39, D38-D51.] is used for this; if a reference sequence is not available, a Blast search finds the best candidate. Using this method, sequences in each genus can be retrieved pre-aligned. The HFV website can be accessed via http://hfv.lanl.gov.

Whole genome pyrosequencing of rare hepatitis C virus genotypes enhances subtype classification and identification of naturally occurring drug resistance variants.

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.

Ancient co-speciation of simian foamy viruses and primates.

Although parasite-host co-speciation is a long-held hypothesis, convincing evidence for long-term co-speciation remains elusive, largely because of small numbers of hosts and parasites studied and uncertainty over rates of evolutionary change. Co-speciation is especially rare in RNA viruses, in which cross-species transfer is the dominant mode of evolution. Simian foamy viruses (SFVs) are ubiquitous, non-pathogenic retroviruses that infect all primates. Here we test the co-speciation hypothesis in SFVs and their primate hosts by comparing the phylogenies of SFV polymerase and mitochondrial cytochrome oxidase subunit II from African and Asian monkeys and apes. The phylogenetic trees were remarkably congruent in both branching order and divergence times, strongly supporting co-speciation. Molecular clock calibrations revealed an extremely low rate of SFV evolution, 1.7 x 10(-8) substitutions per site per year, making it the slowest-evolving RNA virus documented so far. These results indicate that SFVs might have co-speciated with Old World primates for at least 30 million years, making them the oldest known vertebrate RNA viruses.

Genotype 1 and global hepatitis C T-cell vaccines designed to optimize coverage of genetic diversity.

Immunological control of hepatitis C virus (HCV) is possible and is probably mediated by host T-cell responses, but the genetic diversity of the virus poses a major challenge to vaccine development. We considered monovalent and polyvalent candidates for an HCV vaccine, including natural, consensus and synthetic 'mosaic' sequence cocktails. Mosaic vaccine reagents were designed using a computational approach first applied to and demonstrated experimentally for human immunodeficiency virus type 1 (HIV-Delta). Mosaic proteins resemble natural proteins, but are assembled from fragments of natural sequences via a genetic algorithm and optimized to maximize the coverage of potential T-cell epitopes (all 9-mers) found in natural sequences and to minimize the inclusion of rare 9-mers to avoid vaccine-specific responses. Genotype 1-specific and global vaccine cocktails were evaluated. Among vaccine candidates considered, polyvalent mosaic sequences provided the best coverage of both known and potential epitopes and had the fewest rare epitopes. A global vaccine based on conserved proteins across genotypes may be feasible, as a five-antigen mosaic cocktail provided 90, 77 and 70% coverage of the Core, NS3 and NS4 proteins, respectively; protein coverage diminished with increased protein variability, dropping to 38% for NS2. For the genotype 1-specific vaccine, the H77 prototype vaccine sequence matched only 50% of the potential epitopes in the population, whilst a polyprotein three-antigen mosaic cocktail increased potential epitope coverage to 83%. More than 75% coverage of all HCV proteins was achieved with a three-antigen mosaic cocktail, suggesting that genotype-specific vaccines could also include the more variable proteins.

Molecular and epidemiological profiles of hepatitis C virus genotype 4 in Denmark.

The prevalence of hepatitis C virus (HCV) genotype 4 has increased throughout Europe. This is an epidemiological study of patients infected chronically with HCV genotype 4 in Denmark. The HCV strains analyzed originated from patient samples collected between 1999 and 2007 as part of the national Danish hepatitis B and C network, DANHEP. Sequence analyses were based on the envelope 1 region of HCV. Results from a total of 72 patients indicated a high degree of genetic heterogeneity. Fifty-six patients (78%) were infected with one of the three dominating subtypes: 4d, 4a, or 4r. The remaining 16 patients (22%) were infected with subtypes 4h, 4k, 4l, 4n, 4o, or 4Unclassified. Three epidemiological profiles were identified: (1) patients infected with HCV by intravenous drug use were infected solely with subtype 4d. They were all of European origin, and 15 of the 16 patients were ethnic Danes. No single transmission event could be confirmed, but the pairwise nucleotide identity within the patients of Danish origin was relatively high (∼95%), suggesting a recent introduction into Denmark. (2) The 21 patients infected with subtype 4a all came from Northern Africa, Egypt, Pakistan, or the Middle East. (3) Patients from Southern Africa dominated among patients infected with subtype 4r (10 of 12 patients). This study demonstrates that HCV genotype 4d has been introduced in and spread among Danish intravenous drug users. The remaining subtypes show restricted distribution, infecting almost exclusively patients from geographical areas with a relatively high prevalence of HCV genotype 4 infections.

The hepatitis C sequence database in Los Alamos.

The hepatitis C virus (HCV) is a significant public health threat worldwide. The virus is highly variable and evolves rapidly, making it an elusive target for the immune system and for vaccine and drug design. Presently, approximately 50 000 HCV sequences have been published. A central website that provides annotated sequences and analysis tools will be helpful to HCV scientists worldwide. The HCV sequence database collects and annotates sequence data, and provides them to the public via a website that contains a user-friendly search interface and a large number of sequence analysis tools, following the model of the highly regarded and widely used Los Alamos HIV database. The HCV website can be accessed via http://hcv.lanl.gov and http://hcv-db.org.

Web-based design and evaluation of T-cell vaccine candidates.

UNLABELLED: We present a suite of on-line tools to design candidate vaccine proteins, and to assess antigen potential, using coverage of k-mers (as proxies for potential T-cell epitopes) as a metric. The vaccine design tool uses the recently published 'mosaic' method to generate protein sequences optimized for coverage of high-frequency k-mers; the coverage-assessment tools facilitate coverage comparisons for any potential antigens. To demonstrate these tools, we designed mosaic protein sets for B-clade HIV-1 Gag, Pol and Nef, and compared them to antigens used in a recent human vaccine trial. AVAILABILITY: http://hiv.lanl.gov/content/sequence/MOSAIC/.

Nomenclature and numbering of the hepatitis C virus.

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. Consistency in numbering is also increasingly required for functional and clinical studies of HCV. For example, an unambiguous method for referring to amino acid substitutions at specific positions in NS3 and NS5B coding sequences associated with resistance to specific HCV inhibitors is essential in the investigation of antiviral treatment. Inconsistent and inaccurate numbering of locations in DNA and protein sequences is becoming a problem in the HCV scientific literature.A group of experts in the field of HCV genetic variability, and those involved in development of HCV sequence databases, the Hepatitis Virus Database (Japan), euHCVdb (France), and the Los Alamos National Laboratory (United States), convened to reexamine 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. They also discussed how HCV sequence databases could introduce and facilitate a standardized numbering system for HCV nucleotides, proteins, and epitopes.A comprehensive listing of all currently classified variants of HCV incorporates a number of agreed genotype and subtype name reassignments to create consistency in nomenclature. A consensus proposal was drawn up for the classification of new variants into genotypes and subtypes, which recognizes and incorporates new knowledge of HCV genetic diversity and epidemiology. The proposed numbering system was adapted from the Los Alamos HIV database, with elements from the hepatitis B virus numbering system. The system comprises both nucleotides and amino acid sequences and epitopes, and uses the full-length genome sequence of isolate H77 (accession number AF009606) as a reference. It includes a method for numbering insertions and deletions relative to this reference sequence.

Evidence for human leukocyte antigen heterozygote advantage against hepatitis C virus infection.

UNLABELLED: Outcomes of infection with hepatitis C virus (HCV) vary widely, from asymptomatic clearance to chronic infection, leading to complications that include fibrosis, cirrhosis, hepatocellular carcinoma, and liver failure. Previous studies have reported statistical associations between human leukocyte antigen (HLA) heterozygosity and favorable outcomes of infection with either hepatitis B virus (HBV) or human immunodeficiency virus (HIV) (the "heterozygote advantage"). To investigate whether HLA zygosity is associated with outcome of HCV infection, we used data from the United States Organ Procurement and Transplantation Network database of 52,435 liver transplant recipients from 1995 through 2005. Of these, 30,397 were excluded for lack of HLA data, retransplantation, known HIV infection, or insufficient information regarding HBV infection. The remaining cases were analyzed for associations between HCV infection and HLA zygosity with 1-sided Fisher's exact tests. Results show significantly lower proportions of HLA-DRB1 heterozygosity among HCV-infected than uninfected cases. The differences were more pronounced with alleles represented as functional supertypes (P = 1.05 x 10(-6)) than as low-resolution genotypes (P = 1.99 x 10(-3)). No significant associations between zygosity and HCV infection were found for other HLA loci. CONCLUSION: These findings constitute evidence for an advantage among carriers of different supertype HLA-DRB1 alleles against HCV infection progression to end-stage liver disease in a large-scale, long-term study population. Considering HLA polymorphism in terms of supertype diversity is recommended in strategies to design association studies for robust results across populations and in trials to improve treatment options for patients with chronic viral infection. Access to deidentified clinical information relating genetic variation to viral infection improves understanding of variation in infection outcomes and might help to personalize medicine with treatment options informed in part by human genetic variation.

A set of reference sequences for the hepatitis C genotypes 4d, 4f, and 4k covering the full open reading frame.

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.

jpHMM at GOBICS: a web server to detect genomic recombinations in HIV-1.

Detecting recombinations in the genome sequence of human immunodeficiency virus (HIV-1) is crucial for epidemiological studies and for vaccine development. Herein, we present a web server for subtyping and localization of phylogenetic breakpoints in HIV-1. Our software is based on a jumping profile Hidden Markov Model (jpHMM), a probabilistic generalization of the jumping-alignment approach proposed by Spang et al. The input data for our server is a partial or complete genome sequence from HIV-1; our tool assigns regions of the input sequence to known subtypes of HIV-1 and predicts phylogenetic breakpoints. jpHMM is available online at http://jphmm.gobics.de/.

Genomic analysis of HIV type 1 strains derived from a mother and child pair of long-term nonprogressors.

To investigate the viral features of long-term nonprogressive HIV-1 infection and the selection of viral genomes, we studied serial complete HIV-1 sequences obtained from a mother-child pair, both long-term nonprogressors. Analysis of four genomic sequences demonstrated that all viral genes were intact, lacking major deletions or premature stop codons to easily explain the slow disease progression. These data suggest that viral attenuation, if present, was caused by subtle sequence variations or virus-host interactions. Serial sequences from an HIV-1-infected mother-child pair afforded us the opportunity to examine the immune selection of HIV-1 sequences years after transmission between individuals. We demonstrated that the daughter's strains were most likely subjected to immunoselection or immunoediting according to the presence of novel MHC class I alleles that differed between mother and daughter. An analysis of nef-specific cytotoxic T-lymphocyte responses in the child, whose HIV-1 nef sequence differed from the maternal nef, supported this interpretation. This study highlights the potential of full genome analysis in the investigation of pathogenesis and immune selection during HIV-1 evolution.

Los Alamos hepatitis C virus sequence and human immunology databases: an expanding resource for antiviral research.

The hepatitis C virus (HCV) resource at Los Alamos (hcv.lanl.gov) provides access to multiple databases: one containing annotated sequences and the other a repository of immunogenic epitopes. They are derived from databases originally developed for HIV research (hiv.lanl.gov). HCV and HIV are RNA viruses with relatively compact genomes (around 10 kb) that are extraordinarily variable, both within and between hosts. This diversity requires methods to track and exclude variants from an individual infection or from epidemiologically related infections, and tools to analyse the variation. The HCV immunology database contains a curated inventory of immunogenic epitopes and information about their interaction with the host immune system, with associated retrieval and analysis tools. This interactive resource provides flexible retrieval tools for sequences, epitopes, clinical information, and meta-data, as well as utilities for scientific data analysis, to investigators with internet access and a web browser. This paper describes the types of data and the services that these databases offer, the tools they provide, and their configuration and use. Examples of applications to clonal analysis for drug-resistance mutations are shown.

A jumping profile Hidden Markov Model and applications to recombination sites in HIV and HCV genomes.

BACKGROUND: Jumping alignments have recently been proposed as a strategy to search a given multiple sequence alignment A against a database. Instead of comparing a database sequence S to the multiple alignment or profile as a whole, S is compared and aligned to individual sequences from A. Within this alignment, S can jump between different sequences from A, so different parts of S can be aligned to different sequences from the input multiple alignment. This approach is particularly useful for dealing with recombination events. RESULTS: We developed a jumping profile Hidden Markov Model (jpHMM), a probabilistic generalization of the jumping-alignment approach. Given a partition of the aligned input sequence family into known sequence subtypes, our model can jump between states corresponding to these different subtypes, depending on which subtype is locally most similar to a database sequence. Jumps between different subtypes are indicative of intersubtype recombinations. We applied our method to a large set of genome sequences from human immunodeficiency virus (HIV) and hepatitis C virus (HCV) as well as to simulated recombined genome sequences. CONCLUSION: Our results demonstrate that jumps in our jumping profile HMM often correspond to recombination breakpoints; our approach can therefore be used to detect recombinations in genomic sequences. The recombination breakpoints identified by jpHMM were found to be significantly more accurate than breakpoints defined by traditional methods based on comparing single representative sequences.

Comparative analysis of hepatitis C virus phylogenies from coding and non-coding regions: the 5' untranslated region (UTR) fails to classify subtypes.

BACKGROUND: The duration of treatment for HCV infection is partly indicated by the genotype of the virus. For studies of disease transmission, vaccine design, and surveillance for novel variants, subtype-level classification is also needed. This study used the Shimodaira-Hasegawa test and related statistical techniques to compare phylogenetic trees obtained from coding and non-coding regions of a whole-genome alignment for the reliability of subtyping in different regions. RESULTS: Different regions of the HCV genome yield inconsistent phylogenies, which can lead to erroneous conclusions about classification of a given infection. In particular, the highly conserved 5' untranslated region (UTR) yields phylogenetic trees with topologies that differ from the HCV polyprotein and complete genome phylogenies. Phylogenetic trees from the NS5B gene reliably cluster related subtypes, and yield topologies consistent with those of the whole genome and polyprotein. CONCLUSION: These results extend those from previous studies and indicate that, unlike the NS5B gene, the 5' UTR contains insufficient variation to resolve HCV classifications to the level of viral subtype, and fails to distinguish genotypes reliably. Use of the 5' UTR for clinical tests to characterize HCV infection should be replaced by a subtype-informative test.

Integrated sequence and immunology filovirus database at Los Alamos.

The Ebola outbreak of 2013-15 infected more than 28 000 people and claimed more lives than all previous filovirus outbreaks combined. Governmental agencies, clinical teams, and the world scientific community pulled together in a multifaceted response ranging from prevention and disease control, to evaluating vaccines and therapeutics in human trials. As this epidemic is finally coming to a close, refocusing on long-term prevention strategies becomes paramount. Given the very real threat of future filovirus outbreaks, and the inherent uncertainty of the next outbreak virus and geographic location, it is prudent to consider the extent and implications of known natural diversity in advancing vaccines and therapeutic approaches. To facilitate such consideration, we have updated and enhanced the content of the filovirus portion of Los Alamos Hemorrhagic Fever Viruses Database. We have integrated and performed baseline analysis of all family ITALIC! Filoviridaesequences deposited into GenBank, with associated immune response data, and metadata, and we have added new computational tools with web-interfaces to assist users with analysis. Here, we (i) describe the main features of updated database, (ii) provide integrated views and some basic analyses summarizing evolutionary patterns as they relate to geo-temporal data captured in the database and (iii) highlight the most conserved regions in the proteome that may be useful for a T cell vaccine strategy.Database URL:www.hfv.lanl.gov.

Los alamos hepatitis C immunology database.

The Los Alamos Hepatitis C Virus (HCV) Sequence Database (http://hcv.lanl.gov or http://hcv-db.org) was officially launched in September 2003. The sister HCV Immunology Database was made public in September 2004. The HCV Immunology Database is based on the Human Immunodeficiency Virus (HIV) Immunology Database. The HCV Immunology Database contains a curated inventory of immunological epitopes in HCV and their interaction with the immune system, with associated retrieval and analysis tools. This article describes in detail the types of data and services that the new database offers, the tools provided and the database framework. The data and some of the HCV database tools are available for download for non-commercial use.

HIV sequence databases.

Two important databases are often used in HIV genetic research, the HIV Sequence Database in Los Alamos, which collects all sequences and focuses on annotation and data analysis, and the HIV RT/Protease Sequence Database in Stanford, which collects sequences associated with the development of viral resistance against anti-retroviral drugs and focuses on analysis of those sequences. The types of data and services these two databases offer, the tools they provide, and the way they are set up and operated are described in detail.