Michael A. Purdy

Consensus proposals for classification of the family Hepeviridae.

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.

Proposed reference sequences for hepatitis E virus subtypes.

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 (http://talk.ictvonline.org/r.ashx?C). 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.

Chronic Infection With Camelid Hepatitis E Virus in a Liver-transplant Recipient Who Regularly Consumes Camel Meat and Milk.

There have been increasing reports of food-borne zoonotic transmission of hepatitis E virus (HEV) genotype 3, which causes chronic infections in immunosuppressed patients. We performed phylogenetic analyses of the HEV sequence (partial and full-length) from 1 patient from the Middle East who underwent liver transplantation, and compared it with other orthohepevirus A sequences. We found the patient to be infected by camelid HEV. This patient regularly consumed camel meat and milk, therefore camelid HEV, which is genotype 7, might infect human beings. Our finding links consumption of camel-derived food products to post-transplantation hepatitis E, which, if detected at early stages, can be cured with antiviral therapy and reduced administration of immunosuppressive agents.

Genetic Variability and the Classification of Hepatitis E Virus

ABSTRACT 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.

The sequence of hepatitis E virus isolated directly from a single source during an outbreak in China

In this study an IgM antibody-mediated antigen-capture procedure for direct extraction of hepatitis E virus (HEV) RNA from clinical specimens was developed and used with an efficient method for generating viral cDNA that was subsequently sequenced using the dideoxy chain termination method. This is the first time the complete HEV genome has been isolated directly from a single human clinical specimen obtained during an outbreak of enterically transmitted non-A, non-B hepatitis. When the Chinese-derived sequence was compared with the original isolate of Burmese HEV from an experimentally infected cynomolgus macaque, the homology between the two sequences was 94% and 98.5% at the nucleotide and amino acid levels, respectively. The methods we developed for generating and sequencing genomic HEV cDNA dramatically improved the efficiency of cloning the viral genome and should be helpful for continued analysis of this virus as well as other RNA viruses that have proven to be difficult to clone and sequence directly.

Sequence Heterogeneity of TT Virus and Closely Related Viruses

ABSTRACT TT virus (TTV) is a recently discovered infectious agent originally obtained from transfusion-related hepatitis. However, the causative link between the TTV infection and liver disease remains uncertain. Recent studies demonstrated that genome sequences of different TTV strains are significantly divergent. To assess genetic heterogeneity of the TTV genome in more detail, a sequence analysis of PCR fragments (271 bp) amplified from open reading frame 1 (ORF1) was performed. PCR fragments were amplified from 5 to 40% of serum specimens obtained from patients with different forms of hepatitis who reside in different countries (e.g., China, Egypt, Vietnam, and the United States) and from normal human specimens obtained from U.S. residents. A total of 170 PCR fragments were sequenced and compared to sequences derived from the corresponding TTV genome region deposited in GenBank. Genotypes 2 and 3 were found to be significantly more genetically related than any other TTV genotype. Moreover, three sequences were shown to be almost equally related to both genotypes 2 and 3. These observations suggest a merger of genotypes 2 and 3 into one genotype, 2/3. Additionally, five new groups of TTV sequences were identified. One group represents a new genotype, whereas the other four groups were shown to be more evolutionary distant from all known TTV sequences. The evolutionary distances between these four groups were also shown to be greater than between TTV genotypes. The phylogenetic analysis suggested that these four new genetic groups represent closely related yet different viral species. Thus, TTV exists as a “swarm” of at least five closely related but different viruses. These observations suggest a high degree of genetic complexity within the TTV population. The finding of the additional TTV-related species should be taken into consideration when the association between TTV infections and human diseases of unknown etiology is studied.

Evolutionary History and Population Dynamics of Hepatitis E Virus

Background Hepatitis E virus (HEV) is an enterically transmitted hepatropic virus. It segregates as four genotypes. All genotypes infect humans while only genotypes 3 and 4 also infect several animal species. It has been suggested that hepatitis E is zoonotic, but no study has analyzed the evolutionary history of HEV. We present here an analysis of the evolutionary history of HEV. Methods and Findings The times to the most recent common ancestors for all four genotypes of HEV were calculated using BEAST to conduct a Bayesian analysis of HEV. The population dynamics for genotypes 1, 3 and 4 were analyzed using skyline plots. Bayesian analysis showed that the most recent common ancestor for modern HEV existed between 536 and 1344 years ago. The progenitor of HEV appears to have given rise to anthropotropic and enzootic forms of HEV, which evolved into genotypes 1 and 2 and genotypes 3 and 4, respectively. Population dynamics suggest that genotypes 1, 3 and 4 experienced a population expansion during the 20th century. Genotype 1 has increased in infected population size ∼30–35 years ago. Genotype 3 and 4 have experienced an increase in population size starting late in the 19th century until ca.1940-45, with genotype 3 having undergone additional rapid expansion until ca.1960. The effective population size for both genotype 3 and 4 rapidly declined to pre-expansion levels starting in ca.1990. Genotype 4 was further examined as Chinese and Japanese sequences, which exhibited different population dynamics, suggesting that this genotype experienced different evolutionary history in these two countries. Conclusions HEV appears to have evolved through a series of steps, in which the ancestors of HEV may have adapted to a succession of animal hosts leading to humans. Analysis of the population dynamics of HEV suggests a substantial temporal variation in the rate of transmission among HEV genotypes in different geographic regions late in the 20th Century.

The molecular epidemiology of hepatitis E virus infection.

Molecular characterization of various hepatitis E virus (HEV) strains circulating among humans and animals (particularly swine, deer and boars) in different countries has revealed substantial genetic heterogeneity. The distinctive four-genotype distribution worldwide of mammalian HEV and varying degrees of genetic relatedness among local strains suggest a long and complex evolution of HEV in different geographic regions. The population expansion likely experienced by mammalian HEV in the second half of the 20th century is consistent with an extensive genetic divergence of HEV strains and high prevalence of HEV infections in many parts of the world, including developed countries. The rate and mechanisms of human-to-human transmission and zoonotic transmission to humans vary geographically, thus contributing to the complexity of HEV molecular evolution.

Expression of a hepatitis E virus (HEV)-trpE fusion protein containing epitopes recognized by antibodies in sera from human cases and experimentally infected primates

SummaryA 1700 base cDNA fragment coding for the putative structural gene(s) of hepatitis E virus (HEV) was inserted into the pATH 10 expression vector. The fusion protein (C2) expressed by this plasmid was found to contain epitopes recognized by anti-HEV antibodies. C 2 protein was used in a Western blot format to examine its usefulness in detecting anti-HEV antibodies in well documented human cases of HEV and non-human primates infected with HEV. Both IgM and IgG anti-HEV could be detected in our Western blot assay. This Western blot assay was found not to detect antibodies from acute-phase sera from patients with either HAV or HBV. The C 2 protein contains broadly cross-reactive epitopes, and the Western blot assay was able to detect anti-HEV antibodies in patient sera from Asia, Africa, and North America. The optimum serum dilution for the detection of both IgM and IgG was 1:25.

Temporal Variations in the Hepatitis C Virus Intrahost Population during Chronic Infection

ABSTRACT The intrahost evolution of hepatitis C virus (HCV) holds keys to understanding mechanisms responsible for the establishment of chronic infections and to development of a vaccine and therapeutics. In this study, intrahost variants of two variable HCV genomic regions, HVR1 and NS5A, were sequenced from four treatment-naïve chronically infected patients who were followed up from the acute stage of infection for 9 to 18 years. Median-joining network analysis indicated that the majority of the HCV intrahost variants were observed only at certain time points, but some variants were detectable at more than one time point. In all patients, these variants were found organized into communities or subpopulations. We hypothesize that HCV intrahost evolution is defined by two processes: incremental changes within communities through random mutation and alternations between coexisting communities. The HCV population was observed to incrementally evolve within a single community during approximately the first 3 years of infection, followed by dispersion into several subpopulations. Two patients demonstrated this pattern of dispersion for the rest of the observation period, while HCV variants in the other two patients converged into another single subpopulation after ∼9 to 12 years of dispersion. The final subpopulation in these two patients was under purifying selection. Intrahost HCV evolution in all four patients was characterized by a consistent increase in negative selection over time, suggesting the increasing HCV adaptation to the host late in infection. The data suggest specific staging of HCV intrahost evolution.