Swine Hepatitis E Virus Research Articles

Full-Length Sequences of Six Hepatitis E Virus Isolates of Genotypes III and IV from Patients with Sporadic Acute or Fulminant Hepatitis in Japan

Objective: Ranges of variation and conservation in sequence need to be defined for detecting and genotyping hepatitis E virus (HEV). Methods: Six HEV isolates from Japanese patients were sequenced over the entire genome and compared phylogenetically along with 16 reported HEV isolates, including two from pigs. Results: Three of the six HEV isolates were of genotype III, and the remaining three were of genotype IV. Local clusterings of Japanese HEV isolates were observed in the phylogenetic analyses, including a swine HEV isolate reported previously (swJ570). All six HEV isolates possessed three open reading frames (ORFs). The ORF3 in the three isolates of genotype III were in a different reading frame, while that in the three isolates of genotype IV were in the same reading frame as ORF1. A stretch of 46–96 nucleotides was identified, point mutations and deletions in which were specific for the four genotypes (I–IV). A polymerase chain reaction method was developed with 9 nested universal primers, deduced from conserved regions in the 5′-terminal sequences of the 22 HEV genomes. Conclusions: Conserved and genotype-specific variation in HEV sequences, identified in the comparison of 22 full-length genomes, would be useful in designing primers for sensitive detection and specific genotyping of HEV RNA.

Genetic characterization and sequence heterogeneity of a canadian isolate of Swine hepatitis E virus.

Swine hepatitis E virus (HEV) is a newly identified potentially zoonotic agent that is possibly transmitted to humans from pigs. Swine HEV is prevalent in pig populations and does not cause abnormal clinical symptoms in infected pigs, further implicating a likelihood of a risk of transmission to humans by normal contact. To date in North America, only one strain of swine HEV (strain US swine) has been fully sequenced. In the present study, we identified a swine HEV isolate from pigs in Canada, designated the Arkell strain, and determined the full length of the genomic sequence. The genome of Canadian strain Arkell consisted of 7,242 nucleotides, excluding the poly(A) tail of at least 15 A residues. The genome contained three open reading frames (ORFs), ORF1, ORF2, and ORF3, which had coding capacities for proteins of 1,708, 660, and 122 amino acids, respectively. Comparative analysis of the full-length genomic sequence indicated that the sequence of strain Arkell was distinct from those of all other known HEV isolates by 13 to 27% and shared the highest degrees of identity with human HEV isolates US-1 and US-2, HEV isolate US swine, and the human and swine HEV isolates recently isolated in Japan. On the basis of sequence similarities and phylogenetic analyses, HEV strain Arkell was grouped into genotype 3. The sequence of the Arkell swine HEV isolate differed from those of HEV isolate US swine and HEV isolate Japan swine by 13 and 14%, respectively. To date, two isolates of swine HEV (isolates Arkell and SK3 [D. Yoo et al., Clin. Diagn. Lab. Immunol. 8:1213-1219, 2001]) have been identified in Canadian pigs, and their sequences also differ from each other by 11.8%. Our studies indicate that, as with human HEV strains, swine HEV isolates exhibit extensive genetic heterogeneity.

Heterogeneity and seroprevalence of a newly identified avian hepatitis e virus from chickens in the United States.

We recently identified and characterized a novel virus, designated avian hepatitis E virus (avian HEV), from chickens with hepatitis-splenomegaly syndrome (HS syndrome) in the United States. Avian HEV is genetically related to but distinct from human and swine HEVs. To determine the extent of genetic variation and the seroprevalence of avian HEV infection in chicken flocks, we genetically identified and characterized 11 additional avian HEV isolates from chickens with HS syndrome and assessed the prevalence of avian HEV antibodies from a total of 1,276 chickens of different ages and breeds from 76 different flocks in five states (California, Colorado, Connecticut, Virginia, and Wisconsin). An enzyme-linked immunosorbent assay using a truncated recombinant avian HEV ORF2 antigen was developed and used to determine avian HEV seroprevalence. About 71% of chicken flocks and 30% of chickens tested in the study were positive for antibodies to avian HEV. About 17% of chickens younger than 18 weeks were seropositive, whereas about 36% of adult chickens were seropositive. By using a reverse transcription-PCR (RT-PCR) assay, we tested 21 bile samples from chickens with HS syndrome in California, Connecticut, New York, and Wisconsin for the presence of avian HEV RNA. Of the 21 bile samples, 12 were positive for 30- to 35-nm HEV-like virus particles by electron microscopy (EM). A total of 11 of the 12 EM-positive bile samples and 6 of the 9 EM-negative bile samples were positive for avian HEV RNA by RT-PCR. The sequences of a 372-bp region within the helicase gene of 11 avian HEV isolates were determined. Sequence analyses revealed that the 11 field isolates of avian HEV had 78 to 100% nucleotide sequence identities to each other, 79 to 88% identities to the prototype avian HEV, 76 to 80% identities to chicken big liver and spleen disease virus, and 56 to 61% identities to other known strains of human and swine HEV. The data from this study indicated that, like swine and human HEVs, avian HEV isolates are genetically heterogenic and that avian HEV infection is enzoonotic in chicken flocks in the United States.

Spread of hepatitis E virus among different-aged pigs: two-year survey in Taiwan.

Swine are reservoirs of hepatitis E virus (HEV). In this study, a 2-year survey of HEV in feces and sera of swine was conducted to determine if: 1) HEV has circulated among pigs for some time in Taiwan; 2) the spread of HEV among different-aged pigs; and 3) there exists HEV strains possibly imported through trading. From 1998-2000, 521 serum samples and 54 fecal specimens from pigs were examined by reverse transcription polymerase chain reaction. None of the 11 pigs in suckling stage (< 2 months) were serum HEV RNA positive. The highest viremia rate (4.5%) was in pigs of 2 months age, followed by 1.2% and 1.8% in pigs of growing (3-4 months) and finishing stages (5-6 months), and none in pigs older than 6 months. Viremia showed little variation in different years and areas. None of the 20 fecal samples from pigs in suckling stage were HEV RNA positive, whereas 9% of the 34 samples from pigs in growing or finishing stages were positive. Most swine HEV isolates in Taiwan clustered within the genotype 4, whereas the three HEV isolates cloned from pigs imported recently from the U.S. belonged to the genotype 3 HEV in the U.S. The results suggest that HEV may infect pigs at an early growing stage and spread unnoticed among pigs and possibly across countries through trading.

Prevalence of antibodies to hepatitis E virus in veterinarians working with swine and in normal blood donors in the United States and other countries.

Hepatitis E virus (HEV) is endemic in many developing and some industrialized countries. It has been hypothesized that animals may be the source of infection. The recent identification of swine HEV in U.S. pigs and the demonstration of its ability to infect across species have lent credence to this hypothesis. To assess the potential risk of zoonotic HEV infection, we tested a total of 468 veterinarians working with swine (including 389 U.S. swine veterinarians) and 400 normal U.S. blood donors for immunoglobulin G anti-HEV. Recombinant capsid antigens from a U.S. strain of swine HEV and from a human HEV strain (Sar-55) were each used in an enzyme-linked immunosorbent assay. The anti-HEV prevalence assayed with the swine HEV antigen showed 97% concordance with that obtained with the human HEV antigen (kappa = 92%). Among the 295 swine veterinarians tested from the eight U.S. states (Minnesota, Indiana, Nebraska, Iowa, Illinois, Missouri, North Carolina, and Alabama) from which normal blood donor samples were available, 26% were positive with Sar-55 antigen and 23% were positive with swine HEV antigen. In contrast, 18% of the blood donors from the same eight U.S. states were positive with Sar-55 antigen and 17% were positive with swine HEV antigen. Swine veterinarians in the eight states were 1.51 times more likely when tested with swine HEV antigen (95% confidence interval, 1.03 to 2.20) and 1.46 times more likely when tested with Sar-55 antigen (95% confidence interval, 0.99 to 2.17) to be anti-HEV positive than normal blood donors. We did not find a difference in anti-HEV prevalence between veterinarians who reported having had a needle stick or cut and those who had not or between those who spent more time (> or = 80% of the time) and those who spent less time (< or = 20% of the time) working with pigs. Similarly, we did not find a difference in anti-HEV prevalence according to four job categories (academic, practicing, student, and industry veterinarians). There was a difference in anti-HEV prevalence in both swine veterinarians and blood donors among the eight selected states, with subjects from Minnesota six times more likely to be anti-HEV positive than those from Alabama. Age was not a factor in the observed differences from state to state. Anti-HEV prevalence in swine veterinarians and normal blood donors was age specific and paralleled increasing age. The results suggest that swine veterinarians may be at somewhat higher risk of HEV infection than are normal blood donors.

Determination of the nucleotide sequences at the extreme 5' and 3' ends of swine hepatitis E virus genome.

The nucleotide sequences at the extreme 5' and 3' ends of swine hepatitis E virus (swine HEV) genome were determined, and genomic sequence of swine HEV is now complete. Sequence analysis revealed that the 3' and 5' non-coding regions (NCRs) of swine HEV are closely related to that of the US-1 and US-2 strains of human HEV. Like the two U.S. strains of human HEV, an extra G residue immediately proceeding the poly(A) tail was identified in swine HEV. The 5' NCR of swine HEV also differed from many HEV strains: it lacks an A residue at its 5' very end, and the extra 9 nucleotides in the US-2 strain. In the 3' NCR, swine HEV shared 90-91% nucleotide sequence identities with the US-1 and US-2 strains but only about 58-65% identities with other HEV strains. This study further suggests that the US-1 and US-2 strains of human HEV may be of swine origin. The availability of the complete sequence of swine HEV should facilitate the construction of an infectious cDNA clone of swine HEV.

Use of a swine bioassay and a RT-PCR assay to assess the risk of transmission of swine hepatitis E virus in pigs

The objective of this study was to assess the risk of transmission of swine hepatitis E virus (swine HEV) to naı̈ve pigs by inoculation with tissues or feces collected from pigs infected experimentally with swine HEV. Seventy-five, 3-week-old pigs were assigned randomly to 24 groups of 3–4 pigs and inoculated with homogenates of tissues (liver, heart, pancreas, or skeletal muscle) or a suspension of feces from swine HEV-infected pigs collected at 3, 7, 14, 20, 27, or 55 days post inoculation (DPI). Each inoculum was prepared as a 10% suspension (w/v) in PBS buffer and tested by a semi-quantitative RT-PCR for swine HEV RNA and by the swine bioassay. The inoculation route was intravenous for liver, heart and pancreas, and via stomach tube for skeletal muscle and fecal suspension. The liver homogenate inocula and feces collected at 3–7 and 14–20 DPI were positive for swine HEV RNA by RT-PCR. The pigs inoculated with liver homogenates collected at 3–7 and 14–20 DPI developed anti-HEV antibodies and swine HEV RNA was detected in their sera. Pigs inoculated with heart, pancreas, skeletal muscle homogenates or fecal suspensions failed to develop anti-HEV antibodies. These findings suggest that there is a potential risk of transmission of swine HEV via liver tissue from infected pigs in the early stages (3–20 DPI) of infection and the in vitro RT-PCR assay correlates well with the swine bioassay.

Evidence of extrahepatic sites of replication of the hepatitis E virus in a swine model.

Hepatitis E virus (HEV) is the major cause of enterically transmitted non-A, non-B hepatitis in many developing countries and is also endemic in many industrialized countries. Due to the lack of an effective cell culture system and a practical animal model, the mechanisms of HEV pathogenesis and replication are poorly understood. Our recent identification of swine HEV from pigs affords us an opportunity to systematically study HEV replication and pathogenesis in a swine model. In an early study, we experimentally infected specific-pathogen-free pigs with two strains of HEV: swine HEV and the US-2 strain of human HEV. Eighteen pigs (group 1) were inoculated intravenously with swine HEV, 19 pigs (group 2) were inoculated with the US-2 strain of human HEV, and 17 pigs (group 3) were used as uninoculated controls. The clinical and pathological findings have been previously reported. In this expanded study, we aim to identify the potential extrahepatic sites of HEV replication using the swine model. Two pigs from each group were necropsied at 3, 7, 14, 20, 27, and 55 days postinoculation (DPI). Thirteen different types of tissues and organs were collected from each necropsied animal. Reverse transcriptase PCR (RT-PCR) was used to detect the presence of positive-strand HEV RNA in each tissue collected during necropsy at different DPI. A negative-strand-specific RT-PCR was standardized and used to detect the replicative, negative strand of HEV RNA from tissues that tested positive for the positive-strand RNA. As expected, positive-strand HEV RNA was detected in almost every type of tissue at some time point during the viremic period between 3 and 27 DPI. Positive-strand HEV RNA was still detectable in some tissues in the absence of serum HEV RNA from both swine HEV- and human HEV-inoculated pigs. However, replicative, negative-strand HEV RNA was detected primarily in the small intestines, lymph nodes, colons, and livers. Our results indicate that HEV replicates in tissues other than the liver. The data from this study may have important implications for HEV pathogenesis, xenotransplantation, and the development of an in vitro cell culture system for HEV.

HEPATITIS E

Hepatitis E, previously known as enterically transmitted non-A, non-B hepatitis, is an infectious viral disease with clinical and morphologic features of acute hepatitis. Its causative agent, hepatitis E virus, consists of small, 32- to 34-nm diameter, icosahedral, nonenveloped particles with a single-stranded, positive-sense, 7.5-kb RNA. The virus has two main geographically distinct strains, Asian and Mexican; recently, novel isolates from nonendemic areas and a genetically related swine HEV have been described. HEV is responsible for large epidemics of acute hepatitis and a proportion of sporadic hepatitis cases in the Indian subcontinent, southeast and central Asia, the Middle East, parts of Africa, and Mexico. The virus is excreted in feces and is transmitted predominantly by fecal-oral route, usually through contaminated water. Person-to-person transmission is uncommon. Clinical attack rates are the highest among young adults. Recent evidence suggests that humans with subclinical HEV infection and animals may represent reservoirs of HEV; however, further data are needed. Diagnosis of hepatitis E is usually made by detection of specific IgM antibody, which disappears rapidly over a few months; IgG anti-HEV persists for at least a few years. Clinical illness is similar to other forms of acute viral hepatitis except in pregnant women, in whom illness is particularly severe with a high mortality rate. Subclinical and unapparent infections may occur; however, chronic infection is unknown. No specific treatment is yet available. Use of clean drinking water and proper sanitation is currently the most effective method of prevention. Passive immunization has not been proved to be effective, and recombinant vaccines for travelers to disease-endemic areas and for pregnant women currently are being developed.

Clinical and epidemiological implications of swine hepatitis E virus infection

In nonendemic areas, most patients with acute hepatitis E were infected through traveling to endemic areas. However, some patients did not have a history of foreign travel before infection. Furthermore, high seroprevalence rates of antibody to hepatitis E virus (anti-HEV) were found in the general adult population in some countries without any recorded outbreak of hepatitis E. The significance of anti-HEV assay in these subjects remains obscure. To study if swine might be a source of HEV infection, HEV was tested in sera of 235 pigs in Taiwan, and from 5 patients with acute HEV infection who either denied or did not provide any foreign travel history. Three (1.3%) pigs had detectable swine HEV RNA. The swine and human HEV strains from Taiwan formed a monophyletic group, distinct from three previously reported groups: the United States human and swine HEV strains, the Mexico strain, and the largest group composed of the Asian and the African strains. The identity of nucleotide sequences was 84-95% between swine and human HEV strains in Taiwan, and 72-79% between Taiwan strains and those from different areas. The predicted amino acid sequence of a Taiwan swine HEV strain within the peptide 3-2 used in commercial anti-HEV assay showed a high identity (91-94%) with those of other human and swine HEV strains. Swine may be a reservoir of HEV and subclinical swine HEV infection may occur. Cross-reactivity of current anti-HEV assay may account for the high prevalence rate of anti-HEV in the general population in nonendemic areas.

Identity of a novel swine hepatitis E virus in Taiwan forming a monophyletic group with Taiwan isolates of human hepatitis E virus.

Recently, we found that more than 10% of the cases of acute non-A, non-B, non-C hepatitis in Taiwan were caused by a novel strain of hepatitis E virus (HEV). Since none of these patients had a history of travel to areas where HEV is endemic, the source of transmission remains unclear. The recent discovery of a swine HEV in herd pigs in the United States has led us to speculate that HEV may also circulate in herd pigs in Taiwan and may serve as a reservoir for HEV in Taiwan. Of 275 herd pigs obtained from 10 pig farms in Taiwan, 102 (37%) were seropositive for serum anti-HEV immunoglobulin G (IgG). A 185-bp genomic sequence within the ORF-2 of the HEV genome was amplified and cloned from serum samples of an anti-HEV positive pig and subsequently from serum samples of a patient with acute hepatitis E. Sequence comparison revealed that the swine and human isolates of HEV share 97.3% identity. Phylogenetic analyses further showed that the Taiwan swine and human isolates of HEV form a distinct branch divergent from all other known strains of HEV, including the U.S. swine strain. To examine the potential risk of cross-species transmission of swine HEV to humans, the seroprevalences of anti-HEV IgG in 30 swine handlers, 20 pork dealers, and 50 control subjects were assessed and were found to be 26.7, 15, and 8%, respectively (for swine handlers versus controls, P = 0.048). Our findings may help provide an understanding of the modes of HEV transmission and may also raise potential public health concerns for HEV zoonosis.

Prevalence of antibodies to the hepatitis E virus in pigs from countries where hepatitis E is common or is rare in the human populationThis article is a US Government work and, as such, is in the public domain in the United States of America

Hepatitis E virus (HEV) is a very important public health concern in many developing countries where epidemics of hepatitis E are common. Sporadic cases of clinical hepatitis E not only occur in these countries but also occur uncommonly in patients with no known epidemiological exposure to HEV in industrialized countries. The source of infection in industrialized countries is unknown but it has been suggested that animals might serve as a reservoir for HEV in both settings. We recently identified and characterized an HEV strain (swine HEV) that infects large numbers of pigs in the United States. To assess the potential of pigs to serve as a global reservoir of HEV, we measured the prevalence of HEV antibodies in pigs in two countries where hepatitis E is endemic and two countries where it is not. Swine herds in all four countries contained many pigs that were seropositive for IgG anti-HEV, although the percentage of seropositive pigs varied greatly from herd to herd. A very limited number of pig handlers in the two endemic countries were also tested and most of them were found to be seropositive for HEV. The results from this study suggest that hepatitis E is enzootic in pigs regardless of whether HEV is endemic in the respective human population. J. Med. Virol. 59:297–302, 1999. Published 1999 Wiley-Liss, Inc.

Prevalence of antibodies to the hepatitis E virus in pigs from countries where hepatitis E is common or is rare in the human population

Hepatitis E virus (HEV) is a very important public health concern in many developing countries where epidemics of hepatitis E are common. Sporadic cases of clinical hepatitis E not only occur in these countries but also occur uncommonly in patients with no known epidemiological exposure to HEV in industrialized countries. The source of infection in industrialized countries is unknown but it has been suggested that animals might serve as a reservoir for HEV in both settings. We recently identified and characterized an HEV strain (swine HEV) that infects large numbers of pigs in the United States. To assess the potential of pigs to serve as a global reservoir of HEV, we measured the prevalence of HEV antibodies in pigs in two countries where hepatitis E is endemic and two countries where it is not. Swine herds in all four countries contained many pigs that were seropositive for IgG anti-HEV, although the percentage of seropositive pigs varied greatly from herd to herd. A very limited number of pig handlers in the two endemic countries were also tested and most of them were found to be seropositive for HEV. The results from this study suggest that hepatitis E is enzootic in pigs regardless of whether HEV is endemic in the respective human population. J. Med. Virol. 59:297-302, 1999. Published 1999 Wiley-Liss, Inc.

Serological evidence for swine hepatitis E virus infection in Australian pig herds

Hepatitis E virus (HEV) is an enterically transmitted human pathogen, with some similarities to caliciviruses. A variant of HEV was recently identified in pigs in the USA, infecting almost 100% of animals in commercial herds. Phylogenetic analysis suggests that this is a true ‘swine HEV’ distinct from the human virus, but the swine virus may also infect man. Using an in-house ELISA based on a highly conserved, recombinant HEV protein, we have examined collections of sera from Australian pigs for evidence of HEV infection in local pig herds. Sera from one research herd ( n = 32) were uniformly non-reactive, and this was used to establish an assay cut-off (= mean + 3 SD of reference pig serum reactivities). Screening of sera from other herds demonstrates that swine HEV is present in Australia, with reactivity observed in 30% (12/40) of random samples from two piggeries, 92–95% of pigs by the age of 16 weeks in two other piggeries ( n = 45), and 17% (15/59) of wild-caught pigs. Further studies are required to examine whether HEV causes disease in pigs and to determine the risk of swine HEV transmission to man.

Genetic and experimental evidence for cross-species infection by swine hepatitis E virus.

Prior to the recent discovery of the swine hepatitis E virus (swine HEV) in pigs from the midwestern United States, HEV was not considered endemic to this country. Since swine HEV is antigenically and genetically related to human strains of HEV, it was important to characterize this new virus further. The infectivity titer of a pool of swine HEV in pigs was determined in order to prepare a standardized reagent and to evaluate the dose response in pigs. Although the sequence of swine HEV varied extensively from those of most human strains of HEV, it was very closely related to the two strains of human HEV (US-1 and US-2) isolated in the United States. The U.S. strains which were recently recovered from two patients with clinical hepatitis E in the United States shared >/=97% amino acid identity with swine HEV in open reading frames 1 and 2. Phylogenetic analyses of different regions of the genome revealed that swine HEV and the U.S. strains grouped together and formed a distinct branch. These results suggested that swine HEV may infect humans. When we inoculated rhesus monkeys and a chimpanzee, experimental surrogates of humans, with swine HEV, the primates became infected. Furthermore, in a reciprocal experiment, specific-pathogen-free pigs were experimentally infected with the US-2 strain of human HEV that is genetically similar to swine HEV. These results provided experimental evidence for cross-species infection by the swine virus. Thus, humans appear to be at risk of infection with swine HEV or closely related viruses.

A novel virus in swine is closely related to the human hepatitis E virus.

A novel virus, designated swine hepatitis E virus (swine HEV), was identified in pigs. Swine HEV crossreacts with antibody to the human HEV capsid antigen. Swine HEV is a ubiquitous agent and the majority of swine >/=3 months of age in herds from the midwestern United States were seropositive. Young pigs naturally infected by swine HEV were clinically normal but had microscopic evidence of hepatitis, and developed viremia prior to seroconversion. The entire ORFs 2 and 3 were amplified by reverse transcription-PCR from sera of naturally infected pigs. The putative capsid gene (ORF2) of swine HEV shared about 79-80% sequence identity at the nucleotide level and 90-92% identity at the amino acid level with human HEV strains. The small ORF3 of swine HEV had 83-85% nucleotide sequence identity and 77-82% amino acid identity with human HEV strains. Phylogenetic analyses showed that swine HEV is closely related to, but distinct from, human HEV strains. The discovery of swine HEV not only has implications for HEV vaccine development, diagnosis, and biology, but also raises a potential public health concern for zoonosis or xenozoonosis following xenotransplantation with pig organs.