Full‐length sequence of genotype 3 hepatitis E virus derived from a pig in Thailand

Hepatitis E virus (HEV) infection in pigs was investigated in two principal swine farming areas in Thailand. Anti‐HEV antibodies and HEV RNA in sera were examined in 258 pigs reared on five commercial farms from age 1 to 6.5 months and sows. Overall, 167 of 258 (64.7%) pigs were positive for anti‐HEV IgG, while 20 of 258 (7.75%) had detectable HEV RNA. Sequence analysis of 20 HEV isolates obtained from viremic pigs revealed that they were 92.3–100% identical to each other and had 82.2–88.2% nucleotide similarity to other reported genotype 3 isolates in 415 nucleotide sequences within ORF2 region. Further characterization by sequencing the complete genome of the Thai swine HEV isolate (named Thai‐swHEV07) and phylogenetic analysis showed that Thai‐swHEV07 segregated into a cluster consisting of swine isolates from Japan, Mongolia, and Kyrgyzstan within the HEV genotype 3. The Thai‐swHEV07 had a genomic length of 7,229 nt excluding the polyadenylated region at 3′ terminus of the genome. Comparison of Thai‐swHEV07 and 27 reported strains of genotype 3 revealed 80.4–85.9% nucleotide identity, with the highest identity of 85.9% to the novel swHEV strain from Mongolia. These findings suggest that genotype 3 HEV isolates are markedly heterogeneous. J. Med. Virol. 81:657–664, 2009 © 2009 Wiley‐Liss, Inc.     

人、猪、禽戊型肝炎病毒血清学关系的研究

目的研究人、猪、禽戊型肝炎病毒（HEV）的血清学关系。方法应用ELISA分别以人、猪、禽HEVORF2重组蛋白p166human、p166swine、p268avian检测人、猪、鸡血清及其他标本中抗．HEVIs,G，用SAS软件进行统计学分析，同时进行序列同源性比较。结果p166human和p166swine对HEV实验感染动物血清、HEVORF2重组蛋白免疫血清和单克隆抗体均呈阳性反应，而p268avian均呈阴性反应。以p166human、p166swine、p268avian检测人、猪、鸡血清抗．HEVIgG，戊型肝炎患者血清检出率分别为98．5％、97．7％和1．5％，正常人血清为10．0％、10．0％和4．0％，猪血清为26．9％、25．6％和1．3％，鸡血清为4．3％、2．2％和33．3％。p268avian与p166human或p166swine的检出率差异有统计学意义（P〈0．001）。相关性分析表明，p166human和p166swine对不同样本的检测均呈直线正相关，而p268avitm与p166human或p166swine无直线相关性。人和猪HEVpORt2的序列同源性在88．2％。99．2％，而禽HEV与人、猪HEVpORt2的同源性仅为45．5％～46．1％，其中含有多个插入和缺失突变。结论人和猪HEV血清学关系密切，而禽HEV与人、猪HEV抗原性差异有统计学意义，无血清学相关性。因此禽HEV与人、猪HEV的关系应予进一步考证。     

Detection of IgA class antibody to hepatitis E virus in serum samples from patients with hepatitis E virus infection

A newly developed assay for IgA class antibody to hepatitis E virus (IgA anti-HEV) was used to study 145 serum samples collected during an outbreak of an enterically transmitted hepatitis that occurred in 3 villages in the lower Shebeli region of Southern Somalia between January, 1988 and November, 1989. A total of 52.4% of the afflicted patients were found positive for IgA anti-HEV, and 73.1% of these were also positive for IgM. Both antibodies disappeared during the convalescence period. Similar results were also seen in serum obtained from sporadic cases of acute waterborne hepatitis in Pakistan. © 1993 Wiley-Liss, Inc.     

A novel genotype of hepatitis E virus prevalent among farmed rabbits in China

In total, 335 serum samples were collected from rabbits from two farms in Gansu province, China, and tested for anti‐hepatitis E virus (HEV) antibody using EIA and for HEV RNA using nested RT‐ PCR with ORF2 primers. The overall prevalence of anti‐HEV antibody and HEV RNA was 57.0% (191/335) and 7.5% (25/335), respectively. The positivity rate of HEV RNA in the anti‐HEV antibody negative group (7.6% (11/144)) did not differ significantly from that in the positive group (7.3% (14/191)). The concordance between HEV RNA and anti‐HEV antibody was 43.3% with no significant correlation (P < 0.05). All 25 amplicons from the ORF2 region were cloned and sequenced. On the basis of nucleotide sequence comparison, they had 84–99% identity to each other and 73–77%, 70–76%, 75–82%, 71–77%, and 53–65% with the corresponding regions of genotypes 1, 2, 3, 4, and avian HEV, respectively. Samples that were positive with the ORF2 primers were amplified using ORF1 region primers; 17 were positive and shared 71–78%, 73–76%, 74–82%, 72–78%, and 39–58% identity with the corresponding regions of genotypes 1, 2, 3, 4, and avian HEV, respectively, at the nucleotide level. Two representative full‐length sequences were determined. These two sequences shared 85% identity with each other and had 74%, 73%, 78–79%, 74–75%, and 46–47% identity to full‐length genotypes 1, 2, 3, 4, and avian HEV, respectively. Thus, the sequences isolated from the rabbits represent a novel genotype of HEV. This study provides novel information about HEV genotypes infecting rabbits as well as evidence of a new mammalian genotype of HEV. J. Med. Virol. 81:1371–1379, 2009. © 2009 Wiley‐Liss, Inc.     

Prevalence of antibodies to hepatitis E virus in residents of a district in Havana, Cuba

A seroepidemiological study of hepatitis E virus (HEV) infection was conducted in a district of Havana, where hepatitis A virus (HAV) is considered endemic. The levels of anti-HEV antibodies were evaluated by enzyme-linked immunosorbent assay (ELISA) based on the recombinant protein GST-ORF2.1. Anti-HEV antibodies were detected in 11 of 209 (5.3%) of serum samples, compared to 71.3% for anti-HAV antibodies. No risk factors reported previously for HEV infection showed a significant association with the presence of anti-HEV antibodies, whereas anti-HAV antibodies were strongly associated with increasing age. HEV may be considered endemic in this area and is likely to have a significant clinical impact.     

Distinct changing profiles of hepatitis A and E virus infection among patients with acute hepatitis in Mongolia: The first report of the full genome sequence of a novel genotype 1 hepatitis E virus strain

In January 2012, Mongolia started a hepatitis A vaccination program, which has not yet been evaluated. The first occurrence of autochthonous acute hepatitis E in 2013, caused by genotype 4 hepatitis E virus (HEV), suggests the need for a routine study to monitor its prevalence. One hundred fifty‐four consecutive patients who were clinically diagnosed with acute hepatitis between 2014 and 2015 in Ulaanbaatar, Mongolia were studied. By serological and molecular testing followed by sequencing and phylogenetic analysis, only one patient (0.6%) was diagnosed with acute hepatitis A, caused by genotype IA hepatitis A virus (HAV), and 32 (20.8%) patients were diagnosed with acute hepatitis E, caused by genotype 1 HEV. The 32 HEV isolates obtained in this study shared 99.5‐100% nucleotide identity and were grouped into a cluster separated from those of subtypes 1a to 1f. Upon comparison of p‐distances over the entire genome, the distances between one representative HEV isolate (MNE15‐072) and 1a‐1f strains were 0.071‐0.137, while those between 1b and 1c were 0.062‐0.070. In conclusion, the prevalence of acute hepatitis A has decreased in Mongolia since the start of the vaccination program, while the monophyletic genotype 1 HEV strain of a probably novel subtype has been prevalent.     

Comparison of real-time fluorescent RT-PCR and conventional RT-PCR for the detection of hepatitis E virus genotypes prevalent in China

To compare the specificity and sensitivity of a real-time fluorescent RT-PCR assay with conventional RT-PCR, sera from 110 healthy blood donors, 120 patients with a clinical diagnosis of chronic hepatitis B, and 416 patients with non-A-C acute hepatitis, as well as serial dilutions of HEV genotypes 1 and 4, were tested with both assays. All samples from healthy blood donors and patients with chronic hepatitis B were negative by both assays. Real-time RT-PCR could detect the same final dilution of genotype 1 as conventional RT-PCR but could detect a 10-fold lower concentration of genotype 4 than conventional RT-PCR. Of 416 samples from patients with a clinical diagnosis of non-A-C acute hepatitis, 127 (30.5%) and 83 (20.0%) were positive for HEV by real-time and conventional RT-PCR, respectively. The concordance of real-time and conventional RT-PCR was 80.8%. Furthermore, 96 and 57 of 171 samples were positive for anti-HEV IgM by real-time and conventional RT-PCR, respectively, and 31 and 26 of 245 samples negative for anti-HEV IgM, were positive by real-time and conventional RT-PCR, respectively. All amplicons positive by conventional RT-PCR were sequenced. Of 83 isolates, 7 and 76 belonged to genotypes 1 and 4, respectively. Thus, both assays have a high specificity, but the real-time RT-PCR assay is more sensitive than conventional RT-PCR. Furthermore, HEV genotype 4 is responsible for most sporadic cases of hepatitis E in the north of China.     

Human hepatitis E virus circulation in Bulgaria: Deep Bayesian phylogenetic analysis for viral spread control in the country

Hepatitis E virus (HEV) infection in Bulgaria is endemic, as demonstrated by the seroprevalence of antibody against the virus in the general population and by the high prevalence of clinical cases registered. In this study, a deep Bayesian phylogenetic analysis has been performed to provide information on the genetic diversity and the spread of HEV genotypes in Bulgaria. Three different data sets of HEV virus was built for genotyping by the maximum likelihood method, for evolutionary rate estimated by Bayesian Markov Chain Monte Carlo approach, for demographic history investigation and for selective pressure analysis. The evolutionary rate for genotype 3e, was 351 × 10⁻³ substitution/site/year (95% highest posterior density [95% HPD]: 145 × 10 ⁻³-575 × 10 ⁻³). The root of the time to the most recent common ancestor of the Bayesian maximum clade credibility tree of HEV 3e genotype corresponded to 1965 (HPD 95% 1949-1994). The Bulgarian sequences mainly clustered in the main clade (clade A). The monophyletic clade included all Bulgarian genotype 3e sequences. The demographic history showed a slight growth from 1995 to 2000, followed by a sort of bottleneck in 2010s, a peak in 2011 and a new growth to 2015. Selection pressure analysis did not show sites under positive pressure but 64 statistically significant sites under negative selection. Molecular epidemiological surveillance by Bayesian phylogeny of HEV virus can contribute to trace the way of human infection after contact with swine source directly or heating meat improving public health control.     

Seroepidemiology of hepatitis E virus in patients with non-A, non-B, non-C hepatitis in Hungary

Many cases of acute hepatitis remain undiagnosed and the hepatitis E virus (HEV) is emerging in industrialized countries. The aim of this study was to assess the role HEV as causative agent in acute non-A, non-B, and non-C hepatitis patients in Hungary. 10.5% of the 264 acute non-A, non-B, and non-C hepatitis patients tested had anti-HEV IgG and 1.9% had anti-HEV IgM as tested by ELISA. After confirmation by Western blot 6.1% of the acute non-A, non-B, and non-C hepatitis patients had anti-HEV IgG antibodies only and 1.1% of the patients had both IgG and IgM. All 19 patients that were positive for anti-HEV IgG and/or IgM tested negative for HEV RNA by PCR. Only a small proportion of the acute hepatitis cases in the southwest of Hungary are assumed to be attributed to HEV infection, however, hepatitis E should be considered along with hepatitis A, B, and C in the diagnosis of acute hepatitis.     

From barnyard to food table: the omnipresence of hepatitis E virus and risk for zoonotic infection and food safety

Hepatitis E virus (HEV) is an important but extremely understudied pathogen. The mechanisms of HEV replication and pathogenesis are poorly understood, and a vaccine against HEV is not yet available. HEV is classified in the family Hepeviridae consisting of at least four recognized major genotypes. Genotypes 1 and 2 HEV are restricted to humans and associated with epidemics in developing countries, whereas genotypes 3 and 4 HEV are zoonotic and responsible for sporadic cases worldwide. The identification and characterization of a number of animal strains of HEV from pigs, chickens, rabbits, rats, mongoose, deer, and possibly cattle and sheep have significantly broadened the host range and diversity of HEV. The demonstrated ability of cross-species infection by some animal strains of HEV raises public health concerns for zoonotic HEV infection. Pigs are a recognized reservoir for HEV, and pig handlers are at increased risk of zoonotic HEV infection. Sporadic cases of hepatitis E have been definitively linked to the consumption of raw or undercooked animal meats such as pig livers, sausages, and deer meats. In addition, since large amounts of viruses excreted in feces, animal manure land application and runoffs can contaminate irrigation and drinking water with concomitant contamination of produce or shellfish. HEV RNA of swine origin has been detected in swine manure, sewage water and oysters, and consumption of contaminated shellfish has also been implicated in sporadic cases of hepatitis E. Therefore, the animal strains of HEV pose not only a zoonotic risk but also food and environmental safety concerns.     

Cross-protection of hepatitis E virus genotypes 1 and 4 in rhesus macaques

The purpose of this study was to determine cross-protection between HEV genotypes 1 and 4, which are prevalent in China. Fecal suspensions of genotypes 1 and 4 from patients, as well as genotype 4 from swine, were inoculated intravenously into rhesus macaques. Each inoculum contained 5 x 10(4) genome equivalents of HEV. After infection, serum and fecal samples were collected serially and the levels of alanine aminotransferase (ALT) and anti-HEV IgG and IgM in sera, and HEV RNA in fecal samples, were measured. Liver biopsies were carried out. All the infected monkeys (12/12) developed anti-HEV IgG and exhibited fecal shedding of virus. IgM was detected in 11 of 12, and ALT elevation occurred about 2-6 weeks post-inoculation in 10 of 12, infected monkeys. Hepatic histopathology was consistent with acute viral hepatitis and the ORF2 antigen of HEV was detected in the granular cytoplasm of hepatocytes by immunohistochemistry. After recovery from their initial HEV infection, the monkeys were challenged with a heterologous genotype or heterologous source of HEV and monitored for hepatitis and fecal shedding. Previous infection with HEV completely or partially protected against subsequent challenge with a heterologous virus, because 7 of 11 monkeys did not develop HEV infection or shed virus in the feces, and none of them developed hepatitis or exhibited ALT elevation or liver biopsy findings of hepatitis. In conclusion, previous HEV infection may give rise to cross-genotype and cross-host-species protection.     

Valuable antibody detection method for classifying hepatitis E virus genotypes

Nucleotide‐based methods are conventionally used to classify the hepatitis E virus (HEV) genotypes. A serological enzyme immunoassay (EIA) using open reading frame 3 (ORF3) C‐terminal peptides was developed to conveniently and accurately classify and evaluate the genotypes of HEV. The sera of mice immunized with HEV genotype 1, 3, and 4 reacted highly specifically to the peptides of the corresponding genotypes. Most (84.2%) clinical sera infected with HEV genotype 4 were positive for anti‐HEV antibodies when tested with the ORF3 peptides of genotype 4, but were negative for genotypes 1 and 3. Monkey and clinical serial sera infected with HEV reacted strongly to the homologous genotype ORF3 peptides. The indirect EIAs were more sensitive, with stronger reactivity, than commercial anti‐HEV immunoglobulin G assays when serial sera from monkeys infected with HEV genotype 1 or 4 were tested. All our results indicate that the serological typing EIA assays described in this study are more effective and convenient for the classification of HEV genotypes than molecular approaches, and can be used to screen large numbers of serum samples and differentiate genotypes for the diagnosis of HEV infections.     

Antibodies to hepatitis E virus among chinese patients with acute hepatitis in Taiwan

The prevalence of antibodies to hepatitis E virus (anti-HEV) was investigated in patients with acute hepatitis, and correlated with the clinical features. Sera from 110 patients with acute hepatitis and 60 healthy controls were tested for anti-HEV, antibody to hepatitis C virus (anti-HCV), and hepatitis B surface antigen (HBsAg). There were significant differences in the prevalence of anti-HEV, anti-HCV, and HBsAg between patients and controls (21.8% vs. 0%, 16.3% vs. 1.6% and 58.1% vs. 18.0%, respectively). Anti-HEV was detected in 6 (25.0%) of 24 patients with anti-HCV, 6 (9.3%) of 64 patients with HBsAg, and another 6 (22.2%) of 27 patients with acute hepatitis non-A, non-B, non-C. Anti-HEV was found in 15 men and three women, whose ages ranged from 34 to 75 (median, 57) years old. The median age of patients with anti-HEV was older than that in patients without this antibody (57 vs. 38 years; P = 0.001). The prevalence of anti-HEV in patients with anti-HCV alone (35.2%) was higher than that (11.1%) in patients with HBsAg alone (P = 0.03). Compared to patients without anti-HEV, HEV-infected patients had a higher frequency of travel to a foreign country (P = 0.0001), had a lower HBsAg rate (P = 0.019), and had higher serum alkaline phosphatase levels (P = 0.04) and gamma-glutamyl transpeptidase levels (P = 0.01). In conclusion, HEV infection occurs in 22.2% of patients with acute hepatitis non-A, non-B, non-C. HEV superinfection may occur in patients with chronic hepatitis B or C virus infection. © 1994 Wiley-Liss, Inc.     

Cross-species infection of specific-pathogen-free pigs by a genotype 4 strain of human hepatitis E virus

Hepatitis E virus (HEV) is an important pathogen. The animal strain of HEV, swine HEV, is related to human HEV. The genotype 3 swine HEV can infect humans and genotype 3 human HEV can infect pigs. The genotype 4 swine and human HEV strains are genetically related, but it is unknown whether genotype 4 human HEV can infect pigs. A swine bioassay was utilized in this study to determine whether genotype 4 human HEV can infect pigs. Fifteen, 4-week-old, specific-pathogen-free pigs were divided into three groups of five each. Group 1 pigs were each inoculated intravenously with PBS buffer as negative controls, group 2 pigs similarly with genotype 3 human HEV (strain US-2), and group 3 pigs similarly with genotype 4 human HEV (strain TW6196E). Serum and fecal samples were collected at 0, 7, 14, 21, 28, 35, 42, 49, and 56 days postinoculation (dpi) and tested for evidence of HEV infection. All pigs were necropsied at 56 dpi. As expected, the negative control pigs remained negative. The positive control pigs inoculated with genotype 3 human HEV all became infected as evidenced by detection of HEV antibodies, viremia and fecal virus shedding. All five pigs in group 3 inoculated with genotype 4 human HEV also became infected: fecal virus shedding and viremia were detected variably from 7 to 56 dpi, and seroconversion occurred by 28 dpi. The data indicated that genotype 4 human HEV has an expanded host range, and the results have important implications for understanding the natural history and zoonosis of HEV.     

Comparison of tests for antibody to hepatitis E virus

The results of serologic tests for hepatitis E virus have varied widely from laboratory to laboratory, making interpretation of seroepidemiologic studies difficult. The present study compares serologic results with different antigens and tests developed in two laboratories for their ability to diagnose hepatitis E and measure antibody prevalence in a high risk population in Saudi Arabia. The results confirm that tests based upon open reading frame (ORF) 3 of HEV are of limited value for seroepidemiologic studies, whereas ORF2-based antigens have broad utility and yield data that are reproducible in more than one laboratory. J. Med. Virol. 55:134–137, 1998. © 1998 Wiley-Liss, Inc.     

Serological evidence for hepatitis E virus infection in Israel

Israel is suspected to be endemic for hepatitis E virus (HEV) because of its geographic location and the large-scale immigration from endemic countries. Although no cases of local HEV infection have been diagnosed, a serological survey would provide indirect evidence for such infection. We examined sera from 1,416 healthy subjects, including 1,139 Jews from various regions of Israel and 277 Arabs, most of whom reside in the West Bank of the Jordan River. In addition, we tested 13 non-A, non-B, and non-C viral hepatitis patients. Sera were screened for antibody to hepatitis E virus (anti-HEV) by a newly developed enzyme immunoassay (EIA) and by immuno-blots for both IgG and IgM anti-HEV activity. Positive samples were confirmed by neutralization. The seroprevalence found by EIA was 2.81% and 1.81% in the Jewish and Arab populations, respectively. More than a 2-fold higher prevalence in males compared to females and an increase with age were found in both populations. However, these differences were nonsignificant. The geographical distribution was even throughout the country, except for two clusters of 3 and 4 seropositive individuals possibly reflecting past foci of infection. Eight of 37 ElA-positive sera were positive for IgG, and 3 were positive for IgM by the immunoblot assay. Among hepatitis patients (9 acute and 4 chronic), one patient with chronic hepatitis was positive for both IgG and IgM. Our study provides indirect evidence that Israel is endemic for HEV. The lack of outbreaks may be attributed to generally good hygienic conditions and a controlled potable water supply, while unrecognized sporadic cases may be due to the unavailability of diagnostic tests. © 1995 Wiley-Liss, Inc.     

Proteinase 3 and dihydrolipoamide dehydrogenase (E3) are major autoantigens in hepatitis C virus (HCV) infection

Hepatitis C virus (HCV) infection has been found to be strikingly associated with autoimmune phenomena. The aim of the present study was to investigate the presence of various autoantibodies in patients with HCV infection. Anti-neutrophil cytoplamic antibody (ANCA), anti-dihydrolipoamide dehydrogenase (anti-E3), rheumatoid factor (RF), anti-dihydrolipoamide acetyltransferase (anti-E2), anti-SS-A/Ro (60 kD), anti-SS-A/Ro (52 kD), anti-SS-B/La, anti-topoisomerase II (anti-topo II), anti-cardiolipin (aCL), anti-dsDNA, anti-ssDNA, anti-nuclear antibodies (ANA), anti-proteinase 3 (anti-Pr3) and anti-myeloperoxidase (anti-MPO) were determined in sera from 516 patients with HCV infection, 11 with primary biliary cirrhosis (PBC) and 44 healthy controls. Assays employed were indirect immunofluoresence, the particle latex agglutination test, enzyme-linked immunosorbent assay (ELISA) and immunoblotting. ANCA, anti-E3 antibody and RF were positive in 278/516 (55.6%), 276/516 (53.3%) and 288/516 (56%) patients with HCV infection, respectively. Positivity for ANA was present in 15.8%, anti-ssDNA in 15.6%, anti-dsDNA in 8.5%, aCL in 5%, anti-SS-B/La in 4.1%, anti-SS-A/Ro (60 kD) in 3.9%, anti-E2 in 3.3% and anti-SSA/Ro (52 kD) in 1.2%, anti-MPO in 4.8%, anti-Topo II and anti-actinin in 0%. All sera with ANCA showed c-ANCA patterns and contained anti-PR3 specificity. HCV patients with ANCA showed a higher prevalence of skin involvement, anaemia, abnormal liver function and alpha-Fetoprotein (alpha-FP). HCV patients with anti-E3 antibodies showed a higher prevalence of liver cirrhosis, arthritis, abnormal liver function and elevated alpha-FP levels. The prevalence of autoantibodies was not affected by treatment with interferon-alpha (IFN-alpha). In conclusion, autoantibodies are commonly found in patients with HCV infection. There is a high prevalence of anti-E3, ANCA and RF in these patients. Proteinase 3 and E3 are the major target antigens in HCV infection. HCV may be regarded as a possible causative factor in ANCA-related vasculitis.     

Evaluation of antibody-based and nucleic acid-based assays for diagnosis of hepatitis E virus infection in a rhesus monkey model

We have evaluated four hepatitis E virus (HEV) specific antibody assays, using sequential samples taken from 86 rhesus monkeys at intervals for up to 86 weeks after they had been infected with different doses of HEV. The animals are a common experimental model of hepatitis E. The large collection of sequential samples used avoids uncertainties encountered in previous studies regarding the precise infection status of study subjects and minimizes bias due to the individuality of response to infection. One assay (YES IgG) was produced with synthetic peptides; the others (E2 IgM, E2 IgG, and GL IgG) were produced with recombinant antigens. The results were compared with the viral RNA contents of the serum and stool samples and the occurrence of these virological and immunological markers in the course of the infection was temporally related to the development of hepatitis. Diagnostic utility of the markers was assessed according to their response rates and prevalence at different times in the course of infection. All the animals produced E2 IgG and developed viremia and all but one also produced E2 IgM and excreted the virus in stool, whereas response rates for the other antibodies were lower and decreased with virus dose. Hepatitis occurred over a period of 4 weeks between 3 and 7 weeks after infection. Virological activity occurred mainly during the incubation period and the prevalence of viral markers declined rapidly after the onset of hepatitis. Production of the E2 antibodies immediately preceded the onset of hepatitis, and this was followed about one week later by production of the other antibodies. Seroprevalence E2 IgM reached a peak value 3 weeks after the onset of hepatitis, whereas seroprevalence of GL IgG and YES IgG peaked after the disease had subsided. E2 IgG persisted in all animals for the entire duration of the experiment of up to 86 weeks and possibly beyond and, thus, can serve as a useful epidemiological marker of HEV infection.