Human Hepatitis E Virus Strains Research Articles

Hepatitis E virus neutralization by porcine serum antibodies.

The consumption of raw or undercooked meat products poses a serious risk for human hepatitis E virus (HEV) infections. In many high-income countries, domestic pigs and wild boars represent the main animal reservoirs for HEV and are usually identified by reverse transcription-PCR and antibody enzyme-linked immunosorbent assay (ELISA). In order to characterize the humoral immune response in more detail, a cell culture-based serum neutralization assay using a culture-adapted HEV strain was established here. Measurement of neutralizing antibodies was only possible after removing the viral quasi-envelope by detergent treatment. Serum samples of 343 wild boars from Northern Germany were first analyzed for anti-HEV IgG using an in-house ELISA, resulting in 19% positive samples. Subsequently, a subset of 41 representative samples was tested with the neutralization assay, and the results correlated well with those obtained by ELISA. Not only the human HEV strain 47832c but also two porcine HEV strains were shown to be neutralized by porcine serum antibodies. Neutralizing activity was also found in samples containing both HEV-specific antibodies and HEV RNA. Testing of serum samples derived from two experimentally infected domestic pigs showed a steep increase in neutralizing activity at 24 or 51 days post infection, dependent on the used infectious dose. The developed assay can be useful for characterization of the humoral immune response after HEV infection and for assessing the efficiency of HEV vaccine candidates.

Prevalence of hepatitis E virus genotype 4 of probable human origin in Tibetan pigs from the Qinghai-Tibetan Plateau, China.

Hepatitis E virus (HEV) is the most common cause of acute viral hepatitis worldwide. In 2018-2022, we investigated the presence of HEV RNA in 1233 stool samples collected in the Qinghai-Tibetan Plateau, including humans (16), Tibetan pigs (624), yaks (312), sheep (267), and dogs (14). HEV RNA was only detected in Tibetan pig faecal samples (18.27%, 114/624). To perform molecular characterization of HEV strains in Tibetan pigs, we obtained 21 complete HEV genome sequences between 2018 and 2022. Sequence comparisons showed that 21 HEV strains from Tibetan pigs shared the mean nucleotide identities with the reference HEV strains ranging between 82.9% and 94.9% and 89.3% and 92.1% similarities with human HEV strains. Phylogenetic analysis confirmed that all HEV strains were genotype 4, closely related to human HEV strains. Sequence recombinant analysis showed five potential recombinant strains identified in this study, of which SWU/D18/2018 (GenBank No. MK410044) was recombinant with human and swine HEV strains, located 6509-6878 nt from the recombination point. Based on the Bayesian evolutionary trees, we found that most HEV strains diverged later than human HEV (16 Tibetan pig HEV strains diverged later than 1979, and seven human HEV strains diverged earlier than 1979). Therefore, we speculated that the prevalence of HEV 4 in Tibetan pigs possibly originated from humans in the Qinghai-Tibetan Plateau.

Evidence of Circulation and Phylogenetic Analysis of Hepatitis E Virus (HEV) in Wild Boar in South-East Italy.

Hepatitis E virus (HEV) is an important cause of acute viral hepatitis in humans worldwide. The food-borne transmission of HEV appears to be a major route in Europe through the consumption of pork and wild boar meat. HEV epidemiology in wild boars has been investigated mainly in Northern and Central Italian regions, whilst information from Southern Italy is limited. We investigated the occurrence of HEV in wild boar in the Apulia and Basilicata regions (Southern Italy). Thirteen (10.4%) out of one hundred and twenty-five wild boar samples tested positive for HEV using a quantitative reverse transcription PCR. HEV prevalence was 12% in Apulia and 9.3% in Basilicata. Seven samples were genotyped, and different subtypes (c, f, m) of genotype 3 were identified. The complete genome of a 3m strain was determined, and the virus showed the highest nucleotide identity to a human HEV strain identified in France in 2017. These findings demonstrate the substantial circulation of HEV in the wild boar population in Italian Southern regions. Gathering information on the HEV strains circulating in different geographical areas is useful for tracking the origin of HEV outbreaks and assessing the epidemiological role of wild boar as a potential virus reservoir for domestic pigs.

Chirohepevirus from Bats: Insights into Hepatitis E Virus Diversity and Evolution

Homologs of the human hepatitis E virus (HEV) have been identified in more than a dozen animal species. Some of them have been evidenced to cross species barriers and infect humans. Zoonotic HEV infections cause chronic liver diseases as well as a broad range of extrahepatic manifestations, which increasingly become significant clinical problems. Bats comprise approximately one-fifth of all named mammal species and are unique in their distinct immune response to viral infection. Most importantly, they are natural reservoirs of several highly pathogenic viruses, which have induced severe human diseases. Since the first discovery of HEV-related viruses in bats in 2012, multiple genetically divergent HEV variants have been reported in a total of 12 bat species over the last decade, which markedly expanded the host range of the HEV family and shed light on the evolutionary origin of human HEV. Meanwhile, bat-borne HEV also raised critical public health concerns about its zoonotic potential. Bat HEV strains resemble genomic features but exhibit considerable heterogeneity. Due to the close evolutionary relationships, bat HEV altogether has been recently assigned to an independent genus, Chirohepevirus. This review focuses on the current state of bat HEV and provides novel insights into HEV genetic diversity and molecular evolution.

Advances in Hepatitis E Virus Biology and Pathogenesis.

Hepatitis E virus (HEV) is one of the causative agents for liver inflammation across the world. HEV is a positive-sense single-stranded RNA virus. Human HEV strains mainly belong to four major genotypes in the genus Orthohepevirus A, family Hepeviridae. Among the four genotypes, genotype 1 and 2 are obligate human pathogens, and genotype 3 and 4 cause zoonotic infections. HEV infection with genotype 1 and 2 mainly presents as acute and self-limiting hepatitis in young adults. However, HEV infection of pregnant women with genotype 1 strains can be exacerbated to fulminant hepatitis, resulting in a high rate of case fatality. As pregnant women maintain the balance of maternal-fetal tolerance and effective immunity against invading pathogens, HEV infection with genotype 1 might dysregulate the balance and cause the adverse outcome. Furthermore, HEV infection with genotype 3 can be chronic in immunocompromised patients, with rapid progression, which has been a challenge since it was reported years ago. The virus has a complex interaction with the host cells in downregulating antiviral factors and recruiting elements to generate a conducive environment of replication. The virus-cell interactions at an early stage might determine the consequence of the infection. In this review, advances in HEV virology, viral life cycle, viral interference with the immune response, and the pathogenesis in pregnant women are discussed, and perspectives on these aspects are presented.

Serological prevalence and molecular characterization of hepatitis E virus in imported pigs in Singapore (2000-2019).

Hepatitis E is a significant liver disease caused by infection with hepatitis E virus (HEV). The risk factors for hepatitis E in developed countries include blood transfusion and ingestion of undercooked meat or meat products derived from HEV-infected animals. Since 2000, there has been increased human hepatitis E incidence reported in Singapore. Although the causes of this increase have not been established, several studies have linked zoonotic HEV infections in humans to pork consumption. It is therefore important to closely monitor the presence of HEV in food sources for the prevalence and virulence. In this study, we demonstrated the presence of HEV in pigs imported into Singapore for consumption through serological and molecular investigation of live pig and post-slaughter samples collected between 2000 and 2019. Among imported pigs, anti-HEV antibody prevalence remained at a level around 35% until 2017, with a statistically significant increase in 2018. HEV RNA was detected in 8.40% (34/405) of the faecal samples, indicative of an active infection in the pigs. HEV RNA was also detected in 6.67% (4/60) of liver samples obtained post-slaughter. We also report the development of an RT-PCR-based next-generation sequencing (NGS) method that enabled full sequencing of the HEV genome in HEV RNA-positive samples in a relatively short span of time. Phylogenetic analysis identified the HEV in one of the imported pigs (HEV-S28) as genotype 3a, which clustered together with the human HEV strains previously identified in Singapore. We found that the HEV-S28 strain exhibited amino acid substitutions that are associated with reduced HEV replication efficiency. The increase in anti-HEV seroprevalence in the pig population from 2018 is worth further exploration. We will continue to monitor the prevalent HEV strains and assess the genetic diversity of HEV in the imported pigs to confirm the potential association with human infections.

BALB/c Mouse Is a Potential Animal Model System for Studying Acute and Chronic Genotype 4 Hepatitis E Virus Infection.

Hepatitis E virus (HEV) is the main pathogen of hepatitis worldwide. However, its infection biology and pathogenesis remain largely unknown. Suitable small-animal models are required to advance the study of HEV infection. Although an efficient model of genotype 1 (gt1) and gt3 HEV infection has been established in human liver chimeric mice, the infectivity of gt4 HEV infection in mice has not been comprehensively characterized. In this study, immunocompromised BALB/c nude, immunocompetent BALB/c, and C57BL/6 mice were inoculated with either gt3 or gt4 HEV (19 HEV strains, including human, swine, macaque-adapted, and cow HEV strains). Infectivity was identified by viral RNA and antigen detection, inflammation, and histopathological analysis. Then, HEV-infected BALB/c mice were treated with antiviral drugs. Acute HEV infection was established in BALB/c mice inoculated with eight gt4 HEV strains. However, gt3 HEV strains failed to achieve active HEV infection. HEV infection was established in BALB/c nude and regular mice inoculated with gt4 HEV but not in C57BL/6 mice. Gt4 HEV infection resulted in rapid viremia and high titers in feces, sera, and replication sites. HEV infection in mice showed no gender preference. Furthermore, chronic gt4 HEV infection was well imitated in BALB/c mice for 32 weeks and caused liver fibrosis.ConclusionBALB/c mice have a great potential for reproducing the process of gt4 HEV infection. The successful establishment of a gt4 HEV small-animal model provides an opportunity to further understand HEV infection biology and zoonotic transmission and develop anti-HEV vaccine.

Epidemiology of hepatitis E in South-East Europe in the "One Health" concept

The significance of hepatitis E virus (HEV) as an important public health problem is rising. Until a decade ago, cases of HEV infection in Eur-ope were mainly confined to returning travelers, but nowadays, hepatitis E represents an emerging zoonotic infection in many European countries. The aim of this manuscript is to perform a systematic review of the published literature on hepatitis E distribution in humans, animals and environmental samples ("One Health" concept) in the South-Eastern European countries. Comparison of the available data showed that the anti-HEV seroprevalence in the South-Eastern Europe varies greatly, depending on the population studied, geographical area and methods used. The IgG seroprevalence rates in different population groups were found to be 1.1%-24.5% in Croatia, up to 20.9% in Bulgaria, 5.9-%17.1% in Romania, 15% in Serbia, up to 9.7% in Greece and 2%-9.7% in Albania. Among possible risk factors, older age was the most significant predictor for HEV seropositivity in most studies. Higher seroprevalence rates were found in animals. HEV IgG antibodies in domestic pigs were detected in 20%-54.5%, 29.2%-50%, 38.94%-50% and 31.1%-91.7% in Serbia, Bulgaria, Romania and Croatia, respectively. In wild boars seroprevalence rates were up to 10.3%, 30.3% and 31.1% in Romania, Slovenia and Croatia, respectively. A high HEV RNA prevalence in wild boars in some countries (Croatia and Romania) indicated that wild boars may have a key role in the HEV epidemiology. There are very few data on HEV prevalence in environmental samples. HEV RNA was detected in 3.3% and 16.7% surface waters in Slovenia and Serbia, respectively. There is no evidence of HEV RNA in sewage systems in this region. The available data on genetic characterization show that human, animal and environmental HEV strains mainly belong to the genotype 3.

Genetically similar hepatitis E virus strains infect both humans and wild boars in the Barcelona area, Spain, and Sweden.

Hepatitis E virus (HEV) is a hepatotropic virus, endemic in Europe where it infects humans and animals, with domestic pigs and wild boars as main reservoirs. The number of HEV-infected cases with unknown source of infection increases in Europe. There are human HEV strains genetically similar to viruses from domestic pigs, and zoonotic transmission via consumption of uncooked pork meat has been shown. Due to continuous growth of the wild boar populations in Europe, another route may be through direct or indirect contacts with wild boars. In the Collserola Natural Park near Barcelona, Spain, the wild boars have spread into Barcelona city. In Sweden, they are entering into farmlands and villages. To investigate the prevalence of HEV and the risk for zoonotic transmissions, the presence of antibodies against HEV and HEV RNA were analysed in serum and faecal samples from 398 wild boars, 264 from Spain and 134 from Sweden and in sera from 48 Swedish patients with HEV infection without known source of infection. Anti-HEV was more commonly found in Spanish wild boars (59% vs. 8%; p<0.0001) while HEV RNA had similar prevalence (20% in Spanish vs. 15% in Swedish wild boars). Seven Swedish and three Spanish wild boars were infected with subtype 3f, and nine Spanish with subtype 3c/i. There were three clades in the phylogenetic tree formed by strains from wild boars and domestic pigs; another four clades were formed by strains from humans and wild boars. One strain from a Spanish wild boar was similar to strains from chronically infected humans. The high prevalence of HEV infections among wild boars and the similarity between wild boar HEV strains and those from humans and domestic pigs indicate that zoonotic transmission from wild boar may be more common than previously anticipated, which may develop into public health concern.

Identification of a putative novel genotype 3/rabbit hepatitis E virus (HEV) recombinant.

Hepatitis E virus (HEV) is a viral pathogen transmitted by the fecal-oral route and is a major cause of waterborne acute hepatitis in many developing countries. In addition to infecting humans, HEV has been identified in swine, wild boars, rabbits and other mammals; with swine and wild boars being main reservoirs for zoonotic transmission of HEV. There are four major HEV genotypes known to infect humans; genotypes 1 (HEV-1) and 2 (HEV-2) are restricted to humans, and genotypes 3 (HEV-3) and 4 (HEV-4) are zoonotic. Herein, three human HEV strains originating in France were sequenced and near full-length genomes were characterized. Phylogenetic analysis showed that two strains were genotype 3 and closely grouped (a 100% bootstrap value) with subtype 3i reference strains. In percent nucleotide identities, these two strains were 94% identical to each other, 90–93% identical to subtype 3i strains, 82–86% identical to other HEV-3, and 77–79% identical to rabbit HEV strains excluding the two divergent strains KJ013414 and KJ013415 (74%); these two strains were less than 77% identical to strains of HEV genotypes 1, 2 and 4. The third strain was found distinct from any known HEV strains in the database, and located between the clusters of HEV-3 and rabbit HEV strains. This unique strain was 74–75% identical to HEV-1, 73% to HEV-2, 81–82% to HEV-3, 77–79% to rabbit HEV again excluding the two divergent strains KJ013414 and KJ013415 (74%), and 74–75% to HEV-4, suggesting a novel unclassified strain associated with HEV-3 and rabbit HEV. SimPlot and BootScan analyses revealed a putative recombination of HEV-3 and rabbit HEV sequences at four breakpoints. Phylogenetic trees of the five fragments of the genome confirmed the presence of two HEV-3 derived and three unclassified sequences. Analyses of the amino acid sequences of the three open reading frames (ORF1-3) encoded proteins of these three novel strains showed that some amino acid residues specific to rabbit HEV strains were found solely in this unclassified strain but not in the two newly identified genotype 3i strains. The results obtained by SimPlots, BootScans, phylogenetic analyses, and amino acid sequence comparisons in this study all together appear to suggest that this novel unclassified strain is likely carrying a mosaic genome derived from HEV-3 and rabbit HEV sequences, and is thus designated as a putative genotype 3/rabbit HEV recombinant.

Detection and genetic characterization of hepatitis E virus (HEV) genotype 3 subtype c in wild boars in Italy

Hepatitis E virus (HEV) was detected in stools collected from wild boars in Italy, with an overall prevalence of 1.5% (3/196). The sequence of a ~3.0-kb portion at the 3' end of the genome of one such strain, HEV/WB/P6-15/ITA, was determined. In the full-length ORF2, which encodes the capsid protein, the virus was genetically closest to wild boar and human HEV strains currently classified as genotype 3 subtype c. Interestingly, the 3' end of ORF2 of the WB/P6-15/ITA matched the 340-nucleotide (nt) sequence (94.0% nt identity) of the human strain PeGe, identified in 2015 from a patient with acute hepatitis E in Genoa, Italy, suggesting that similar HEV strains are circulating in the same geographical setting in humans and animals.

Distribution and Molecular Characterization of Hepatitis E virus in Domestic Animals and Wildlife in Croatia.

Hepatitis E is becoming a growing health concern in European countries as an increase of sporadic human cases of unknown origin has been recorded lately. Its causative agent, Hepatitis E virus (HEV), is known to have zoonotic potential and thus the role of domestic and wild animals in the chain of viral spread should be considered when investigating risk factors and the epidemiology of the disease. A comprehensive survey based on viral RNA detection was carried out in Croatia including blood, spleen and liver samples originating from 1816 different domestic and wild animals and digestive gland samples from 538 molluscs. A high HEV prevalence was detected in domestic pigs (24.5%) and wild boars (12.3%), whereas cattle, molluscs, ruminant and carnivore wildlife samples tested negative. Molecular characterization of both ORF1 and ORF2 genomic regions confirmed the phylogenetic clustering of the obtained sequences into genotype 3, previously reported in Europe. Furthermore, our results proved the presence of identical sequence variants in different samples, regardless of their origin, age or habitat of the host, suggesting transmission events between domestic swine, as well as between domestic swine and wild boars in the country. Moreover, a close genetic relationship of Croatian animal strains and known human HEV strains from GenBank opens the question of possible cross-species HEV transmission in Croatia, especially in the areas with an intensive swine production.

Experimental infection of Z:ZCLA Mongolian gerbils with human hepatitis E virus.

To investigate whether Z:ZCLA Mongolian gerbils are readily susceptible to infection by human hepatitis E virus (HEV). Z:ZCLA Mongolian gerbils were infected with a clinical HEV strain isolated from an acute hepatitis E patient, and virus pathogenesis was assessed in this host. Non-infected gerbils served as the control group. Feces samples from gerbils were collected weekly for reverse transcription-nested polymerase chain reaction. Serum anti-HEV IgG and alanine aminotransferase (ALT) were detected by enzyme linked immunosorbent assay. At sacrifice, each animal's liver, spleen and kidney were collected for histopathologic examination. HEV-infected gerbils showed fatigue, with histopathological changes observed in the liver, spleen and kidney. HEV RNA was detected in fecal samples taken at day 7 after inoculation and the detectable levels lasted out to day 42 after inoculation. Interestingly, ALT levels were only moderately increased in the HEV-infected animals compared with the non-infected control group. Z:ZCLA Mongolian gerbils are susceptible to human HEV.

Rabbit and human hepatitis E virus strains belong to a single serotype

Hepatitis E virus (HEV) is a zoonotic pathogen and all four established genotypes of HEV belong to a single serotype. The recently identified rabbit HEV is antigenically and genetically related to human HEV. It is unclear whether rabbit HEV belongs to the same serotype as human HEV. The purpose of this study was to determine the serotypic relationship between rabbit and human HEVs. HEV ORF2 recombinant capsid protein p166 (amino acids 452–617) of four known HEV genotypes and rabbit HEV were used to induce immune serum, which were evaluated for their ability to neutralize human HEV genotype 1, 4, and rabbit HEV strains by an in vitro PCR-based HEV neutralization assay. Immune sera of five kinds of p166 proteins were all found to neutralize or cross-neutralize the three different HEV strains, suggesting a common neutralization epitope(s) existing between human and rabbit HEV. Rabbit models of a second-passage rabbit HEV strain, JS204-2, and a genotype 4 human HEV strain, NJ703, were established as evidenced by fecal virus shedding, viremia and anti-HEV IgG seroconversion. Six rabbits, recovered from JS204 infection, were challenged with NJ703, and another six recovered from NJ703 infection were challenged with JS204-2. After challenge, viremia was not detected, shorter fecal virus shedding durations and obvious early stage declines in anti-HEV IgG values were observed. The results from this study indicate that rabbit HEV belongs to the same serotype as human HEV.

Genetic heterogeneity and subtyping of human Hepatitis E virus isolates from Uruguay

Hepatitis E virus (HEV) infection is an important public health concern in many developing countries causing waterborne outbreaks, as well as sporadic autochthonous hepatitis. It is transmitted primarily by the fecal–oral route. However, zoonotic transmission from animal reservoirs to human has also been suggested.Genotype 3 is the most frequent genotype found in South America and the HEV epidemiology in this region seems to be very complex. However, data about the molecular characterization of HEV isolates of the region is still lacking and further investigation is needed.Our study characterized human HEV strains detected in a 1-year period in Uruguay, by extensive sequence analysis of three regions of the HEV genome.Uruguayan strains were closely related to a set of European strains and in turn, were dissimilar to Brazilian, Argentinean and Bolivian isolates. Additionally, the co-circulation of viral subtypes 3i and 3h was observed. Circulation of subtype 3i had been reported in Argentina and Bolivia whereas sequences of subtype 3h are rare and had never been reported in Latin America. In order to contribute to shedding light over the molecular epidemiology of this emergent infection in the region, we thoroughly analyzed the genetic variability of HEV strains detected in Uruguay, providing the largest dataset of sequences of HEV ever reported in a country in South America.

Detection and molecular characterization of hepatitis E virus in clinical, environmental and putative animal sources

Putative animal reservoirs and environmental samples were studied to investigate potential routes of transmission for indigenous hepatitis E virus (HEV) infection in Hokkaido, Japan. A total of 468 liver samples and 954 environmental samples were collected from 2003 to 2011 for this study. Four swine livers (1%) were positive for HEV RNA; two strains belonged to genotype 3 and the other two strains were genotype 4. Genotype 3 HEV was detected in a sewage sample and a seawater sample. HEV strains derived from swine liver, seawater and raw sewage samples shared 93-100% sequence similarity with human HEV strains.

Human and Porcine Hepatitis E Viruses, Southeastern Bolivia

To the Editor: Hepatitis E virus (HEV) genotypes 3 and 4 are considered to be primarily zoonotic (1). However, recent data indicate that both genotypes can be transmitted among humans through other routes (2,3). Observations of genetic distinctiveness between swine and human HEV strains circulating within the same region argue against exclusivity of zoonotic transmission (4). A recent report presented a remarkable example of such distinction between genotype 3 isolates in rural communities in southeastern Bolivia (5). We examined HEV sequences obtained in that study to show the independent genetic origin of swine and human variants. Findings suggest disjunction between human and swine HEV strains in this epidemiologic setting, despite the potential for extensive cross-species exposure. Using reference sequences from Lu et al. (6), we conducted subtype analysis of HEV open reading frame 2 sequences at nucleotide positions 826–1173 (GenBank accession no. {type:entrez-nucleotide,attrs:{text:AF060668,term_id:4321753}}AF060668) from isolates from 2 rural communities in southeastern Bolivia (5). Analysis showed that swine sequences belonged to subtype 3i and that the human sequences belonged to 3e. We collected all available GenBank genotype 3 sequences covering this genomic region for which the dates of collection were documented. Sequences were used to estimate the time from the most recent common ancestor (tMRCA) by using BEAST version 1.6.1 (7). Estimated tMRCA for GenBank sequences was longer than for sequences from Bolivia alone (Table) or for all genotype 3 sequences together (Table). Table Model estimates of time to most common recent ancestor for HEV ORF2 nucleotide sequences, southeastern Bolivia* To reduce the effect of close relatedness among human or swine HEV sequences from Bolivia on the tMRCA estimate, we used only 1 representative sequence per species from each community in the final analysis. This analysis identified an estimated tMRCA similar to that seen for GenBank sequences alone (Table, model F vs. model D). This estimate indicates that human and swine HEV isolates from southeastern Bolivia last shared a common ancestor ≈275 years ago (Table, model F). Thus, swine HEV strains from both rural communities belonged to subtype 3i, and the human HEV strains identified from the community of Bartolo, Bolivia, belonged to subtype 3e and shared an ancestor with swine strains almost 3 centuries ago. This finding is surprising because the community of Bartolo has several potential risk factors for zoonotic transmission of HEV. There are ≈200 humans and ≈70 swine in Bartolo (8). Residents are mainly native Quechua and Guarani with some of mixed Spanish ancestry who subsist at a low socioeconomic level. Their main livelihood activities are agriculture and breeding of animals. Free-range pig farms are family owned. Because of its impoverished state, the community has no running water, and few houses have toilets. No facilities are suitable for safely slaughtering animals (5,9). These conditions appear to create a setting in which zoonotic transmission of HEV should be common, and infection should be caused by a strain shared between swine and humans. However, the data suggest host-specific infection with distinct HEV subtypes. Although specimens were collected from 172 humans (≈86%) and 67 swine (≈96%) in Bartolo (8), zoonotically transmitted isolates may have been missed because of the sample-pooling technique used (5). Nevertheless, detection of distinct HEV strains in human and swine populations indicates possible nonzoonotic, human-to-human transmission in this community. Detection of antibodies against HEV among 7% of residents and HEV genomes in persons without serologic markers of HEV infection indicate a higher HEV prevalence in Bartolo (5). Subclinical infection detected by PCR among Bartolo residents (5), rapid decrease of HEV antibody, and uncertain sensitivity of commercial serologic assays (10) suggest that the reported extent of HEV infection is most likely an underestimate. High prevalence may generate conditions in this community that effectively prevent cross-species transmission because of frequent exposure to HEV early in life when contacts between humans and animals are limited, thus promoting host-specific transmission. This supposition is supported by the higher seropositivity seen among children 1–5 years of age and adults 41–50 years of age in Bartolo (5). Implications of these observations for understanding HEV evolution and epidemiology of HEV infections warrant further research on genetic heterogeneity of HEV strains in this region and other epidemiologic settings.

Genetic characteristics and pathogenicity of human hepatitis E virus in Nanjing, China

To investigate the genetic characteristics and pathogenicity of hepatitis E virus (HEV) and assess the potential risk factors for sporadic hepatitis E. Sixty-two serum samples from the patients with acute hepatitis E were collected, including 23 cases coinfected with hepatitis B virus. Anti-HEV detection and partial HEV RNA amplification were performed by enzyme immunoassays and reverse transcription-nested polymerase chain reaction (RT-nPCR) method, respectively, and PCR products were sequenced. The isolated human HEV sequences were analyzed phylogenetically. The positive rate of serum HEV RNA were 21.0% (13/62), including 5 cases of liver failure. All the 13 isolates shared a 82.1%-98.0% nucleotide homology with each other and had identities of 74.7%-81.0%, 75.3%-78.6%, 75.3%-80.0% and 82.1%-96.1% with the corresponding regions of HEV genotypes 1-4, respectively. The human HEV strain GS-NJ-12 shared a 100% nucleotide identity with the swine HEV strain swIM6-43 isolated from Inner Mongolia, China. Swine may be a principal risk factor for occurrence of sporadic hepatitis E in eastern China, and genotype 4 HEV can induce acute liver failure.

A549 and PLC/PRF/5 cells can support the efficient propagation of swine and wild boar hepatitis E virus (HEV) strains: demonstration of HEV infectivity of porcine liver sold as food

Recent evidence has indicated the cross-species transmission of hepatitis E virus (HEV) from pigs and wild boars to humans, causing zoonosis, mostly via consumption of uncooked or undercooked animal meat/viscera. However, no efficient cell culture system for swine and boar HEV strains has been established. We inoculated A549 cells with 12 swine and boar HEV strains of liver, feces, or serum origin at an HEV load of ≥2.0 × 10(4) copies per well and found that the HEV progeny replicated as efficiently as human HEV strains, with a maximum load of ~10(8) copies/ml. However, the HEV load in the culture medium at 30 days post-inoculation differed markedly by inoculum, ranging from 1.0 × 10(2) to 1.1 × 10(7) copies/ml upon inoculation at a lower load of approximately 10(5) copies per well. All progeny were passaged successfully onto A549 and PLC/PRF/5 cells. In sharp contrast, no progeny viruses were detectable in the culture supernatant upon inoculation with 13 swine and boar HEV strains at an HEV load of <1.8 × 10(4) copies per well. The present study also demonstrates that swine liver sold as food can be infectious, supporting the risk of zoonotic food-borne HEV infection.

Hepatitis E virus in domestic pigs and surface waters in Slovenia: Prevalence and molecular characterization of a novel genotype 3 lineage

A novel hepatitis E virus (HEV) genotype 3 lineage was identified in Slovenian pig herds. Stool samples from six Slovenian pig farms were collected and tested for the presence of HEV RNA. Of 85 individual samples 15 (20.3%) were positive for HEV RNA of which 2/38 (5.3%), 6/21 (28.6%) and 7/26 (26.9%) were from suckling, weanling and fattening pigs, respectively. Additionally, 51 pooled porcine stool samples were tested in one of the biggest pig farm and the estimated infection rate of individual pig was calculated, resulting in 7.8%, 10.6% and 24.2% for suckling, weanling and fattening pigs, respectively. The majority of HEV positive porcine samples were from the same pig farm. Out of 17 Slovenian patients with confirmed recent hepatitis E in the period 1999–2011, the serum samples of 10 patients were tested and 3 samples turned out to be HEV RNA positive. Furthermore, 60 surface water samples were tested throughout the country, of which 2 (3.3%) were positive for HEV RNA, one of them in the near vicinity of a pig farm. All HEV strains were analysed at 5’ ORF1 and 5’ ORF2 regions and both genome regions confirmed that Slovenian HEV strains represent a distinct genotype 3 lineage, diverse from all other genotype 3 lineages available in GenBank and described in the literature to date. All but one HEV strains detected in pigs in Slovenia represent a monophyletic branch in phylogenetic trees, with a high degree of sequence identity. One human HEV strain belonged to genotype 1 and two to genotype 3 but did not match the new genotype 3 lineage detected in Slovenian pig herds.