Recent decline in hepatitis E virus prevalence among wild boars in Japan: Probably due to countermeasures implemented in response to outbreaks of classical swine fever virus infection.

Previous studies have emphasized the necessity of surveillance and control measures for hepatitis E virus (HEV) infection in wild boars, an important reservoir of HEV. To assess the current situation of HEV infection in wild boars in Japan, this study investigated the prevalence and genetic diversity of HEV among wild boars captured in 16 prefectures of Japan during 2018-2023. Serum samples from 968 wild boars were examined for anti-HEV IgG antibodies and HEV RNA. The prevalence of anti-HEV IgG varied geographically from 0 % to 35.0 %. HEV RNA was detected in 3.6 % of boars, with prevalence varying by prefecture from 0 % to 22.2 %. Genotype 3 was the most prevalent genotype (91.9 %), followed by genotype 4 (5.4 %), with one strain closely related to genotype 6. The prevalence of HEV infection among wild boars decreased from 2018/2019 to 2022/2023 with significant declines in levels of anti-HEV IgG antibodies (14.5 % vs. 6.2 %, P < 0.0001) and HEV RNA (7.6 % vs. 1.5 %, P < 0.0001). Regional analysis showed varying trends, with no HEV RNA-positive boars found in several regions in recent years. A plausible factor contributing to the decline in HEV infection is the application of countermeasures, including installing fences to prevent intrusion into pig farms, implemented in response to the emergence of classical swine fever virus (CSFV) infection in wild boars and domestic pigs, with incidents reported annually since 2018. Further investigation is warranted to explore the association between countermeasures to CSFV infection and the decrease in HEV infection among wild boars.

The Full-Genome Analysis and Generation of an Infectious cDNA Clone of a Genotype 6 Hepatitis E Virus Variant Obtained from a Japanese Wild Boar: In Vitro Cultivation in Human Cell Lines.

Hepatitis E virus (HEV) can cause self-limiting acute and chronic hepatitis infections, particularly in immunocompromised individuals. In developing countries, HEV is mainly transmitted via drinking contaminated water, whereas zoonotic transmission dominates the route of infection in developed countries, including Japan. Pigs are an important reservoir for HEV infection. Wild boars, which share the same genus and species as domestic pigs, are also an HEV reservoir. During our nationwide study of HEV infection in wild boar populations in Japan, a genotype 6 (HEV-6) strain, wbJHG_23, was isolated in Hyogo Prefecture in 2023. The genomic length was 7244 nucleotides, excluding the poly(A) tract. The wbJHG_23 strain exhibited the highest nucleotide identity throughout its genome with two previously reported HEV-6 strains (80.3-80.9%). Conversely, it displayed lower similarity (73.3-78.1%) with the HEV-1-5, HEV-7, and HEV-8 strains, indicating that, although closely related, the wbJHG_23 strain differs significantly from the reported HEV-6 strains and might represent a novel subtype. The wbJHG_23 strain successfully infected the human-derived cancer cell lines, PLC/PRF/5 and A549 1-1H8 cells, suggesting that HEV-6 has the potential for zoonotic infection. An infectious cDNA clone was constructed using a reverse genetics system, and a cell culture system supporting the efficient propagation of the HEV-6 strain was established, providing important tools for further studies on this genotype. Using this cell culture system, we evaluated the sensitivity of the wbJHG_23 strain to ribavirin treatment. Its good response to this treatment suggested that it could be used to treat human infections caused by HEV-6.

Recent advances in hepatitis E virus research and the Japanese clinical practice guidelines for hepatitis E virus infection.

Acute hepatitis E was considered rare until reports emerged affirming the existence of hepatitis E virus (HEV) genotypes 3 and 4 infections in Japan in the early 2000s. Extensive studies by Japanese researchers have highlighted the pivotal role of pigs and wild animals, such as wild boars and deer, as reservoirs for HEV, linking them to zoonotic infections in Japan. Currently, when hepatitis occurs subsequent to the consumption of undercooked or grilled pork, wild boar meat, or offal (including pig liver and intestines), HEV infection should be considered. Following the approval of anti-HEV immunoglobulin A antibody as a diagnostic tool for hepatitis E by Japan's Health Insurance System in 2011, the annual number of diagnosed cases of HEV infection has surged. Notably, the occurrence of post-transfusion hepatitis E promoted nationwide screening of blood products for HEV using nucleic acid amplification tests since 2020. Furthermore, chronic hepatitis E has been observed in immunosuppressed individuals. Considering the significance of hepatitis E, heightened preventive measures are essential. The Japan Agency for Medical Research and Development Hepatitis A and E viruses (HAV and HEV) Study Group, which includes special virologists and hepatologists, held a virtual meeting on February 17, 2024. Discussions encompassed pathogenesis, transmission routes, diagnosis, complications, severity factors, and ongoing and prospective vaccination or treatments for hepatitis E. Rigorous assessment of referenced studies culminated in the formulation of recommendations, which are detailed within this review. This comprehensive review presents recent advancements in HEV research and Japanese clinical practice guidelines for HEV infection.

Three Distinct Reporter Systems of Hepatitis E Virus and Their Utility as Drug Screening Platforms.

The hepatitis E virus (HEV) is increasingly acknowledged as the primary cause of acute hepatitis. While most HEV infections are self-limiting, cases of chronic infection and fulminant hepatitis necessitate the administration of anti-HEV medications. However, there is a lack of specific antiviral drugs designed for HEV, and the currently available drug (ribavirin) has been associated with significant adverse effects. The development of innovative antiviral drugs involves targeting distinct steps within the viral life cycle: the early step (attachment and internalization), middle step (translation and RNA replication), and late step (virus particle formation and virion release). We recently established three HEV reporter systems, each covering one or two of these steps. Using these reporter systems, we identified various potential drug candidates that target different steps of the HEV life cycle. Through rigorous in vitro testing using our robust cell culture system with the genotype 3 HEV strain (JE03-1760F/P10), we confirmed the efficacy of these drugs, when used alone or in combination with existing anti-HEV drugs. This underscores their significance in the quest for an effective anti-HEV treatment. In the present review, we discuss the development of the three reporter systems, their applications in drug screening, and their potential to advance our understanding of the incompletely elucidated HEV life cycle.

Development of a HiBiT-tagged reporter hepatitis E virus and its utility as an antiviral drug screening platform.

Previously, we developed an infectious hepatitis E virus (HEV) harboring the nanoKAZ gene in the hypervariable region of the open reading frame 1 (ORF1) of the HEV3b (JE03-1760F/P10) genome and demonstrated the usefulness for screening anti-HEV drugs that inhibit the early infection process. In the present study, we constructed another reporter HEV (HEV3b-HiBiT) by placing a minimized HiBiT tag derived from NanoLuc luciferase at the 3'-end of the viral capsid (ORF2) coding sequence. It replicated efficiently in PLC/PRF/5 cells, produced membrane-associated particles identical to those of the parental virus, and was genetically stable and infectious. The HiBiT tag was fused to both secreted ORF2s (ORF2s-HiBiT) and ORF2c capsid protein (ORF2c-HiBiT). The ORF2c-HiBiT formed membrane-associated HEV particles (eHEV3b-HiBiT). By treating these particles with digitonin, we demonstrated that the HiBiT tag was expressed on the surface of capsid and was present inside the lipid membrane. To simplify the measurement of luciferase activity and provide a more convenient screening platform, we constructed an ORF2s-defective mutant (HEV3b-HiBiT/ΔORF2s) in which the secreted ORF2s are suppressed. We used this system to evaluate the effects of introducing small interfering RNAs and treatment with an inhibitor or accelerator of exosomal release on HEV egress and demonstrated that the effects on virus release can readily be analyzed. Therefore, HEV3b-HiBiT and HEV3b-HiBiT/ΔORF2s reporters may be useful for investigating the virus life cycle and can serve as a more convenient screening platform to search for candidate drugs targeting the late stage of HEV infection such as particle formation and release. IMPORTANCE The construction of recombinant infectious viruses harboring a stable luminescence reporter gene is essential for investigations of the viral life cycle, such as viral replication and pathogenesis, and the development of novel antiviral drugs. However, it is difficult to maintain the stability of a large foreign gene inserted into the viral genome. In the present study, we successfully generated a recombinant HEV harboring the 11-amino acid HiBiT tag in the ORF2 coding region and demonstrated the infectivity, efficient virus growth, particle morphology, and genetic stability, suggesting that this recombinant HEV is useful for in vitro assays. Furthermore, this system can serve as a more convenient screening platform for anti-HEV drugs. Thus, an infectious recombinant HEV is a powerful approach not only for elucidating the molecular mechanisms of the viral life cycle but also for the screening and development of novel antiviral agents.

Infection Dynamics and Genomic Mutations of Hepatitis E Virus in Naturally Infected Pigs on a Farrow-to-Finish Farm in Japan: A Survey from 2012 to 2021.

Hepatitis E virus (HEV) causes acute or chronic hepatitis in humans. Pigs are the primary reservoir for zoonotic HEV genotypes 3 and 4 worldwide. This study investigated the infection dynamics and genomic mutations of HEV in domestic pigs on a farrow-to-finish pig farm in Japan between 2012 and 2021. A high prevalence of anti-HEV IgG antibodies was noted among pigs on this farm in 2012, when the survey started, and persisted for at least nine years. During 2012-2021, HEV RNA was detected in both serum and fecal samples, indicating active viral replication. Environmental samples, including slurry samples in manure pits, feces on the floor, floor and wall swabs in pens, and dust samples, also tested positive for HEV RNA, suggesting potential sources of infection within the farm environment. Indeed, pigs raised in HEV-contaminated houses had a higher rate of HEV infection than those in an HEV-free house. All 104 HEV strains belonged to subgenotype 3b, showing a gradual decrease in nucleotide identities over time. The 2012 (swEJM1201802S) and 2021 (swEJM2100729F) HEV strains shared 97.9% sequence identity over the entire genome. Importantly, the swEJM2100729F strain efficiently propagated in human hepatoma cells, demonstrating its infectivity. These findings contribute to our understanding of the prevalence, transmission dynamics, and genetic characteristics of HEV in domestic pigs, emphasizing the potential risks associated with HEV infections and are crucial for developing effective strategies to mitigate the risk of HEV infection in both animals and humans.

Development and Characterization of Efficient Cell Culture Systems for Genotype 1 Hepatitis E Virus and Its Infectious cDNA Clone.

Hepatitis E virus (HEV) is a major cause of acute viral hepatitis globally. Genotype 1 HEV (HEV-1) is responsible for multiple outbreaks in developing countries, causing high mortality rates in pregnant women. However, studies on HEV-1 have been hindered by its poor replication in cultured cells. The JE04-1601S strain recovered from a Japanese patient with fulminant hepatitis E who contracted HEV-1 while traveling to India was serially passaged 12 times in human cell lines. The cell-culture-generated viruses (passage 12; p12) grew efficiently in human cell lines, but the replication was not fully supported in porcine cells. A full-length cDNA clone was constructed using JE04-1601S_p12 as a template. It was able to produce an infectious virus, and viral protein expression was detectable in the transfected PLC/PRF/5 cells and culture supernatants. Consistently, HEV-1 growth was also not fully supported in the cell culture of cDNA-derived JE04-1601S_p12 progenies, potentially recapitulating the narrow tropism of HEV-1 observed in vivo. The availability of an efficient cell culture system for HEV-1 and its infectious cDNA clone will be useful for studying HEV species tropism and mechanisms underlying severe hepatitis in HEV-1-infected pregnant women as well as for discovering and developing safer treatment options for this condition.

Ritonavir Blocks Hepatitis E Virus Internalization and Clears Hepatitis E Virus In Vitro with Ribavirin.

Hepatitis E virus (HEV) is increasingly recognized as the leading cause of acute hepatitis. Although HEV infections are mostly self-limiting, a chronic course can develop especially in those with immunocompromised state. Ribavirin is currently used to treat such patients. According to various reports on chronic HEV infections, a sustained virological response (SVR) was achieved in approximately 80% of patients receiving ribavirin monotherapy. To increase the SVR rate, drug combination might be a viable strategy, which we attempted in the current study. Ritonavir was identified in our previous drug screening while searching for candidate novel anti-HEV drugs. It demonstrated potent inhibition of HEV growth in cultured cells. In the present study, ritonavir blocked HEV internalization as shown through time-of-addition and immunofluorescence assays. Its combination with ribavirin significantly increased the efficiency of inhibiting HEV growth compared to that shown by ribavirin monotherapy, even in PLC/PRF/5 cells with robust HEV production, and resulted in viral clearance. Similar efficiency was seen for HEV genotypes 3 and 4, the main causes of chronic infection. The present findings provide insight concerning the advantage of combination therapy using drugs blocking different steps in the HEV life cycle (internalization and RNA replication) as a potential novel treatment strategy for chronic hepatitis E.

Development of Recombinant Infectious Hepatitis E Virus Harboring the nanoKAZ Gene and Its Application in Drug Screening.

Hepatitis E virus (HEV) is a quasi-enveloped virus with a single-stranded positive-sense RNA genome belonging to the family Hepeviridae. Studies of the molecular aspects of HEV and drug screening have benefited from the discovery of bioluminescent reporter genes. However, the stability of large foreign genes is difficult to maintain after insertion into the viral genome. Currently, ribavirin is used to treat HEV-infected patients who require antiviral therapy. This has several major drawbacks. Thus, the development of novel anti-HEV drugs is of great importance. We developed a system consisting of recombinant infectious HEV harboring a small luciferase gene (nanoKAZ) in the hypervariable region (HVR) of the open reading frame 1 (ORF1) (HEV-nanoKAZ). It replicated efficiently in cultured cells, was genetically stable, and had morphological characteristics similar to those of the parental virus. Both membrane-associated (eHEV-nanoKAZ) and membrane-unassociated (neHEV-nanoKAZ) particles were infectious. HEV particles circulating in the bloodstream and attaching to hepatocytes in HEV-infected patients are membrane-associated; thus, eHEV-nanoKAZ was applied in drug screening. The eHEV-nanoKAZ system covers at least the inhibitor of HEV entry and inhibitor of HEV RNA replication. Four drugs with anti-HEV activity were identified. Their effectiveness in cultured cells was confirmed in naive and HEV-producing PLC/PRF/5 cells. Two hit drugs (azithromycin and ritonavir) strongly inhibited HEV production in culture supernatants, as well as intracellular expression of ORF2 protein, and may therefore be candidate novel anti-HEV drugs. The HEV-nanoKAZ system was developed and applied in drug screening and is expected to be useful for investigating the HEV life cycle. IMPORTANCE Bioluminescent reporter viruses are essential tools in molecular virological research. They have been widely used to investigate viral life cycles and in the development of antiviral drugs. For drug screening, the use of a bioluminescent reporter virus helps shorten the time required to perform the assay. A system, consisting of recombinant infectious HEV harboring the nanoKAZ gene in the HVR of ORF1 (HEV-nanoKAZ), was developed in this study and was successfully applied to drug screening in which four hit drugs with anti-HEV activity were identified. The results of this study provide evidence supporting the use of this system in more variable HEV studies. In addition, both forms of viral particles (eHEV-nanoKAZ and neHEV-nanoKAZ) are infectious, which will enable their application in HEV studies requiring both forms of viral particles, such as in the investigation of unknown HEV receptors and the elucidation of host factors important for HEV entry.

Production of capsid proteins of rat hepatitis E virus in Escherichia coli and characterization of self-assembled virus-like particles.

Rat hepatitis E virus (HEV) has been isolated from wild rats worldwide and the potential of zoonotic transmission has been documented. Escherichia coli (E. coli) is utilized as an effective system for producing HEV-like particles. However, the production of rat HEV ORF2 proteins in E. coli forming virus-like particles (VLPs) has not yet been reported. In this study, nine rat HEV ORF2 proteins of the ratELOMB-131L strain with truncated N- and C-termini (amino acids 339-594, 349-594, 351-594, 354-594, 357-594, 357-599, 357-604, 357-609, and 357-614 of ORF2 protein) were expressed in E. coli and the 357-614 protein self-assembled most efficiently. A bioanalyzer showed that the purified 357-614 protein has a molecular weight of 33.5 kDa and a purity of 93.2%. Electron microscopy revealed that the purified 33.5 kDa protein formed VLPs with a diameter of 21-52 (average 32) nm, and immunoelectron microscopy using an anti-rat HEV ORF2 monoclonal antibody (TA7014) indicated that the observed VLPs were derived from rat HEV ORF2. The VLPs attached to and entered the PLC/PRF/5 cells and blocked the neutralization of rat HEV by TA7014, suggesting that the VLPs possess the antigenic structure of infectious rat HEV particles. In addition, rat HEV VLPs showed high immunogenicity in mice. The present results would be useful for future studies on the development of VLP-based vaccines for HEV prevention in a rat model and for the prevention of rat HEV infection in humans.

Mechanism of Cross-Species Transmission, Adaptive Evolution and Pathogenesis of Hepatitis E Virus.

Hepatitis E virus (HEV) is the leading cause of acute hepatitis worldwide. While the transmission in developing countries is dominated by fecal-oral route via drinking contaminated water, the zoonotic transmission is the major route of HEV infection in industrialized countries. The discovery of new HEV strains in a growing number of animal species poses a risk to zoonotic infection. However, the exact mechanism and the determinant factors of zoonotic infection are not completely understood. This review will discuss the current knowledge on the mechanism of cross-species transmission of HEV infection, including viral determinants, such as the open reading frames (ORFs), codon usage and adaptive evolution, as well as host determinants, such as host cellular factors and the host immune status, which possibly play pivotal roles during this event. The pathogenesis of hepatitis E infection will be briefly discussed, including the special forms of this disease, including extrahepatic manifestations, chronic infection, and fulminant hepatitis in pregnant women.

Multivesicular body sorting and the exosomal pathway are required for the release of rat hepatitis E virus from infected cells.

Recent reports have shown that rat hepatitis E virus (HEV) is capable of infecting humans. We also successfully propagated rat HEV into human PLC/PRF/5 cells, raising the possibility of a similar mechanism shared by human HEV and rat HEV. Rat HEV has the proline-rich sequence, PxYPMP, in the open reading frame 3 (ORF3) protein that is indispensable for its release. However, the release mechanism remains unclear. The overexpression of dominant-negative (DN) mutant of vacuolar protein sorting (Vps)4A or Vps4B decreased rat HEV release to 23.9 % and 18.0 %, respectively. The release of rat HEV was decreased to 8.3 % in tumor susceptibility gene 101 (Tsg101)-depleted cells and to 31.5 % in apoptosis-linked gene 2-interacting protein X (Alix)-depleted cells. Although rat HEV ORF3 protein did not bind to Tsg101, we found a 90-kDa protein capable of binding to wild-type rat HEV ORF3 protein but not to ORF3 mutant with proline to leucine mutations in the PxYPMP motif. Rat HEV release was also decreased in Ras-associated binding 27A (Rab27A)- or hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs)-depleted cells (to 20.1 % and 18.5 %, respectively). In addition, the extracellular rat HEV levels in the infected PLC/PRF/5 cells were increased after treatment with Bafilomycin A1 and decreased after treatment with GW4869. These results indicate that rat HEV utilizes multivesicular body (MVB) sorting for its release and that the exosomal pathway is required for rat HEV egress. A host protein alternative to Tsg101 that can bind to rat HEV ORF3 should be explored in further study.

Antiviral candidates against the hepatitis E virus (HEV) and their combinations inhibit HEV growth in in vitro.

Hepatitis E is a global public health problem. Ribavirin (RBV) and pegylated interferon alpha are currently administered to cure hepatitis E. Recently, in combination with RBV, sofosbuvir (SOF), an anti-hepatitis C virus nucleotide analog, is also given to patients with chronic hepatitis E. However, this combinatorial therapy sometimes fails to achieve a sustained virological response. In this study, we used 27 antiviral compounds, including 15 nucleos(t)ide analogs, for in vitro screening against a genotype 3 HEV strain containing a Gaussia luciferase reporter. RBV, SOF, 2'-C-methyladenosine, 2'-C-methylcytidine (2CMC), 2'-C-methylguanosine (2CMG), and two 4'-azido nucleoside analogs (R-1479 and RO-9187) suppressed replication of the reporter genome, while only RBV, SOF, 2CMC and 2CMG inhibited the growth of genotype 3 HEV in cultured cells. Although 2CMG and RBV (2CMG/RBV) exhibited a synergistic effect while SOF/RBV and 2CMC/RBV showed antagonistic effects on the reporter assay, these three nucleos(t)ide analogs acted additively with RBV in inhibiting HEV growth in cultured cells. Furthermore, SOF and 2CMG, with four interferons (IFN-α2b, IFN-λ1, IFN-λ2 and IFN-λ3), inhibited HEV growth efficiently and cleared HEV in cultured cells. These results suggest that, in combination with RBV or interferons, SOF and 2CMG would be promising bases for developing anti-HEV nucleos(t)ide analogs.

Genetic Variability and Evolution of Hepatitis E Virus.

Hepatitis E virus (HEV) is a single-stranded positive-sense RNA virus. HEV can cause both acute and chronic hepatitis, with the latter usually occurring in immunocompromised patients. Modes of transmission range from the classic fecal-oral route or zoonotic route, to relatively recently recognized but increasingly common routes, such as via the transfusion of blood products or organ transplantation. Extrahepatic manifestations, such as neurological, kidney and hematological abnormalities, have been documented in some limited cases, typically in patients with immune suppression. HEV has demonstrated extensive genomic diversity and a variety of HEV strains have been identified worldwide from human populations as well as growing numbers of animal species. The genetic variability and constant evolution of HEV contribute to its physiopathogenesis and adaptation to new hosts. This review describes the recent classification of the Hepeviridae family, global genotype distribution, clinical significance of HEV genotype and genomic variability and evolution of HEV.

The identification and characterization of novel rat hepatitis E virus strains in Bali and Sumbawa, Indonesia.

All three genetic groups of ratHEV have been found in Indonesia, suggesting the presence of additional variants of ratHEV in unexamined areas of Indonesia. A total of 242 wild rats were captured in Bali and Sumbawa, Indonesia, during 2014-2016. Among them, 4.1% were seropositive for anti-ratHEV IgG and two (0.8%) had detectable ratHEV RNA: ratESUMBAWA-140L and ratEBali2016D-047L, sharing 84.9-85.4% and 86.9-92.1% nucleotide identity with the reported G2 strains, respectively. The provisional criteria supported the notion that the ratEBali2016D-047L and ratESUMBAWA-140L strains were novel G2 variants. These results suggested the spatial distribution of further divergent ratHEV strains in Indonesia.

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.

Full-length genomic sequences of new subtype 1g hepatitis E virus strains obtained from four patients with imported or autochthonous acute hepatitis E in Japan.

Hepatitis E virus (HEV) causes acute or chronic hepatitis in humans worldwide and can be transmitted via the fecal-oral route. Four HEV strains (HE-JA14-2173, HE-JA15-1335, HE-JA15-1920 and HE-JA16-0610) obtained from patients with imported (from Pakistan or India) or autochthonous acute hepatitis E in Japan were most closely related to the Nepalese and Mongolian genotype 1 HEV strains of unassigned subtype within the partial ORF2 sequence. To investigate whether a putative novel subtype (1g) of genotype 1 can be assigned, full-length genomic sequences were determined for the four HEV strains. They shared 95.4-99.2% nucleotide identity over the entire genome, and differed by 6.3-11.7% from the reported HEV strains of subtypes 1a-1f and by only 0.6-4.7% from a Mongolian genotype 1 HEV strain (MNE15-072) of unassigned subtype. A phylogenetic analysis showed that the four HEV strains obtained in the present study formed a cluster with MNE15-072, with a bootstrap value of 100%. Although the p-distance between subtypes 1a and 1f was 0.048-0.083, these five strains showed a higher nucleotide p-distance value of 0.068-0.138 with the genotype 1 HEV strains of subtypes 1a-1f. A BLAST search revealed the presence of candidate members of subtype 1g HEV in at least five other countries, including France, Israel, the Netherlands, Portugal, and the UK, sharing identities of 95.4-99.6% with the HE-JA16-0610 strain within the common sequence of 294-867 nucleotides. These results support the assignment of a new subtype 1g within genotype 1 and suggest a global distribution of subtype 1g strains. Subtype 1g strains found in Europe can be imported from Asia. Further studies are needed to confirm the global distribution of HEV subtype 1g.

Characterization of the Quasi-Enveloped Hepatitis E Virus Particles Released by the Cellular Exosomal Pathway.

Our previous studies demonstrated that membrane-associated hepatitis E virus (HEV) particles-now considered "quasi-enveloped particles"-are present in the multivesicular body with intraluminal vesicles (exosomes) in infected cells and that the release of HEV virions is related to the exosomal pathway. In this study, we characterized exosomes purified from the culture supernatants of HEV-infected PLC/PRF/5 cells. Purified CD63-, CD9-, or CD81-positive exosomes derived from the culture supernatants of HEV-infected cells that had been cultivated in serum-free medium were found to contain HEV RNA and the viral capsid (ORF2) and ORF3 proteins, as determined by reverse transcription-PCR (RT-PCR) and Western blotting, respectively. Furthermore, immunoelectron microscopy, with or without prior detergent and protease treatment, revealed the presence of virus-like particles in the exosome fraction. These particles were 39.6 ± 1.0 nm in diameter and were covered with a lipid membrane. After treatment with detergent and protease, the diameter of these virus-like particles was 26.9 ± 0.9 nm, and the treated particles became accessible with an anti-HEV ORF2 monoclonal antibody (MAb). The HEV particles in the exosome fraction were capable of infecting naive PLC/PRF/5 cells but were not neutralized by an anti-HEV ORF2 MAb which efficiently neutralizes nonenveloped HEV particles in cell culture. These results indicate that the membrane-wrapped HEV particles released by the exosomal pathway are copurified with the exosomes in the exosome fraction and suggest that the capsids of HEV particles are individually covered by lipid membranes resembling those of exosomes, similar to enveloped viruses.IMPORTANCE Hepatitis E, caused by HEV, is an important infectious disease that is spreading worldwide. HEV infection can cause acute or fulminant hepatitis and can become chronic in immunocompromised hosts, including patients after organ transplantation. The HEV particles present in feces and bile are nonenveloped, while those in circulating blood and culture supernatants are covered with a cellular membrane, similar to enveloped viruses. Furthermore, these membrane-associated and -unassociated HEV particles can be propagated in cultured cells. The significance of our research is that the capsids of HEV particles are individually covered by a lipid membrane that resembles the membrane of exosomes, similar to enveloped viruses, and are released from infected cells via the exosomal pathway. These data will help to elucidate the entry mechanisms and receptors for HEV infection in the future. This is the first report to characterize the detailed morphological features of membrane-associated HEV particles.

Full-length genome of a novel genotype 3 hepatitis E virus strain obtained from domestic pigs in Japan.

Hepatitis E virus (HEV) causes acute or chronic hepatitis in humans and can be transmitted via the fecal-oral route. Pigs are one of the main reservoirs for this infection. Sixty pigs, 4-5 months of age, on a swine herd in Japan had detectable anti-HEV IgG antibodies, and five (8.3%) of them had ongoing infection of genotype 3 HEV. Five HEV strains obtained from the viremic pigs shared 98.8-100% nucleotide identity, and one representative strain (swHE1606845), whose entire genomic sequence was determined in this study, differed by 14.1-19.6% from the reported HEV strains of subtypes 3a-3k and by 14.7-19.1% from other genotype 3 HEV strains whose subtypes have not yet been assigned. swHE1606845 showed a higher nucleotide p-distance value of ≥0.143 with the genotype 3 HEV strains of subtypes 3a-3k and ≥0.152 with other genotype 3 strains of unassigned subtypes. A SimPlot analysis revealed a lack of recombination events. These results indicate that swHE1606845 is a candidate member of a novel subtype of genotype 3. Further efforts to identify the swHE1606845-like novel strain are warranted to clarify the origin of this strain and to determine the complete nucleotide sequences of two additional swHE1606845-like strains for assigning a new subtype.