長嶋 茂雄

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 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.

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 & PRIME;-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/& UDelta;ORF2s) 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/& UDelta;ORF2s 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.

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.

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.

A woman in her late 70 s was diagnosed with liver injury at a health examination. Despite treatment with ursodeoxycholic acid at a nearby hospital, her transaminase levels elevated in two peaks. She was transferred to our hospital 77 days after the health examination. She weighed 42 kg and had a low body mass index of 19.8 kg/m2. Viral markers, including immunoglobulin A (IgA) against hepatitis E virus (anti-HEV IgA), were negative. Drug-induced liver injury was negligible. We suspected autoimmune hepatitis because of the patient's female gender and positive antinuclear antibody. However, prednisolone and azathioprine failed to completely improve her hepatitis. On day 643, anti-HEV IgA was re-evaluated and found to be positive. She was diagnosed with autochthonous chronic hepatitis E because the virus strains in the preserved serum on day 77 and the serum on day 643 had identical nucleotide sequences (genotype 3a). Following prednisolone and azathioprine discontinuation, ribavirin (RBV) was administered for 3 months. HEV RNA disappeared and remained negative for more than 6 months after the cessation of RBV. The HEV RNA titer of 6.2 log10 copies/mL on day 77 was unusually high 2.5 months after the onset, suggesting that hepatitis E had already been chronic before immunosuppressive treatment for possible autoimmune hepatitis. After getting married at 23 years old, she had been a housewife and had no comorbidities that might deteriorate her immunity. Chronicity should be kept in mind when encountering HEV infection in elderly and underweight patients.

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.

Rat hepatitis E virus (HEV-C1) in the Orthohepevirus C species has been reported to cause zoonotic infection and hepatitis in humans. HEV-C1 strains have been detected from wild rats in many countries in Europe, Asia, and North America. However, in Japan, no HEV-C1 strains have been identified. In the present study, 5 (1.2%) of 428 wild rats (Rattus norvegicus or R. rattus) were positive for anti-HEV-C1 IgG. Although all 428 rat sera were negative for HEV-C1 RNA, it was detectable in 20 (19.8%) of 101 rat fecal samples collected on a swine farm, where HEV (genotype 3b, HEV-3b) was prevalent and wild rats were present. In addition, HEV-C1 RNA was detectable in the intestinal contents and liver tissues of 7 (18.9%) of 37 additional rats captured on the same farm. The HEV-C1 strain (ratEJM1703495L) obtained in this study shared only 75.8-84.7% identity with reported HEV-C1 strains over the entire genome but propagated efficiently in cultured cells. HEV-3b strains were detected in the rats' intestinal contents, with 97.3-99.5% identity to those in pigs on the same farm, but were undetectable in rat liver tissues, suggesting that wild rats do not support the replication of HEV-3b of swine origin.

A case of subclinical hepatitis E virus (HEV) infection was detected by nucleic acid amplification test on blood donation. The patient was followed-up until day 220 after the blood donation but showed no symptoms throughout the observation period. Aspartate aminotransferase and alanine aminotransferase levels reached the maximum values on day 37 with a slight increase but remained in normal ranges from day 67 to 220. The quantity of HEV RNA at the initial examination on day 13 was 1.1 × 102 copies/mL, which increased to 2.8 × 103 copies/mL by day 37. It was not detected from day 67 to 220. Immunoglobulin G class antibody to HEV (anti-HEV IgG) was below the cut-off value until day 37 and exceeded the cut-off value to positive on day 67, accompanied by normalization of liver function and negative conversion of HEV RNA. Thereafter, the titer decreased gradually, falling below the cut-off value on day 163, and continuing negative until day 220. Although the persistent duration of anti-HEV IgG positive is believed to be generally long, it was within only 126 days for this subclinical case. Further investigation is needed to determine whether short-term positivity for anti-HEV IgG is typical in subclinical HEV infection.

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.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.

Hepatitis E virus (HEV) is a zoonotic agent mainly transmitted through the consumption of uncooked or undercooked meat products derived from infected animals. In Japan, domestic pigs and wild boars are the major animal reservoirs, and whether or not deer are an HEV reservoir remains controversial. We analyzed 395 serum and 199 liver samples from 405 sika deer (Cervus nippon) caught in the wild between 1997 and 2020 in 11 prefectures of Japan for markers of HEV infection. Overall, 17 deer had anti-HEV IgG (4.3%), while 1 (0.2%) had HEV RNA (genotype 3b), indicating the occurrence of ongoing HEV infection in wild deer in Japan. An analysis of the complete HEV genome (deJOI_14) recovered from a viremic deer in Oita Prefecture revealed only 88.8% identity with the first HEV strain in sika deer (JDEER-Hyo03L) in Japan, being closest (96.3%) to the HEV obtained from a hepatitis patient living in the same prefecture. Of note, the deJOI_14 strain was 8.7-9.0% different from the wild boar HEV strains obtained in the same habitat and the same year, suggesting that difference in infected HEV strains between boar and deer may be explained by the limited possibility of close contact with each other, although boars are a known source of HEV infection. Increased numbers of hepatitis E cases after consumption of raw or undercooked meat products of wild deer have been reported in Japan. These results suggest a low but nonnegligible zoonotic risk of HEV infection in wild deer in this country.

岐阜県近郊で発生したE型肝炎13例のE型肝炎ウイルス(HEV)の感染源・感染経路を、問診と原因HEVの遺伝子配列の解析から推測した。全員孤発例であった。2例は生のブタ内臓肉、1例は調理したブタ内臓肉、7例は調理したブタ肉、2例は調理不十分なブタ肉の喫食歴があった。HEVゲノムのORF2領域の412塩基長の配列をもとに近隣結合法で系統樹を作成した。1例はORF1領域を解析した。原因HEVの遺伝子型は、3aが5例、3bが6例、3fが1例、4iが1例であった。13例中2例は原因HEV株に近縁株の登録はなかったが、11例には97%以上の相同性を持つ株が見つかり、地域内拡散8例、国内広域拡散2例、国際拡散1例の3つのパターンに類別された。

A 66-year-old man, who had undergone plasma exchange 30 years previously in Egypt for the treatment of falciparum malaria, was referred to our hospital for treatment of chronic hepatitis C (HCV). An analysis of the 655-nucleotide 5'-untranslated region-core region sequence revealed infection with HCV subtype 1g. A phylogenetic analysis of the full-length HCV genome confirmed that the patient's HCV was subtype 1g, which was the first case identified in Japan. Although his HCV possessed several naturally occurring resistance-associated substitutions in the nonstructural (NS) 3 and NS5A regions, he was successfully treated by combination therapy with glecaprevir/pibrentasvir.

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.

A 76-year-old woman with spontaneous reactivation of hepatitis B virus (HBV) without any immunosuppressants who had been successfully treated with tenofovir alafenamide fumarate (TAF) was reported. The patient was admitted to our hospital because of acute exacerbation of the liver function and jaundice. She had been found to have chronic HBV infection with a normal liver function and had been treated for lifestyle-related diseases, such as diabetes mellitus, dyslipidemia and hypertension, for over 10 years at a local clinic. At admission, her serum HBV DNA was high (7.3 log IU/mL), and anti-hepatitis B core protein immunoglobulin M was slightly elevated (1.47 S/CO). Due to the absence of known risk factors for HBV reactivation, the reactivation was regarded as "spontaneous". After the initiation of the nucleotide analog TAF, her liver function gradually improved with a decrease in the HBV DNA load. Her HBV genome was typed as subgenotype B1 and possessed a frameshift mutation due to an insertion of T after nucleotide (nt) 1817 and G to A mutations at nt 1896 and nt 1899 (G1896A/G1899A) in the precore region as well as serine to glutamine substitution of amino acid 21 in the core protein. In addition to these viral mutations, aging and complications of lifestyle-related diseases in the present case may have been responsible for the spontaneous HBV reactivation. Careful observation and management of aged HBV carriers with underlying diseases are needed even when persistent HBV infection is free from symptoms and liver dysfunction and no immunosuppressive conditions are involved.

Hepatitis E virus (HEV) infects humans and a wide variety of other mammalian hosts. Recently, HEV strains belonging to genotype 8 (G8) within the Orthohepevirus A species of the Hepeviridae family, were identified in Bactrian camels (Camelus bactrianus) in China. The Bactrian camel (also known as the Mongolian camel) is native to the steppes of Central Asia. However, the HEV strains of Mongolian camels have not been examined. Among 200 serum samples from domestic Bactrian camels raised on 6 farms, in 6 soums in 3 provinces; 71 (35.5 %) were positive for anti-HEV IgG, with prevalence differing by farm (soum) (4.2-75.0 %); and 2 camels (1.0 %) that had been raised in Bogd, Bayankhongor Province, which had the highest seroprevalence among the six studied areas, were positive for HEV RNA. The two HEV strains (BcHEV-MNG140 and BcHEV-MNG146) obtained from the viremic camels in the present study shared 97.7 % nucleotide identity. They were closest to the reported G8 Chinese camel HEV strains but differed from them by 13.9-14.3 % over the entire genome, with a nucleotide difference of 24.0-26.5 % from the reported G1-G7 HEV strains. A phylogenetic tree indicated that the BcHEVMNG140 and BcHEV-MNG146 strains were located upstream of a clade consisting of the Chinese camel HEV strains and formed a cluster with them, with a bootstrap value of 100 %, suggesting that they may represent a novel subtype within G8. These results indicate a high prevalence of HEV infection in Mongolian camels and suggest that the variability of camel HEV genomes is markedly high.

In Mie Prefecture, autochthonous hepatitis E cases occurred via homologous hepatitis E virus subgenotype 3e (HEV-3e), namely, Mie indigenous HEV-3e, from 2004 through 2014. We experienced three cases of autochthonous hepatitis E in 2020 in Mie Prefecture, and analyzed the partial HEV genomes recovered from them. The three HEV isolates clustered with Mie indigenous HEV-3e in the phylogenetic tree and exhibited high similarities to it. Mie indigenous HEV-3e had thus reemerged for the first time in six years. One affected patient often consumed meat of wild animals, and another ate undercooked pork liver, although the third case had no clear risk factors. Mie indigenous HEV-3e appears to be maintained in wild animals or domestic pigs.

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.

The clinical and virologic features of hepatitis E virus (HEV) infection seem to vary among regions even in developed countries. However, we have little information on the diversity of HEV infection. Here, we investigated the characteristics of 26 patients in our hospital located in Tochigi prefecture, 90 km north of Tokyo, between 2000 and 2019. The reported number of patients with acute hepatitis E is increasing in Japan because measurement of IgA-class anti-HEV antibody was commercially available from 2011. In contrast, the numbers at our hospital were 1.5/y and 1.0/y in 2000 to 2011 and 2012 to 2019, respectively. This is attributed to the fact that we have been investigating HEV as a cause of unknown hepatitis before 2011. Among isolated HEV subgenotypes, including 3a, 3b, 4b, 4c, and 4d, all three patients with subgenotype 4c infection presented acute liver failure. Four HEV strains shared more than or equal to 99% identity within the 412-nucleotide partial sequence, in which the time and place of HEV infection varied, except for one intrafamilial infection. In addition, some strains were similar to HEV strains isolated far from Tochigi prefecture. In conclusion, the number of patients with acute hepatitis E was not increasing at Jichi Medical University Hospital and some strains were found to circulate in Japan.

To further investigate the prevalence of hepatitis E virus (HEV) infection and characterize HEV genomes among Japanese wild boars (Sus scrofa leucomystax), 1880 boars captured in 17 prefectures in Japan from 2013 to 2019 were studied. Overall, anti-HEV IgG was detected in 8.9 % and HEV RNA was detected in 3.9 % of boars, which was comparable with our previous studies during 2003-2013 (10.3 % and 3.5 %, respectively). Among 74 boar HEV strains obtained from infected boars in the present study, 50 (68 %) were classified into genotype 3 (3a and 3b), 23 (31 %) were classified into genotype 4 (4i), and the remaining strain (wbJGF_19-1) was classified into genotype 5. The wbGF_19-1 strain shared 92.7 % identity over the entire genome with the prototype genotype 5 strain (JBOAR135-Shiz09). The identification of the second genotype 5 HEV strain in a place that is located only 100 km from the site at which JBOAR135-Shiz09 was identified, suggests that genotype 5 HEV circulates within a relatively close range in Japan. Genetically similar HEV strains forming a clade were identified from wild boars living in each area during the observation period of 11-13 years, although the nucleotide sequence changed gradually, accounting for up to 3.4-3.6 % within the 412-nucleotide ORF2 sequence. Eight groups of boars with a cluster of HEV infections were observed, consisting of two, three or four infected offspring, presumably born to the same mother or offspring with their mother. These results suggest that wild boars continue to be important reservoirs for HEV infection in humans in Japan.

A 64-year-old woman was infected with hepatitis E virus (HEV) during chemotherapy for leukemia. By retrospective analyses of stored serum from the blood products and the patient, the source of the infection was determined to be platelet concentration (PC) transfused during chemotherapy. The partial nucleotide sequence of the HEV strain isolated from the donated PC and that from the patient's sera was identical and was subgenotype 3b. Clinical indicators such as alanine aminotransferase, HEV RNA titer, and anti-HEV antibodies in the serum were investigated from the beginning of the infection until 1 year after the termination of HEV infection. HEV RNA had propagated over 6 months and then cleared spontaneously after the completion of chemotherapy. Anti-HEV antibodies appeared in the serum just before the clearance of HEV RNA. Interestingly, HEV RNA was detected in the patient's urine, spinal fluid, and saliva. The HEV RNA titers in those samples were much lower than in the serum and feces. No renal, neurological, or salivary gland disorders appeared during the follow-up. We observed virological and biochemical progress and cure of transfusion-transmitted chronic hepatitis E in the patient despite an immunosuppressive status during and after chemotherapy against hematological malignancy.

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.

BACKGROUND: Recently, chronic hepatitis E has been reported in solid organ transplant (SOT) recipients in European countries. Previously, we clarified the prevalence of hepatitis E virus (HEV) infection in Japanese liver transplant recipients and identified 2 chronic hepatitis E patients infected by blood transfusion. However, the rate of HEV infection in recipients of SOTs other than liver in Japan remains unclear, so we conducted a nationwide survey to clarify the prevalence of chronic HEV infection in Japanese heart and kidney transplant recipients. METHODS: A total of 99 heart and 2526 kidney transplant recipients in 17 hospitals in Japan were examined for the presence of the IgG class of anti-HEV antibodies as well as for serum HEV RNA. RESULTS: The prevalence of anti-HEV IgG among heart and kidney transplant recipients was 7.07% (7/99) and 4.08% (103/2526), respectively. One heart transplant patient (1.01%) and 11 kidney transplant patients (0.44%) were found to be positive for HEV RNA. The HEV isolates from all viremic patients were typed as genotype 3. Four patients developed chronic hepatitis E after transplantation. Three patients were treated with ribavirin; their liver enzymes normalized, and HEV RNA became negative immediately. Sustained virologic response was achieved in all cases. CONCLUSIONS: This is the first nationwide survey of HEV infection in Japanese heart and kidney transplant recipients. The prevalence of anti-HEV IgG and HEV RNA in heart and kidney transplant recipients in Japan was lower than that in European countries. Of note, 42% of viremic transplant patients developed chronic hepatitis.

We experienced a case of autochthonous hepatitis E by hepatitis E virus (HEV) genotype 4. The phyloge-netic tree analysis indicated that the HEV isolate ob-tained from the patient was segregated into the HEV subgenotype 4a (HEV-4a) cluster. The patient had no history of travel abroad, e.g., to China, where HEV-4a strains are endemic. The source and the route of the HEV-4a infection were unknown. Strains of HEV-4a may be indigenous to Japan despite remaining unde-tected to date, either in swine or wild animals. Addi-tional HEV subgenotypes circulating in swine and wild animals in Japan require investigation to reveal the characteristics of HEV-4a infection in autochthonous cases in Japan.

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.

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.

Aim The major transmission mode of hepatitis A virus (HAV) in Japan is the fecal-oral route by contaminated foods. In contrast, HAV infection is well documented as a sexually transmitted disease in Europe and North America. The present study was undertaken to determine the full-genome sequence of HAV and trace the transmission route of HAV in Japanese men who have sex with men (MSM). Methods In 2018, we encountered three Japanese MSM with acute hepatitis A co-infected with HIV for 4-12 years. Serum samples obtained from these patients were used for HAV full-genome analyses. Results Isolated HAV strains were segregated into subgenotype IA. The three HAV strains shared 100% identity within the 481-nucleotide partial sequence. The entire nucleotide sequence showed that the three strains were 99.97% similar to each other with only two nucleotide substitutions. At the amino acid level, the three strains differed from each other by only one or two amino acids. All three strains obtained in the present study were >99.6% identical to the 66 reported strains isolated from Taiwan and European countries during 2015-2017. In addition, these 66 strains include the RIVM-HAV16-090 (EuroPride) strain, which has been involved in HAV outbreaks among MSM worldwide. Conclusions We determined for the first time the full-genome sequence of HAV isolated from Japanese MSM with acute hepatitis A and found that the strains were identical to those from MSM worldwide. Thus, these HAV strains were imported to Japan from foreign countries through MSM.

A 65-year-old man presented with acute liver failure and grade IV coma caused by hepatitis B virus (HBV) infection in 2017. The patient died on day 12 from the disease onset. The HBV isolated from the patient was genotype/subgenotype B/B1 and had multiple genomic mutations. The patient's wife was hepatitis B surface antigen (HBsAg)-positive when she delivered her first daughter in 1979. The HBV isolates of the patient and the wife shared 100% similarity over the entire genome. Because the patient's HBsAg value had been negative one year earlier, we considered the source of HBV transmission to be his wife.

著者らは三重県と岐阜県を中心にE型肝炎の発生状況を調査し、患者の喫食歴などを問診するとともに、原因となったHEVの遺伝子配列を決定し、症例相互の原因HEV株の関係や、データベースから探究した近縁株との関係を調べることで、感染源・感染経路の解明に努めている。今回、問診では互いに接点がないと思われたが、発症時期と遺伝子解析結果から感染源が同一と推測された3組6症例が存在したので、各症例の概要を報告した。6例とも同一経済圏内の発生であり、感染にはブタ肉などの食品の流通や人の往来が関与していると考えられた。

症例は73歳男性で、進行胃癌+肝転移に対する化学療法にて薬剤性間質性肺炎を発症し、プレドニゾロンを12日間投与され軽快傾向で退院した。定期受診にて急性肝障害が指摘され緊急入院となり、グリチルリチン製剤投与にて経過観察中IgAクラスE型肝炎ウイルス陽性が判明し急性E型肝炎と診断した。血小板減少を伴ったが、入院13日目には肝障害が改善したため退院し、血小板数も正常に復した。退院後52日目に再入院して胃癌に対しニボルマブを投与したところ血小板が0.2×10^4/μlまで減少し、血小板輸血とデキサメサゾン投与を行ったが、再入院15日目に上部消化管出血のため死亡した。骨髄穿刺検査の結果と経過から、ニボルマブ投与による免疫性の血小板減少症が考えられた。

Rat hepatitis E virus (ratHEV) genome has four open reading frames (ORFs: ORF1, ORF2, ORF3 and ORF4). The functions of ORF3 and ORF4 are unknown. An infectious cDNA clone (pUC-ratELOMB-131L_wt, wt) and its derivatives including ORF3-defective (ΔORF3) and ORF4-defective (ΔORF4) mutants, were constructed and their full-length RNA transcripts transfected into PLC/PRF/5 cells. ΔORF3 replicated as efficiently as wt in cells. However, ≤1/1000 of the number of progenies were detectable in the culture supernatant of ΔORF3-infected cells compared with wt-infected cells. ORF4 protein was not detectable in ratHEV-infected cells or in the liver tissues of ratHEV-infected rats. No marked differences were noted between wt and ΔORF4 regarding the viral replication and protein expression. ORF3 mutants with proline-to-leucine mutations at amino acids (aa) 93, 96 and/or 98 in ORF3 were constructed and transfected into PLC/PRF/5 cells. Wt and an ORF3 mutant with leucine at aa 98 (ORF3-L98) replicated efficiently (density 1.15–1.16 g/cm3), while ORF3-L93 + L96 exhibited a decreased viral release and banded at 1.26–1.27 g/cm3, similar to ΔORF3. In conclusion, the ORF3 protein, especially its proline residues at aa 93 and 96, is essential for the release of membrane-associated ratHEV particles, and ORF4 is unnecessary for the replication of ratHEV.

Positive-strand RNA viruses, including picornaviruses, utilize cellular machinery for genome replication. Previously, we reported that each of the 2B, 2BC, 2C, 3A, and 3AB proteins of Aichi virus (AiV), a picornavirus, forms a complex with the Golgi apparatus protein ACBD3 and phosphatidylinositol 4-kinase IIIβ (PI4KB) at viral RNA replication sites (replication organelles [ROs]), enhancing PI4KB-dependent phosphatidylinositol 4-phosphate (PI4P) production. Here, we demonstrate AiV hijacking of the cellular cholesterol transport system involving oxysterol-binding protein (OSBP), a PI4P-binding cholesterol transfer protein. AiV RNA replication was inhibited by silencing cellular proteins known to be components of this pathway, OSBP, the ER membrane proteins VAPA and VAPB (VAP-A/B), the PI4P-phosphatase SAC1, and PItransfer protein β. OSBP, VAP-A/B, and SAC1 were present at RNA replication sites. We also found various previously unknown interactions among the AiV proteins (2B, 2BC, 2C, 3A, and 3AB), ACBD3, OSBP, VAP-A/B, and SAC1, and the interactions were suggested to be involved in recruiting the component proteins to AiV ROs. Importantly, the OSBP-2B interaction enabled PI4P-independent recruitment of OSBP to AiV ROs, indicating preferential recruitment of OSBP among PI4P-binding proteins. Protein-protein interaction-based OSBP recruitment has not been reported for other picornaviruses. Cholesterol was accumulated at AiV ROs, and inhibition of OSBPmediated cholesterol transfer impaired cholesterol accumulation and AiV RNA replication. Electron microscopy showed that AiV-induced vesicle-like structures were close to ER membranes. Altogether, we conclude that AiV directly recruits the cholesterol transport machinery through protein-protein interactions, resulting in formation of membrane contact sites between the ER and AiV ROs and cholesterol supply to the ROs.

We found three pairs of six sporadic acute hepatitis E cases infected with three different genotype 3b hepatitis E virus (HEV) strains, which were 99.7%-100% identical between two cases in each pair within the partial nucleotide sequences of ORF1 and ORF2 regions of the HEV genome. While no common source of infection was recognized within each pair, HEV infections in these cases occurred within the same economic bloc in the central region of Japan. Moreover, nucleotide sequences of the HEV strains from one pair were 99.8% identical to those obtained from pig liver sold in the same economic bloc. These results suggest the importance of continued molecular epidemiologic studies on HEV strains from humans and food products.

A 73-year-old man contracted acute hepatitis E with decreased platelet count in the course of chemotherapy against advanced gastric cancer and administration of corticosteroid for adverse effects of chemotherapy. After the recovery of hepatitis, he underwent the first administration of Nivolumab, and subsequently died of bleeding from the cancer by immune-mediated severe thrombocytopenia. The relationship between the thrombocytopenia after Nivolumab therapy and prior hepatitis E virus (HEV) infection cannot be excluded. The patient ate pig offal before hepatitis E, and the HEV strain obtained from the patient was of genotype 3b, which was prevalent in Japanese pigs. HEV infection should be considered as the cause of liver dysfunction and thrombocytopenia during chemotherapy or treatment by immuno-suppressive agents.

A 79-year-old Japanese woman was diagnosed with acute hepatitis B based on laboratory tests showing positivity for IgM-class antibody against hepatitis B virus (HBV) core and hepatitis B surface antigen (HBsAg) as well as elevated transaminases. A phylogenetic analysis revealed that the HBV strain obtained from the patient belonged to genotype D/subgenotype D1, similar to strains circulating in foreign countries but different from those in Japan. The clinical course was favorable. HBsAg became negative within 10 weeks after the onset. To our knowledge, this is the first report of acute hepatitis B caused by subgenotype D1 HBV in Japan.

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.

Hepatitis E virus subtype 3f (HEV-3f) strains are usually isolated in Europe and Thailand. Recently, HEV-3f strains were detected from six acute hepatitis E patients in Japan, none of whom had a history of travel to endemic areas. We inferred the origin and transmission route of the six HEV-3f strains. A time-scaled phylogenetic tree of the six strains with reference strains was constructed using a Bayesian statistical inference framework. The time-scaled tree indicated that the six strains independently derived from similar European strains between 2008 and 2014. The pattern suggested recent inflow of multiple HEV-3f strains from Europe to Japan. Japan imports a substantial amount of pork from European countries every year. The emergence of acute hepatitis cases caused by HEV-3f strains in Japan, in patients with no history of travel abroad, might be influenced by the increased opportunities to consume pork products imported from European countries.

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.

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.

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 &gt;= 0.143 with the genotype 3 HEV strains of subtypes 3a-3k and &gt;= 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.

Full-length genomic sequences of hepatitis E virus (HEV) obtained from two consecutive cases of acute self-limiting hepatitis E in a city in northeast Japan were determined. Interestingly, two HEV isolates from each patient shared nucleotide identity of 99.97% in 7225 nucleotides, and a phylogenetic analysis showed that they formed a cluster of Japanese isolates that is considered as a new HEV subtype 3k. The high similarity of HEV sequences of two isolates from these patients in this study suggested that a subtype 3k HEV strain had spread via a commonly distributed food in the city, possibly pig liver.

AimTo evaluate the clinical and virological features of acute hepatitisE (AH-E) in Gunma prefecture and focus on the hepatitisE virus (HEV) infection in immunocompromised patients. MethodsA total of 30 patients with AH-E diagnosed at our Gunma University Hospital, and located in 3-39-15 Showa-machi, Maebashi, Gunma 371-8511 Japan, and its affiliated hospitals from 2004 to 2015, were studied. We evaluated the detailed medical histories, laboratory examinations and virological features of these participants. ResultsOf the 30 patients, 21 patients were men, with a median age of 61years. Three of these patients had a history of recent oversea travel. A total of 14 patients had eaten raw or undercooked meat/viscera from animals, and two patients had contracted transfusion-transmitted AH-E. Eight patients were immunocompromised, including those with hematological disease, cancer receiving systemic chemotherapy and kidney transplant or connective tissue disease undergoing immunosuppressive medications. The alanine aminotransferase and total bilirubin levels were more significantly reduced in these immunocompromised patients than in the non-immunocompromised patients. Severe thrombocytopenia, an extra-hepatic manifestation of AH-E, occurred in one case. Among the 22 HEV strains whose subgenotype was determined, two were imported strains (1a and 1f), and 11 strains formed four distinct phylogenetic clusters within subgenotype3b. The remaining nine strains differed from each other by 9.8-22.4%, and were classified into four subgenotypes (3a, 3b, 3e and 3f). ConclusionMarkedly divergent HEV strains (3a, 3b, 3e and 3f) were found to circulate in Gunma. Although immunosuppression appears to play a crucial role in establishing chronic sequels, AH-E in eight immunocompromised patients, including transfusion-transmitted HEV infection in two patients, did not become chronic.