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.

TRIM26 positively affects hepatitis B virus replication by inhibiting proteasome-dependent degradation of viral core protein.

Chronic hepatitis B virus (HBV) infection is a major medical concern worldwide. Current treatments for HBV infection effectively inhibit virus replication; however, these treatments cannot cure HBV and novel treatment-strategies should be necessary. In this study, we identified tripartite motif-containing protein 26 (TRIM26) could be a supportive factor for HBV replication. Small interfering RNA-mediated TRIM26 knockdown (KD) modestly attenuated HBV replication in human hepatocytes. Endogenous TRIM26 physically interacted with HBV core protein (HBc), but not polymerase and HBx, through the TRIM26 SPRY domain. Unexpectedly, TRIM26 inhibited HBc ubiquitination even though TRIM26 is an E3 ligase. HBc was degraded by TRIM26 KD in Huh-7 cells, whereas the reduction was restored by a proteasome inhibitor. RING domain-deleted TRIM26 mutant (TRIM26ΔR), a dominant negative form of TRIM26, sequestered TRIM26 from HBc, resulting in promoting HBc degradation. Taking together, this study demonstrated that HBV utilizes TRIM26 to avoid the proteasome-dependent HBc degradation. The interaction between TRIM26 and HBc might be a novel therapeutic target against HBV infection.

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.

Genomic characterization and the prevalence of a novel copiparvovirus in wild sika deer (Cervus nippon) in Japan.

A preliminary metagenomic analysis of the virome of wild sika deer (Cervus nippon) blood in Japan resulted in the identification of a novel parvovirus. The virus was closest, but only 44.7-60.7% identical to 17 reported strains belonging to the genus Copiparvovirus within the subfamily Parvovirinae, over the near-entire genomic sequence. The sika deer copiparvovirus DNA was detected in 15% (31/206) of sika deer captured in 7 prefectures of Japan, and a region-dependent prevalence of 0-66.7% was noted, with a biased distribution in the southern part of Japan. The observed biased distribution of sika deer copiparvovirus may be due to the habitat density of deer and the number of ticks, which might play a role in the transmission of the virus.

Chronic Infection with Hepatitis C Virus Subtype 1g in a Japanese Patient Successfully Treated with Glecaprevir/Pibrentasvir.

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.

Regulatory function of interferon-inducible 44-like for hepatitis B virus covalently closed circular DNA in primary human hepatocytes.

AIM: Curing hepatitis B virus (HBV) infection requires elimination of covalently closed circular DNA (cccDNA). Interferon (IFN)-γ has noncytolytic antiviral potential; however, elimination of cccDNA could not be achieved. To enhance the regulatory effect, we comprehensively analyzed the host factors associated with cccDNA amplification and IFN-γ and IFN-α effects using an in vitro HBV infection system showing various transcription levels. METHODS: Primary human hepatocytes were infected with HBV using genomic plasmids carrying the basic core promoter mutation A1762T/G1764A and/or the precore mutation G1896A and treated with IFN-γ and IFN-α. Comprehensive and functional studies involving microarray and small interfering RNA analysis revealed the host factors related to cccDNA regulation. RESULTS: The HBV infection system reproduced the HBV life cycle and showed various propagation levels. Microarray analysis revealed 53 genes correlated with the cccDNA levels. Of the 53 genes, expression of IFN-induced protein 44-like (IFI44L) was significantly upregulated by IFN-γ and IFN-α. The anti-HBV effect of IFI44L is exerted regardless of IFN-γ or IFN-α by inhibiting the activation of nuclear factor-κB and signal transducer and activator of transcription 1 pathways. CONCLUSIONS: Using the in vitro HBV infection system, an IFN-inducible molecule, IFI44L, associated with cccDNA amplification, was identified. These results suggest an innovative molecular strategy for the regulation of HBV cccDNA by controlling a novel host factor, IFI44L.

Identification of a novel pegivirus in pet cats (Felis silvestris catus) in Japan.

We report a novel pegivirus in pet cats (Felis silvestris catus) in Japan. This virus was only 44.0-49.6 % identical to the reported viruses in the 11 current Pegivirus species and an unclassified pegivirus in dolphins within the entire protein-coding nucleotide sequence and was detected in 1.6 % of pet cats.

Complete Genome Sequences of Two Rat Pegivirus Strains in Indonesia.

The entire genome sequences of two pegivirus strains recovered from serum samples of wild rats (Rattus rattus) in Indonesia were determined. They possessed 11,013 to 11,014 nucleotides and differed from the reported rodent pegivirus strains within the Pegivirus J species of the genus Pegivirus by 12.7% to 40.9% in the near-entire coding region sequences.

Identification and a full genome analysis of novel camel hepatitis E virus strains obtained from Bactrian camels in Mongolia.

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 BcHEV-MNG140 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.

Prevalence and genotype/subtype distribution of hepatitis E virus (HEV) among wild boars in Japan: Identification of a genotype 5 HEV strain.

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.

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.

Molecular signature of hepatitis B virus regulation by interferon-γ in primary human hepatocytes.

AIM: A complete cure for chronic hepatitis B virus (HBV) infection requires elimination of covalently closed circular DNA; however, this remains to be clinically achieved. Interferon (IFN)-γ, a type II IFN, is produced by intrahepatic cytotoxic T lymphocytes and has non-cytolytic antiviral potential. However, the mechanism by which IFN-γ regulates HBV infection has not been fully elucidated. Thus, we developed an in vitro HBV infection assay system and analyzed the molecular signature of HBV regulation by IFN-γ. METHODS: The in vitro HBV infection assay system was established in primary human hepatocytes infected with HBV derived from the plasmid containing 1.3-mer HBV genome, and treated with IFN-γ. The antiviral effects and signaling pathways of IFN-γ were examined using microarray, and assessed by siRNA knockdown experiments of the related genes. RESULTS: IFN-γ treatment suppressed both HBV propagation and transcription as efficiently as IFN-α. Microarray analysis showed that IFN-γ stimulation induced the activation of both IFN-γ and IFN-α signaling, regulating HBV covalently closed circular DNA. HBV production was decreased by IFN-γ through Janus kinase/signal transducer and activator of transcription signaling and interferon-stimulated genes, such as 2'-5'-oligoadenylate synthase 2 and apolipoprotein B mRNA editing enzyme catalytic subunit 3G. CONCLUSIONS: IFN-γ can suppress HBV propagation and transcription in hepatocytes by activating specific intracellular signaling pathways in hepatocytes, and suggests the future application of these particular signaling pathways or genes for the complete elimination of HBV.

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.

Screening of novel drugs for inhibiting hepatitis E virus replication.

Hepatitis E, which is caused by hepatitis E virus (HEV), is generally a self-limiting, acute, and rarely fatal disease. It is sometimes fulminant and lethal, especially during pregnancy. Indeed, it occasionally takes a chronic course in immunocompromised individuals. To cure hepatitis E patients, the broad-spectrum antivirals (ribavirin and pegylated interferon α) are used. However, this treatment is insufficient and unsafe in some patients due to embryoteratogenic effects, leukopenia, and thrombocytopenia. In this study, we constructed an HEV replication reporter system with Gaussia luciferase for comprehensively screening anti-HEV drug candidates, and developed a cell-culture system using cells robustly producing HEV to validate the efficacy of anti-HEV drug candidates. We screened anti-HEV drug candidates from United States Food and Drug Administration-approved drugs using the established HEV replication reporter system, and investigated the selected candidates and type III interferons (interferon λ1-3) using the cell-culture system. In conclusion, we constructed an HEV replicon system for anti-HEV drug screening and a novel cell-culture system to strictly evaluate the replication-inhibitory activities of the obtained anti-HEV candidates. Our findings suggested that interferon λ1-3 might be effective for treating hepatitis E.

Identification and full-genome characterization of novel circoviruses in masked palm civets (Paguma larvata).

The family Circoviridae comprises a large group of small, circular, single-stranded DNA viruses and is classified into two genera: Circovirus and Cyclovirus. They have marked genetic diversity and a broad host range. In this study, three novel circovirus genomes were identified from wild-caught masked palm civets (Paguma larvata) in Japan and classified as a new species within the genus Circovirus based on the demarcation criteria of the International Committee on the Taxonomy of Viruses. Of note, the presence of two predicted introns at the 5'-terminus of the rep gene was suggested in the Paguma larvata circovirus genomes.

Identification and whole genome characterization of novel anelloviruses in masked palm civets (Paguma larvata): Segregation into four distinct clades.

The members of the family Anelloviridae are small and single-stranded DNA viruses with marked diversity in sequence and length, which ubiquitously infect many vertebrates, including mammals, birds and reptiles. The anelloviruses isolated from mammals are currently classified into 11 assigned and four proposed genera; some anelloviruses remain unassigned. The present study was conducted to identify anelloviruses in wild-caught masked palm civets (Paguma larvata) in Japan using a rolling-circle amplification method. Thirteen novel anellovirus strains were identified from 8 of 10 masked palm civets and their entire genomic sequences (2039-2535 nucleotides) were determined; they were classifiable into four distinct clades. Comparative analyses of all reported anelloviruses for which the entire or near-entire genomic sequences have been determined, including the 13 strains obtained in the present study, revealed that anelloviruses can provisionally be classified into 20 clades, which may correspond to 20 genera (including 11 assigned and four proposed genera) by a >70% amino acid sequence difference in open reading frame 1 (ORF1). This study suggested that novel anelloviruses of marked diversity are circulating in animals worldwide, and that the rolling-circle amplification method would be useful for identifying novel anelloviruses and other viruses with a circular DNA genome.

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.

An analysis of two open reading frames (ORF3 and ORF4) of rat hepatitis E virus genome using its infectious cDNA clones with mutations in ORF3 or ORF4.

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.

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.