Suljid Jirintai

Tumour susceptibility gene 101 and the vacuolar protein sorting pathway are required for the release of hepatitis E virions.

We have previously demonstrated that an intact PSAP motif in the ORF3 protein is required for the formation and release of membrane-associated hepatitis E virus (HEV) particles with ORF3 proteins on their surface. In this study, we investigated the direct interaction between the ORF3 protein and tumour susceptibility gene 101 (Tsg101), a cellular factor involved in the budding of viruses containing the P(T/S)AP late-domain, in PLC/PRF/5 cells expressing the wild-type or PSAP-mutated ORF3 protein and Tsg101 by co-immunoprecipitation. Tsg101 bound to wild-type ORF3 protein, but not to the PSAP-inactive ORF3 protein. To examine whether HEV utilizes the multivesicular body (MVB) pathway to release the virus particles, we analysed the efficiency of virion release from cells upon introduction of small interfering RNA (siRNA) against Tsg101 or dominant-negative (DN) mutants of Vps4 (Vps4A and Vps4B). The relative levels of virus particles released from cells depleted of Tsg101 decreased to 6.4 % of those transfected with negative control siRNA. Similarly, virion egress was significantly reduced by the overexpression of DN forms (Vps4AEQ or Vps4BEQ). The relative levels of virus particles released from cells expressing Vps4AEQ and Vps4BEQ were 19.2 and 15.6 %, respectively, while the overexpression of wild-type Vps4A and Vps4B did not alter the levels of virus release. These results indicate that the ORF3 protein interacts with Tsg101 through the PSAP motifs in infected cells, and that Tsg101 and the enzymic activities of Vps4A and Vps4B are involved in HEV release, thus suggesting that HEV requires the MVB pathway for egress of virus particles.

Hepatitis E virus egress depends on the exosomal pathway, with secretory exosomes derived from multivesicular bodies.

Our previous studies indicated that hepatitis E virus (HEV) forms membrane-associated particles in the cytoplasm, most likely by budding into intracellular vesicles, and requires the multivesicular body (MVB) pathway to release virus particles, and the released HEV particles with a lipid membrane retain the trans-Golgi network protein 2 on their surface. To examine whether HEV utilizes the exosomal pathway to release the virus particles, we analysed whether the virion release from PLC/PRF/5 cells infected with genotype 3 HEV (strain JE03-1760F) is affected by treatment with bafilomycin A1 or GW4869, or by the introduction of a small interfering RNA (siRNA) against Rab27A or Hrs. The extracellular HEV RNA titre was increased by treatment with bafilomycin A1, but was decreased by treatment with GW4869. The relative levels of virus particles released from cells depleted of Rab27A or Hrs were decreased to 16.1 and 11.5 %, respectively, of that released from cells transfected with negative control siRNA. Electron microscopic observations revealed the presence of membrane-associated virus-like particles with a diameter of approximately 50 nm within the MVB, which possessed internal vesicles in infected cells. Immunoelectron microscopy showed positive immunogold staining for the HEV ORF2 protein on the intraluminal vesicles within the MVB. Additionally, immunofluorescence analysis indicated the triple co-localization of the ORF2, ORF3 and CD63 proteins in the cytoplasm, as specific loculated signals, supporting the presence of membrane-associated HEV particles within the MVB. These findings indicate that membrane-associated HEV particles are released together with internal vesicles through MVBs by the cellular exosomal pathway.

Molecular analysis of hepatitis E virus from farm rabbits in Inner Mongolia, China and its successful propagation in A549 and PLC/PRF/5 cells☆

Rabbit hepatitis E virus (HEV) strains have recently been isolated in several areas of China and in the US and France. However, the host range, distribution and zoonotic potential of these HEV strains remain unknown and their propagation in cultured cells has not yet been reported. A total of 211 4-month-old rabbits raised on a farm in Inner Mongolia were tested for the presence of anti-HEV antibodies and HEV RNA. Overall, 121 rabbits (57.3%) tested positive for anti-HEV antibodies, and 151 (71.6%) had detectable HEV RNA. The 174 HEV strains recovered from these viremic rabbits, including two distinct strains each from 23 rabbits, differed from each other by up to 13.6% in a 412-nucleotide (nt) sequence within ORF2, and were 89.3-95.9% identical to the reported rabbit HEV strains in other provinces of China. Three representative Inner Mongolian strains, one each from three phylogenetic clusters, whose entire genomic sequences were determined, shared 79.6-96.7% identities with reported rabbit HEV strains within the entire or 242- to 1349-nt partial genomic sequence. Rabbit HEV strains recovered from liver tissues of rabbits with a high HEV load propagated efficiently in human cell lines (A549 and PLC/PRF/5 cells), suggesting the potential zoonotic risk of rabbit HEV.

Molecular characterization of a novel hepatitis E virus (HEV) strain obtained from a wild boar in Japan that is highly divergent from the previously recognized HEV strains.

Although a consensus classification system for hepatitis E virus (HEV) genotypes is currently unavailable, HEV variants (JBOAR135-Shiz09 and wbJOY_06) from wild boars (Sus scrofa leucomystax) have provisionally been classified into two novel genotypes (5 and 6). While performing a survey of HEV infections among 566 wild boars that were captured in Japan between January 2010 and August 2013, we found 24 boars (4.2%) with ongoing HEV infections: 13 had genotype 3 HEV, 10 had genotype 4 HEV and the remaining boar possessed a novel HEV variant (designated wbJNN_13). The entire wbJNN_13 genome comprised 7247 nucleotides excluding the poly(A) tail, and was highly divergent from known genotype 1 to 4 HEV isolates derived from humans, swine, wild boars, deer, mongoose and rabbits by 22.4-28.2%, JBOAR135-Shiz09 and wbJOY_06 by 19.6-21.9% and rat, ferret, bat and avian HEV isolates by 40.9-46.1% over the entire genome. Phylogenetic trees confirmed that wbJNN_13 is distantly related to all known HEV isolates. A Simplot analysis revealed no significant recombination among the existing HEV strains. These results indicate the presence of at least three genetic lineages of presumably boar-indigenous HEV strains. Further studies to fully understand the extent of the genomic heterogeneity of HEV variants infecting wild boars are warranted.

The membrane on the surface of hepatitis E virus particles is derived from the intracellular membrane and contains trans-Golgi network protein 2

Our previous studies demonstrated that hepatitis E virus (HEV) requires the multivesicular body (MVB) pathway to release virus particles, suggesting that HEV utilizes the cellular ESCRT machinery in the cytoplasm, not at the cell surface, to be released from infected cells. In this study, we generated a murine monoclonal antibody (mAb) against the membrane-associated HEV particles to examine whether the membrane is derived from intracellular vesicles or the cell surface. An established mAb, TA1708, was found to capture the membrane-associated HEV particles, but not the membrane-dissociated particles or fecal HEV, in an immunocapture RT-PCR assay. Furthermore, digitonin treatment confirmed that the membrane on the surface of cell-culture-generated HEV particles was a lipid membrane. Double immunofluorescence staining revealed that mAb TA1708 specifically recognizes trans-Golgi network protein 2 (TGOLN2), an intracellular antigen derived from the trans-Golgi network. Supporting these findings, HEV particles with lipid membranes and ORF3 proteins on their surface were found abundantly in the lysates of HEV-infected cells. These results indicate that HEV forms membrane-associated particles in the cytoplasm, most likely by budding into intracellular vesicles, and that the released HEV particles with a lipid membrane retain the antigenicity of TGOLN2 on their surface.

Marked genomic heterogeneity of rat hepatitis E virus strains in Indonesia demonstrated on a full-length genome analysis.

Rat hepatitis E virus (HEV) strains have recently been isolated in several areas of Germany, Vietnam, the United States, Indonesia and China. However, genetic information regarding these rat HEV strains is limited. A total of 369 wild rats (Rattus rattus) captured in Central Java (Solo) and on Lombok Island, Indonesia were tested for the presence of rat HEV-specific antibodies and RNA. Overall, 137 rats (37.1%) tested positive for rat anti-HEV antibodies, while 97 (26.3%) had rat HEV RNA detectable on reverse transcription-PCR with primers targeting the ORF1-ORF2 junctional region. The 97 HEV strains recovered from these viremic rats were 76.3-100% identical to each other in an 840-nucleotide sequence and 75.4-88.4% identical to the rat HEV strains reported in Germany and Vietnam. Five representative Indonesian strains, one from each of five phylogenetic clusters, whose entire genomic sequence was determined, were segregated into three genetic groups (a German type, Vietnamese type and novel type), which differed from each other by 19.5-23.5 (22.0 ± 1.7)% over the entire genome. These results suggest the presence of at least three genetic groups of rat HEV and indicate the circulation of polyphyletic strains of rat HEV belonging to three distinct genetic groups in Indonesia.

Identification and characterization of a natural inter-genotypic (2b/1b) recombinant hepatitis C virus in Japan.

A hepatitis C virus (HCV) strain (HC10-0804) recovered from a 12-year-old Japanese female with chronic hepatitis C segregated into discordant genotypes, 2b and 1b, in the 5’UTR/core and NS5B regions, respectively, thus suggesting an inter-genotypic recombination. The HC10-0804 isolate had a genomic length of 9,423 nucleotides (nt), excluding the poly(U) tract at the 3’ terminus, and encoded a single open reading frame (ORF) for a polyprotein of 3,014 amino acids (aa). Based on Simplot and Bootscan analyses, the crossover point from 2b to 1b was estimated at nt 3443/3444 (aa 1034/1035), just after the beginning of the NS3 region. Comparison of the entire genomic sequence showed that the HC10-0804 strain was only 90.2% identical to the previously reported 2b/1b recombinant strain (SE-03-07-1689) from the Philippines, whose putative crossover point was 24 nt downstream of that of HC10-0804. These results indicate the circulation of a novel inter-genotypic (2b/1b) recombinant HCV in Japan.

Characterization and epitope mapping of monoclonal antibodies raised against rat hepatitis E virus capsid protein: An evaluation of their neutralizing activity in a cell culture system

Hepatitis E virus (HEV) is the causative agent of acute hepatitis. Rat HEV is a recently discovered virus related to, but distinct from, human HEV. Since laboratory rats can be reproducibly infected with rat HEV and a cell culture system has been established for rat HEV, this virus may be used as a surrogate virus for human HEV, enabling studies on virus replication and mechanism of infection. However, monoclonal antibodies (MAbs) against rat HEV capsid (ORF2) protein are not available. In this study, 12 murine MAbs were generated against a recombinant ORF2 protein of rat HEV (rRatHEV-ORF2: amino acids 101-644) and were classified into at least six distinct groups by epitope mapping and a cross-reactivity analysis with human HEV ORF2 proteins. Two non-cross-reactive MAbs recognizing the protruding (P) domain detected both non-denatured and denatured rRatHEV-ORF2 protein and efficiently captured cell culture-produced rat HEV particles that had been treated with deoxycholate and trypsin, but not those without prior treatment. In addition, these two MAbs were able to efficiently neutralize replication of cell culture-generated rat HEV particles without lipid membranes (but not those with lipid membranes) in a cell culture system, similar to human HEV.

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 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.