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.

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.

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

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

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.

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.

Full-length genomic sequence analysis of new subtype 3k hepatitis E virus isolates with 99.97% nucleotide identity obtained from two consecutive acute hepatitis patients in a city in northeast Japan.

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 7 225 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.

Prognostic predictive value of gene mutations in Japanese patients with hypertrophic cardiomyopathy.

Although some studies have attempted to find useful prognostic factors in hypertrophic cardiomyopathy (HCM), those results are not fully helpful for use in actual clinical practice. Furthermore, several genetic abnormalities associated with HCM have been identified. However, the genotype-phenotype correlation in HCM remains to be elucidated. Here, we attempted to assess patients with different types of gene mutations causing HCM and investigate the prognosis. A total of 140 patients with HCM underwent a screening test for myofilament gene mutations by direct sequencing of eight sarcomeric genes. Patients with a single mutation in cardiac troponin T, cardiac troponin I, α-tropomyosin, and regulatory and essential light chains were excluded from the study because the number of cases was too small. The clinical presentations and outcomes of the remaining 127 patients with HCM, 31 ß-myosin heavy chain (MYH7) mutation carriers, 19 cardiac myosin-binding protein C (MYBPC3) mutation carriers, and 77 mutation non-carriers were analyzed retrospectively. MYBPC3 mutation carriers had a high frequency of ventricular arrhythmia and syncope. Kaplan-Meier curves revealed no significant difference in prognosis among the three groups, but a lack of family history of sudden death (SD) and a past history of syncope were significantly related to poor prognosis. An absence of family history of SD and past history of syncope are useful prognostic factors in patients with HCM. MYH7 and MYBPC3 mutations did not significantly influence prognosis compared to non-carriers. However, patients with the MYBPC3 mutation should be closely followed for the possibility of SD.

Enhanced pregenomic RNA levels and lowered precore mRNA transcription efficiency in a genotype A hepatitis B virus genome with C1766T and T1768A mutations obtained from a fulminant hepatitis patient.

The viral factors associated with the development of fulminant hepatitis B are not fully understood. We recently found four unique mutations [G to A at nucleotide 1742 (G1742A), C1766T, T1768A and T1809C] in the basal core promoter (BCP) region of a genotype A hepatitis B virus (HBV) strain (FH) obtained from a 53-year-old man with fatal fulminant hepatitis. To elucidate the association of the mutations of the FH genome with the disease, we constructed a 1.3-fold FH genome and its five variants by replacing one or two mutated nucleotides with wild-type nucleotide(s) via site-directed mutagenesis, and transfected human hepatoma cells (HepG2/C3A) with the constructs. There were no discernible differences between FH and two variants (FH_A1742G and FH_C1809T) with regard to viral replication and protein expression. However, in comparison to three other variants (FH_T1766C, FH_A1768T and FH_T1766C/A1768T) with wild-type nucleotide(s) at 1766 and/or 1768, the FH genome exhibited a 2.5-5-fold enhancement of viral replication by heightened pregenomic RNA synthesis and a 1.5-2.5-fold reduction in the hepatitis B e antigen (HBeAg) synthesis by the downregulation of the precore mRNA level. An immunofluorescence analysis revealed the increased and predominant cytoplasmic localization of the core protein in the FH genome. The present study demonstrates that the C1766T/T1768A mutations in the BCP region of genotype A HBV enhance viral replication, downregulate HBeAg expression and are responsible for the predominant localization of the core protein in the cytoplasm, which are likely associated with the development of fulminant hepatitis.

Molecular analysis of hepatitis A virus strains obtained from patients with acute hepatitis A in Mongolia, 2004-2013.

Despite the high endemicity of hepatitis A virus (HAV) in Mongolia, the genetic information on those HAV strains is limited. Serum samples obtained from 935 patients with acute hepatitis in Ulaanbaatar, Mongolia during 2004-2013 were tested for the presence of HAV RNA using reverse transcription-PCR with primers targeting the VP1-2B region (481 nucleotides, primer sequences at both ends excluded). Overall, 180 patients (19.3%) had detectable HAV RNA. These 180 isolates shared 94.6-100% identity and formed four phylogenetic clusters within subgenotype IA. One or three representative HAV isolates from each cluster exhibited 2.6-3.9% difference between clusters over the entire genome. Cluster 1 accounted for 65.0% of the total, followed by Cluster 2 (30.6%), Cluster 3 (3.3%), and Cluster 4 (1.1%). Clusters 1 and 2 were predominant throughout the observation period, whereas Cluster 3 was undetectable in 2009 and 2013 and Cluster 4 became undetectable after 2009. The Mongolian HAV isolates were closest to those of Chinese or Japanese origin (97.7-98.5% identities over the entire genome), suggesting the evolution from a common ancestor with those circulating in China and Japan. Further molecular epidemiological analyses of HAV infection are necessary to investigate the factors underlying the spread of HAV and to implement appropriate prevention measures in Mongolia.

Production of monoclonal antibodies against the ORF3 protein of rat hepatitis E virus (HEV) and demonstration of the incorporation of the ORF3 protein into enveloped rat HEV particles.

Eight murine monoclonal antibodies (MAbs) against a synthetic peptide corresponding to the C-terminal 15-amino-acid portion of the ORF3 protein of rat hepatitis E virus (ratHEV) were produced and characterized. Immunofluorescence assays using the anti-ratHEV ORF3 MAbs revealed the accumulation of ORF3 protein in the cytoplasm of PLC/PRF/5 cells transfected with ORF3-expressing plasmids or inoculated with cell-culture-generated ratHEV strains. Anti-ORF3 MAbs could capture ratHEV particles in culture supernatant and serum following treatment with 0.5 % deoxycholate, but not those without prior detergent treatment or fecal ratHEV particles. Following treatment with 0.5 % deoxycholate and 0.5 % trypsin, the buoyant density of ratHEV particles in culture supernatant with ORF3 protein on the surface shifted from 1.15 g/cm3 to 1.26 g/cm3 in a sucrose gradient; the resulting particles were capturable by an anti-ORF2 MAb but not by an anti-ORF3 MAb. This indicates that the ORF3 protein (at least its C-terminal portion) is incorporated into the enveloped ratHEV virions released from infected cells but that it is not found in the virions in the feces, supporting the hypothesis that the ratHEV ORF3 protein is associated with the egress of virions from infected cells, similar to human HEV, despite the fact that the ratHEV ORF3 protein lacks a PSAP amino acid motif.

Compound Mutations Cause Increased Cardiac Events in Children with Long QT Syndrome: Can the Sequence Homology-Based Tools be Applied for Prediction of Phenotypic Severity?

Long QT syndrome (LQTS) can cause syncope, ventricular fibrillation, and death. Recently, several disease-causing mutations in ion channel genes have been identified, and compound mutations have also been detected. It is unclear whether children who are carriers of compound mutations exhibit a more severe phenotype than those with single mutations. Although predicting phenotypic severity is clinically important, the availability of prediction tools for LQTS is unknown. To determine whether the severity of the LQTS phenotype can be predicted by the presence of compound mutations in children is needed. We detected 97 single mutations (Group S) and 13 compound mutations (Group C) between 1998 and 2012, age at diagnosis ranging 0-19 years old (median age is 9.0) and 18.0 years of follow-up period. The phenotypes and Kaplan-Meier event-free rates of the two groups were compared for cardiac events. This study investigated phenotypic severity in relation to the location of mutations in the protein sequence, which was analyzed using two sequence homology-based tools. In results, compound mutations in children were associated with a high incidence of syncope within the first decade (Group S: 32 % vs. Group C: 61 %), requiring an ICD in the second decade (Group S: 3 % vs. Group C: 56 %). Mortality in these patients was high within 5 years of birth (23 %). Phenotypic prediction tools correctly predicted the phenotypic severity in both Groups S and C, especially by using their coupling method. The coupling prediction method is useful in the initial evaluation of phenotypes both with single and compound mutations of LQTS patients. However, it should be noted that the compound mutation makes more severe phenotype.

Germline gain-of-function mutations in RAF1 cause Noonan syndrome.

Noonan syndrome is characterized by short stature, facial dysmorphia and a wide spectrum of congenital heart defects. Mutations of PTPN11, KRAS and SOS1 in the RAS-MAPK pathway cause approximately 60% of cases of Noonan syndrome. However, the gene(s) responsible for the remainder are unknown. We have identified five different mutations in RAF1 in ten individuals with Noonan syndrome; those with any of four mutations causing changes in the CR2 domain of RAF1 had hypertrophic cardiomyopathy (HCM), whereas affected individuals with mutations leading to changes in the CR3 domain did not. Cells transfected with constructs containing Noonan syndrome-associated RAF1 mutations showed increased in vitro kinase and ERK activation, and zebrafish embryos with morpholino knockdown of raf1 demonstrated the need for raf1 for the development of normal myocardial structure and function. Thus, our findings implicate RAF1 gain-of-function mutations as a causative agent of a human developmental disorder, representing a new genetic mechanism for the activation of the MAPK pathway.

Spontaneous reactivation of hepatitis B virus (HBV) infection in patients with resolved or occult HBV infection.

Reactivation of a former hepatitis B virus (HBV) infection can be triggered by immunosuppressive therapy, diseases associated with an immunocompromised state, organ transplantation or the withdrawal of antiviral drugs. Despite the absence of such risk factors, a spontaneous reactivation of HBV replication occurred in two elderly patients with resolved or occult HBV infection. A 73-year-old male underwent coronary artery bypass grafting in October 2008, and was negative for HBsAg but positive for anti-HBs. In July 2009, his serum became positive for HBsAg, HBeAg and HBV DNA (6.4 log copies/ml; genotype C), but negative for anti-HBc IgM, with abrupt elevation of the liver enzymes. The entire genomic sequence of HBV recovered from this patient revealed no mutations in the core promoter and precore regions that interfere with HBeAg production. A 76-year-old male with a history of endoscopic mucosal resection for esophageal cancer in 2002 and an initial diagnosis of diabetes mellitus in 2009, at which time he was negative for HBsAg. He was found to be positive for HBsAg in September 2012 during a laboratory examination performed prior to the resection of recurrent esophageal cancer, despite a low HBV load (2.1 log copies/ml). Three months later, without the administration of any anticancer drugs, the HBV DNA (genotype B) level increased to 5.1 log copies/ml. A precore G1896A variant with high quasispecies diversity was recovered from the patient. Aging, surgical stress and complication of disease(s) associated with compromised immunity, such as cancer, arteriosclerosis and diabetes mellitus may trigger spontaneous HBV reactivation.

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.

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.

The complete genomes of subgenotype IA hepatitis A virus strains from four different islands in Indonesia form a phylogenetic cluster.

Despite the high endemicity of hepatitis A virus (HAV) in Indonesia, genetic information on those HAV strains is limited. Serum samples obtained from 76 individuals during outbreaks of hepatitis A in Jember (East Java) in 2006 and Tangerang (West Java) in 2007 and those from 82 patients with acute hepatitis in Solo (Central Java), Denpasar on Bali Island, Mataram on Lombok Island, and Makassar on Sulawesi Island in 2003 or 2007 were tested for the presence of HAV RNA by reverse transcription PCR with primers targeting the VP1-2B region (481 nucleotides, primer sequences at both ends excluded). Overall, 34 serum samples had detectable HAV RNA, including at least one viremic sample from each of the six regions. These 34 strains were 96.3-100 % identical to each other and formed a phylogenetic cluster within genotype IA. Six representative HAV isolates from each region shared 98.3-98.9 % identity over the entire genome and constituted a IA sublineage with a bootstrap value of 100 %, consisting of only Indonesian strains. HAV strains recovered from Japanese patients who were presumed to have contracted HAV infection while visiting Indonesia were closest to the Indonesian IA HAV strains obtained in the present study, with a high identity of 99.5-99.7 %, supporting the Indonesian origin of the imported strains. These results indicate that genetic analysis of HAV strains indigenous to HAV-endemic countries, including Indonesia, are useful for tracing infectious sources in imported cases of acute hepatitis A and for defining the epidemiological features of HAV infection in that country.

Characterization of sporadic acute hepatitis E and comparison of hepatitis E virus genomes in acute hepatitis patients and pig liver sold as food in Mie, Japan.

AIM: To characterize hepatitis E in Mie prefecture and to investigate whether raw pig liver sold as food in Mie is contaminated with hepatitis E virus (HEV) strains similar to those recovered from patients. METHODS: Seventeen patients with sporadic acute hepatitis E treated from 2004 to 2012 were studied. A total of 243 packages of raw pig liver from regional grocery stores were tested for the presence of HEV RNA. The partial genomic sequences of human and swine HEV isolates were determined and subjected to the phylogenetic analyses. RESULTS: The HEV isolates recovered from the 17 patients segregated into genotype 3 (n = 15) and genotype 4 (n = 2), and 15 genotype 3 isolates further segregated into 3e (n = 11) and 3b (n = 4). Pig liver specimens from 12 (4.9%) of the 243 packages had detectable HEV RNA. All 12 swine HEV isolates were grouped into genotype 3 (3a or 3b). Although no 3e strains were isolated from pig liver specimens, two 3b swine strains were 99.5-100% identical to two HEV strains recovered from hepatitis patients, within 412-nt partial sequences. CONCLUSION: The 3e HEV was prevalent among hepatitis E patients. HEV RNA was detected in approximately 5% of pig liver sold as food. The presence of identical HEV strains between hepatitis patients and pig liver indicated that pigs play an important role as reservoirs for HEV in humans in Mie. Further studies are needed to clarify the source of 3e HEV in the animal and environmental reservoirs.

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.

Frequent detection and characterization of hepatitis E virus variants in wild rats (Rattus rattus) in Indonesia.

One hundred sixteen rats (Rattus rattus) captured in Indonesia from 2011 to 2012 were investigated for the prevalence of hepatitis E virus (HEV)-specific antibodies and HEV RNA. Using an ELISA based on HEV genotype 4 with an ad hoc cutoff value of 0.500, 18.1 % of the rats tested positive for anti-HEV IgG. By nested RT-PCR, 14.7 % of the rats had rat HEV RNA, and none were positive for HEV genotype 1-4. A high HEV prevalence among rats was associated with lower sanitary conditions in areas with a high population density. Sixteen of the 17 HEV isolates obtained from infected rats showed >93.0 % nucleotide sequence identity within the 840-nucleotide ORF1-ORF2 sequence and were most closely related to a Vietnamese strain (85.9-87.9 % identity), while the remaining isolate differed from known rat HEV strains by 18.8-23.3 % and may belong to a novel lineage of rat HEV. These results suggest a wide distribution of rat HEV with divergent genomes.

Mutations in the cardiac troponin T gene show various prognoses in Japanese patients with hypertrophic cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) is an autosomal dominant disorder resulting from mutations in genes for at least 15 various sarcomere-related proteins including cardiac ß-myosin heavy chain, cardiac myosin-binding protein C, and cardiac troponin T. The troponin T gene (TNNT2) mutation has the third incidence of familial HCM, and the genotype-phenotype correlation of this gene still remains insufficient in Japanese familial HCM. Therefore, in the present study, we focused on screening the TNNT2 mutation in 173 unrelated Japanese patients with familial HCM, and found three reported mutations and a new mutation of TNNT2 in 11 individuals from four families. In these families, two individuals from one family had double mutations, Arg130Cys and Phe110Ile, six individuals from two other families had an Arg92Trp mutation, and one individual of another family had a new mutation, Ile79Thr, of TNNT2. The phenotype of each family was often different from reported cases, even if they had the same genetic mutation. In addition, families with the same genetic mutation showed a similar trend in the phenotype, but it was not exactly the same. However, sudden death in youth was observed in all of these families. Although the type of genetic mutation is not useful for predicting prognosis in HCM, the possibility of sudden cardiac death remains. Therefore, the prognosis of individuals bearing the TNNT2 mutation with familial HCM should be more carefully observed from birth.