Tsutomu Nishizawa

Analysis of the full-length genome of a hepatitis E virus isolate obtained from a wild boar in Japan that is classifiable into a novel genotype.

While performing a nationwide survey of hepatitis E virus (HEV) infection among 450 wild boars (Sus scrofa leucomystax) that had been captured in Japan between November 2005 and March 2010, we found 16 boars (3.6%) with ongoing HEV infection: 11 had genotype 3 HEV, four had genotype 4 HEV and the remaining boar was infected with HEV of an unrecognized genotype (designated wbJOY_06). The entire wbJOY_06 genome was sequenced and was found to comprise 7246 nt excluding the poly(A) tail. The wbJOY_06 isolate was highly divergent from known genotype 1-4 HEV isolates derived from humans, swine, wild boars, deer, mongoose and rabbits (n=145) by 22.6-27.7%, rat HEV isolates (n=2) by 46.0-46.2%, and avian HEV isolates (n=5) by 52.5-53.1% over the entire genome. A Simplot analysis revealed no significant recombination between the existing HEV strains of genotypes 1-4. Therefore, we propose that the wbJOY_06 isolate is the first member of a previously unidentified genotype.

GATA6 mutations cause human cardiac outflow tract defects by disrupting semaphorin-plexin signaling

Congenital heart diseases (CHD) occur in nearly 1% of all live births and are the major cause of infant mortality and morbidity. Although an improved understanding of the genetic causes of CHD would provide insight into the underlying pathobiology, the genetic etiology of most CHD remains unknown. Here we show that mutations in the gene encoding the transcription factor GATA6 cause CHD characteristic of a severe form of cardiac outflow tract (OFT) defect, namely persistent truncus arteriosus (PTA). Two different GATA6 mutations were identified by systematic genetic analysis using DNA from patients with PTA. Genes encoding the neurovascular guiding molecule semaphorin 3C (SEMA3C) and its receptor plexin A2 (PLXNA2) appear to be regulated directly by GATA6, and both GATA6 mutant proteins failed to transactivate these genes. Transgenic analysis further suggests that, in the developing heart, the expression of SEMA3C in the OFT/subpulmonary myocardium and PLXNA2 in the cardiac neural crest contributing to the OFT is dependent on GATA transcription factors. Together, our data implicate mutations in GATA6 as genetic causes of CHD involving OFT development, as a result of the disruption of the direct regulation of semaphorin-plexin signaling.

A nationwide survey of hepatitis E virus (HEV) infection in wild boars in Japan: identification of boar HEV strains of genotypes 3 and 4 and unrecognized genotypes

To investigate the nationwide prevalence of hepatitis E virus (HEV) infection and to characterize HEV genomes among Japanese wild boars (Sus scrofa leucomystax), 578 boars captured in 25 prefectures from 2003 to 2010 were studied. Anti-HEV IgG was detected in 8.1%, and HEV RNA in 3.3% of boars. Among the 19 boar HEV isolates obtained from infected boars, 14 isolates (74%) were classified as genotype 3, 4 isolates (21%) as genotype 4, and the remaining isolate (wbJOY_06) was distantly related to all known HEV isolates of genotypes 1-4, differing by 18.4-25.0% and 18.0-24.3% within the 412-nucleotide sequence of ORF1 and ORF2, respectively. A genotype 4 boar HEV isolate (wbJGF_08-1) obtained herein shared 98.6% identity over the entire genome with a human HEV isolate obtained from a patient who developed acute hepatitis after consuming undercooked wild boar meat, suggesting that wild boars are also reservoirs for genotype 4 HEV in humans.

A PSAP motif in the ORF3 protein of hepatitis E virus is necessary for virion release from infected cells

We have previously demonstrated that the release of hepatitis E virus (HEV) from infected cells depended on ORF3 protein, which harbours one or two PSAP motifs. To elucidate the PSAP motif(s) in the ORF3 protein during virion egress, five PSAP mutants derived from an infectious genotype 3 cDNA clone of pJE03-1760F/wt that can grow efficiently in PLC/PRF/5 cells were analysed. Four mutants, including mutLSAP, mutPSAL, mutLSAL (the substituted amino acids in the authentic PSAP motif are underlined) and mutPLAP/PSAP (the changed amino acid in the additional PSAP motif is underlined) generated progenies as efficiently as the wild-type virus. Conversely, the HEV RNA level in the culture supernatant of mutPLAP/LSAL RNA-transfected cells was significantly lower than in cells transfected with the wild-type RNA, similar to an ORF3-null mutant. Consistent with the ORF3-deficient mutant, the mutPLAP/LSAL mutant with no intact PSAP motifs banded at 1.26-1.27 g ml(-1) in sucrose, and was captured by anti-ORF2, but not by anti-ORF3, with or without prior treatment with detergent (0.1u200a% sodium deoxycholate). The absence of the ORF3 protein on the mutant particles in the culture supernatant was confirmed by Western blotting, despite the expression of ORF3 protein in the RNA-transfected cells, as detected by immunofluorescence and Western blotting. Therefore, at least one of the two intact PSAP motifs in the ORF3 protein is required for the formation of membrane-associated HEV particles possessing ORF3 proteins on their surface, thus suggesting that the PSAP motif plays a role as a functional domain for HEV budding.

Presence of Antibodies to Hepatitis E Virus in Japanese Pet Cats

Recently, it has become a growing consensus that hepatitis E is a zoonosis and swine act as a reservoir for hepatitis E virus (HEV) infection in humans, as suggested by the close genetic relationship of swine and human virus and crossspecies infection of HEV [1–4]. In an earlier study, we found a high prevalence of swine anti-HEV IgG among 2 to 6 month-old Japanese pigs (58% or 1,448/2,500) [5]. However, in Japan, patients with sporadic acute or fulminant hepatitis who had not traveled abroad and were infected with presumably Japan-indigenous HEV strains had no contact with pigs [6, 7]. Antibodies to HEV (anti-HEV) have been detected in a number of animal species other than swine, such as rodents, cows, sheep, goats and dogs [8]. Although some of our patients with hepatitis E had regular contact with pet cats, it remains unknown whether cats are seropositive for HEV. We analyzed serum samples from 135 Japanese pet cats of 0.5 to 18 (6.5 ± 5.3 [mean ± standard deviation]) years of age. The serum samples had been stored below –20 °C and were tested for the IgG class of anti-HEV by in-house enzyme immunoassay, using purified recombinant open reading frame 2 (ORF2) protein which had been expressed in the pupae of silkworm [6] as the antigen probe, and for HEV RNA by reverse transcription PCR by the method described previously with primers targeting the ORF2 region [6]. The A450 values of anti-HEV antibodies ranged from 0.026 to 2.917, and 44 (33%)of the 135 samples had an A450 value of ≥ 0.600.The A450 value of the 44 samples decreased to less than 30% of the original value after absorption with the same recombinant ORF2 protein used as the antigen probe, but it remained greater than 70% of the original value after absorption with a mock protein obtained from the pupae of silkworm infected with nonrecombinant baculovirus, confirming the specificity of the assay. Therefore, these 44 samples were conservatively regarded as being positive for anti-HEV in this study. The prevalence and mean A450 value of feline anti-HEV generally increased with the cat’s age (Table 1).A high A450 value of ≥ 2.500 was seen in 22 cats of ≥ 5 years of age. However, HEV RNA was not detectable in the sera from any of the 135 cats; a larger study with more cats is needed to draw a definitive conclusion. To be exact, demonstration of antibodies in cats that react with the ORF2 protein of HEV is not proof of existence of HEV in cats. Until recently, HEV was considered to be closely related to or even a member of the Caliciviridae family. Feline calicivirus (FCV) is wide spread in the domestic cat population and most cats are vaccinated against this agent, suggesting that the antibodies detected in the present study might have been induced by FCV infection or vaccination.To disprove immunological cross-reInfection Correspondence

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 6.4u200a% 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 15.6u200a%, 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.

Production of monoclonal antibodies against hepatitis E virus capsid protein and evaluation of their neutralizing activity in a cell culture system

Nine murine monoclonal antibodies (mAbs) generated against a recombinant ORF2 protein (amino acids 111–660) of a genotype 4 hepatitis E virus (HEV) strain recognized four sets of epitopes by pairwise competitive ELISA. One mAb (H6225) was able to capture HEV efficiently regardless of genotype and was tested for its ability to neutralize a genotype 3 HEV strain (JE03-1760F) in a recently developed cell culture system for HEV in a hepatocarcinoma cell line (PLC/PRF/5). When PLC/PRF/5 cells were inoculated with HEV (4.0xa0×xa0105 or 4.0xa0×xa0106 copies/ml) incubated with 100xa0μg/ml of a negative control mAb, HEV RNA in the culture medium continued to be detectable after day 14 or 12 post-inoculation (dpi), respectively. However, when cells were inoculated with the two distinct concentrations of HEV that had been mixed with 100xa0μg/ml of H6225, the harvested culture supernatants were negative for HEV RNA throughout the 60-day observation period. Upon prior mixing of the virus with 10xa0μg/ml of H6225, HEV RNA in culture supernatant continued to be undetectable until 46 or 28xa0dpi, respectively. In conclusion, one mAb (H6225) against HEV capsid protein that can efficiently neutralize HEV in vitro was obtained in the present study.

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 11.5u200a%, 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.

Clinical manifestations of the deletion of Down syndrome critical region including DYRK1A and KCNJ6

A relatively small region of human chromosome 21 (Hsa21) is considered to play a major role in Down syndrome (DS) phenotypes, and the concept of a Down syndrome critical region (DSCR) has been proposed. The goal of the phenotype–genotype correlation study is to discover which genes are responsible for each DS phenotype. Loss of the genomic copy numbers of Hsa21 can give us important suggestion to understand the functions of the involved genes. Genomic copy number aberrations were analyzed by micro‐array‐based comparative genomic hybridization (aCGH) in 300 patients with developmental delay. Partial deletions of Hsa21 were identified in three patients with developmental delay, epilepsy, microcephaly, and distinctive manifestations. Two of the patients had mosaic deletions of 21q22‐qter including a part of DSCR; one of whom whose mosaic ratio was higher than the other showed more severe brain morphogenic abnormality with colpocephaly, which was similar to the previously reported patients having pure deletions of 21q22‐qter, indicating the critical region for cortical dysplasia at this region. The remaining patient had the smallest microdeletion with 480u2009kb in DSCR including DYRK1A and KCNJ6. Although we could not identify any nucleotide alteration in DYRK1A and KCNJ6 in our cohort study for 150 patients with mental retardation with/without epilepsy, this study underscores the clinical importance of DSCR not only for DS but also for developmental disorders.

RAF1 mutations in childhood-onset dilated cardiomyopathy

Dilated cardiomyopathy (DCM) is a highly heterogeneous trait with sarcomeric gene mutations predominating. The cause of a substantial percentage of DCMs remains unknown, and no gene-specific therapy is available. On the basis of resequencing of 513 DCM cases and 1,150 matched controls from various cohorts of distinct ancestry, we discovered rare, functional RAF1 mutations in 3 of the cohorts (South Indian, North Indian and Japanese). The prevalence of RAF1 mutations was ∼9% in childhood-onset DCM cases in these three cohorts. Biochemical studies showed that DCM-associated RAF1 mutants had altered kinase activity, resulting in largely unaltered ERK activation but in AKT that was hyperactivated in a BRAF-dependent manner. Constitutive expression of these mutants in zebrafish embryos resulted in a heart failure phenotype with AKT hyperactivation that was rescued by treatment with rapamycin. These findings provide new mechanistic insights and potential therapeutic targets for RAF1-associated DCM and further expand the clinical spectrum of RAF1-related human disorders.