Surveillance of poliovirus-isolates in Japan, 2000.
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Adult↗
Biomedical subjects
T Miyamura
Publications and source records attributed to T Miyamura.
[The present studies of the development of HCV vaccine].
After isolation of a hepatitis C virus genome in 1989, all donated blood samples have been analyzed by a sensitive screening system, which brought us 'safety transfusion' (free from HCV). However it is likely that about 170 million people around the world, more than 2 million people in Japan have already infected with HCV. Despite the numerous efforts, the lack of efficient cell culture system makes it difficult to develop HCV vaccine. Some novel strategies are engineered day by day that might be useful tools. Hereafter we must clarify the mechanisms of replication and infection in depth, to develop a vaccine that clear HCV from carrier.
Animals↗
Neurovirulence of Sabin 1-derived polioviruses isolated from an immunodeficient patient with prolonged viral excretion.
We have analysed the neurovirulence of Sabin 1-derived isolates which persisted more than nine years in an immunodeficient patient in the U.S.A. Samples were collected from stool specimens at days 11 (St1), 23 (St2), 48 (St3), 126 (St5), and 200 (St7) after the onset of paralysis. Critical nucleotides associated with the reversion of virulence were examined. All the isolates had the substitutions at nucleotide positions 480 (G to A) in the 5'-non-coding region (NCR), 2438 (A to U) in VP3, 2795 (A to G) in VP1, and 6203 (C to U) in 3D. Serially diluted samples were injected intracerebrally to transgenic mice harbouring the human poliovirus receptor gene. Samples St2, 3, 5 and 7 showed typical virulent characters in transgenic mice, whereas the sample ST1 showed intermediate neurovirulence. It seemed that there were two variant viruses providing for a major (M) and a minor (m) populations. After disappearance of the m-variant, samples obtained at the later stages showed neurovirulence almost equivalent to that of the Mahoney strain. Thus, the Sabin 1 strain evolved towards full neuropathogenicity after long-term replication in humans by accumulating mutations. Therefore, OPV-vaccination of immunodeficient persons should be avoided.
Animals↗
Necessity of two-stool sample test for sensitive detection of poliovirus.
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Feces↗
Complete nucleotide sequence of the chiba virus genome and functional expression of the 3C-like protease in Escherichia coli.
We cloned the genome RNA of the Chiba virus (ChV; Hu/NLV/Chiba 407/1987/JP) and determined its complete nucleotide sequence. The genome is predicted to be a positive-sense, single-stranded RNA of 7697 bases, excluding a poly(A) tract. Comparison of the nucleotide and amino acid sequences with those of other members of the species Norwalk virus (NV) revealed that ChV belongs to genogroup I NV. The ChV genome contains three open reading frames (ORFs). A large 5'-terminal ORF (ORF1) encodes a polyprotein with 1785 amino acids that are likely processed into functional proteins, including RNA helicase, VPg, protease, and RNA-dependent RNA polymerase. ORF2 encodes the capsid protein with 544 amino acids, and a small 3'-terminal ORF (ORF3) encodes a basic protein with 208 amino acids. The amino acid sequences of five cleavage sites in ORF1 are highly conserved compared with those of other members of NV. When expressed in Escherichia coli, the glutathione-S-transferase (GST) fusion protein of the ChV protease connected via a short peptide containing a human rhinovirus 3C protease cleavage site was cleaved into GST and the protease; however, this cleavage did not occur when the Cys mutation was introduced into the putative active site of the protease. Moreover, the ChV protease recognized and cleaved the predicted proteolytic sites between VPg and protease and between protease and RNA polymerase. Therefore, the ChV protease expressed in E. coli retained an enzymatic activity and a substrate specificity similar to that of the human rhinovirus 3C protease.
3C Viral Proteases↗
Characterisation of vaccine-derived polioviruses isolated from sewage and river water in Japan.
BACKGROUND: A nucleotide change from U to C at position 472 in the 5' non-coding region of the type 3 poliovirus is associated with increased neurovirulence. Moreover, the proportion of type 3 polioviruses containing this mutation (472-C revertants) correlates with the neurovirulence of a particular sample. We used mutant analysis by PCR and restriction-enzyme cleavage (MAPREC) to estimate the neurovirulence of environmental samples obtained from Toyama prefecture, Japan. METHODS: Sewage and river water were collected between October, 1993, and September, 1995, and concentrated samples were inoculated into three different cell types. Isolated type 3 viruses were analysed to determine whether they were derived from the live oral poliovirus vaccine strain; they were then tested for neurovirulence by MAPREC. RESULTS: 29 type 3 strains were isolated--all of which were vaccine-derived. 16 (55%) comprised between 2% and 91% 472-C revertants by MAPREC and were expected to have high neurovirulence. The remaining strains included less than 0.25% revertants, and were regarded as attenuated viruses. Both types were isolated about 3 months after routine oral poliovirus vaccine administrations in May and October. Three strains isolated from river water were of the virulent type. INTERPRETATION: Our results emphasise that there is an environmental risk of vaccine-associated paralytic poliomyelitis as long as live oral poliovirus vaccine is not replaced by inactivated polio vaccine.
DNA, Complementary↗
Role of the DExH motif of the Japanese encephalitis virus and hepatitis C virus NS3 proteins in the ATPase and RNA helicase activities.
The role of the conserved DExH motif of the Japanese encephalitis virus (JEV) NS3 protein in the ATPase and RNA helicase activities was compared with that of the hepatitis C virus (HCV) NS3 protein. In the DExH motif of JEV NS3, Asp-285 and Glu-286 were essential for both ATPase and RNA helicase activities. Cys-287 was critical for the RNA helicase activity of JEV NS3 but not for ATPase activity. A His-288-to-Ala substitution in the DExH motif of HCV NS3 resulted in an increase in ATPase activity which was suppressed by poly(U). In contrast, alanine substitution at the same site in JEV NS3 did not increase basal ATPase activity which remained to be stimulated by poly(U). Thus, the mutational effect at His in motif II was different in the HCV and JEV NS3 proteins. Mutagenesis at His-288 of JEV NS3 revealed that His was the most preferable amino acid for ATPase activity and Ala, Gly, Asn, Gln, Ser, or Arg could partly substitute for it. However, any other mutation at His-288 completely disrupted the RNA helicase activity of JEV NS3. The results suggest that Cys-287 and His-288 are essential residues especially for the RNA helicase activity of JEV NS3 and the ATPase and helicase activities are separable enzymatic functions.
Adenosine Triphosphatases↗
Induction of hepatitis C virus-specific cytotoxic T lymphocytes in mice by an intrahepatic inoculation with an expression plasmid.
We assessed the possibility of intrahepatic inoculation with a plasmid encoding hepatitis C virus (HCV) proteins to elicit HCV-specific cytotoxic T lymphocytes (CTL) in mice as a conventional animal model of HCV infection. BALB/c mice were intrahepatically or intramuscularly inoculated with an expression plasmid DNA encoding HCV structural proteins under the control of the elongation factor 1-alpha promoter. Expressions of HCV-core protein and envelope proteins (E1 and E2) in hepatocytes were detected immunohistochemically 6 days after inoculation. CTL responses were examined using target cells either pulsed with a specific peptide or infected with a recombinant vaccinia virus expressing HCV structural protein. Both intrahepatically and intramuscularly DNA-inoculated mice developed CD8(+), MHC class I-restricted CTL responses that recognized the peptide pulsed as well as HCV proteins expressing target cells. These studies demonstrated the usefulness of a murine model of HCV infection induced by direct intrahepatic DNA inoculation for understanding the immunopathogenic mechanisms in HCV infection.
Animals↗
Selective binding of hepatitis C virus core protein to synthetic oligonucleotides corresponding to the 5' untranslated region of the viral genome.
Although it is assumed that hepatitis C virus (HCV) core protein binds with viral RNA to form a nucleocapsid, little is known about the resulting molecular interactions. We utilized surface plasmon resonance technology to study the structural basis of the affinity and the preference of the interaction between HCV core protein and oligonucleotides derived from the viral genome. Among the 10 oligonucleotides corresponding to the 5' untranslated region (5'UTR) of the tested HCV genome, the real-time analysis of sensorgrams indicated that the core protein binds most efficiently and stably to the 31-nucleotide-long sequence of the loop IIId domain, whose secondary structure is highly conserved not only among different HCV genotypes but also among pestiviruses. There also could be some interactions of the core protein with the loop I domain and the region of nt 23-41. The kinetic profiles, together with those obtained in experiments using single- and double-stranded polymeric oligonucleotides, suggest a multimerization of the core protein in solution. These newly characterized properties could provide a basis for understanding the pathway of the viral nucleocapsid assembly.
5' Untranslated Regions↗
Identification and characterization of the RNA helicase activity of Japanese encephalitis virus NS3 protein.
The NS3 protein of Japanese encephalitis virus (JEV) contains motifs typical of RNA helicase/NTPase but no RNA helicase activity has been reported for this protein. To identify and characterize the RNA helicase activity of JEV NS3, a truncated form of the protein with a His-tag was expressed in Escherichia coli and purified. The purified JEV NS3 protein showed an RNA helicase activity, which was dependent on divalent cations and ATP. An Asp-285-to-Ala substitution in motif II of the JEV NS3 protein abolished the ATPase and RNA helicase activities. These results indicate that the C-terminal 457 residues are sufficient to exhibit the RNA helicase activity of JEV NS3.
Adenosine Triphosphatases↗
Recombinant subunit ORF2.1 antigen and induction of antibody against immunodominant epitopes in the hepatitis E virus capsid protein.
A recombinant subunit antigen (ORF2.1), representing the carboxy-terminal 267 amino acids of the 660-amino-acid hepatitis E virus (HEV) capsid protein, was expressed in Escherichia coli and used for the immunisation of rats. Purified antigen formulated with either Aluminium Hydroxide Gel Adjuvant (Alum) or Titermax gave high and equivalent levels of antibody after three doses. Responses to two doses of 15, 75, or 150 microg antigen, formulated with Alum and given at 0 and 4 weeks, were also equivalent by 17 weeks after immunisation. Rats initially developed antibody to a wide range of linear epitopes in the ORF2.1 region, but by 27 weeks the predominant response detected by Western immunoblotting was restricted to the conformational epitope unique to ORF2.1 [Li et al. (1997) Journal of Medical Virology 52:289-300], a pattern that was also observed when comparing acute-phase patient serum samples with serum samples from convalescing patients. Antibody from immunised rats blocked the majority of patients' serum reactivity in enzyme-linked immunosorbent assay against both ORF2.1 (57-92% inhibition) and virus-like particles of HEV produced using the baculovirus system (74-97% inhibition). Together, these results suggest that the ORF2.1 subunit vaccine induces an antibody response against immunodominant, conformational epitopes in the viral capsid, which largely mimics that seen in convalescent patients, who are presumed to be immune to HEV infection.
Adjuvants, Immunologic↗
Serotype-specific antigen ELISA for detection of Chiba virus in stools.
Chiba virus (CV), a Norwalk-like virus (NLV), was first identified as a cause of oyster-associated outbreak of gastroenteritis that occurred in Chiba prefecture, Japan, in 1987. An enzyme-linked immunosorbent assay (ELISA), based on hyperimmune antisera to recombinant baculovirus-expressed capsid proteins of CV (rCV), was developed to detect CV antigen in stools. No cross-reactions were observed with other enteric viruses including enteroviruses, rotaviruses, astroviruses, or enteric adenoviruses. The ELISA was used to screen 101 stools collected from 16 oyster-associated outbreaks of acute gastroenteritis. Twelve stools (11.9%) from seven outbreaks were positive for CV antigen. Ten rCV ELISA-positive strains were confirmed by RT-PCR and nucleotide sequencing. ELISA-positive strains showed 96-100% nucleotide sequence identity to each other, though they were obtained nine years apart. Phylogenetic analysis demonstrated that all ten strains clustered with the prototype CV in genogroup I viruses. We concluded that the antigen ELISA described in this study is highly type-specific, and that this method should be useful for epidemiological surveys of Chiba virus infections.
Animals↗
Empty virus-like particle-based enzyme-linked immunosorbent assay for antibodies to hepatitis E virus.
Hepatitis E, an enterically transmitted non-A, non-B hepatitis, is a serious viral infection that occasionally causes large epidemics in developing countries. In developed countries, the disease only appears sporadically due to the transmission routes, and it is considered to be less important. The hepatitis E virus (HEV) cannot grow in cultured cells and no reliable assay system has ever been developed. In addition, the present diagnostic are not perfect, and actual rates of HEV infection may be underestimated. Highly purified empty virus-like particles (VLPs) of HEV have been produced by the use of a recombinant baculovirus vector in insect cells. Using these VLPs as an antigen, an enzyme-linked immunosorbent assay (ELISA) for antibodies to HEV was developed. A panel of 164 sera that were randomized and coded, and sera collected periodically from three patients with hepatitis E were used for the evaluation. The sensitivity of the assay was shown to be equal to or better than that obtained in previous research that used the same serum panel. The ELISA demonstrated that the serum IgM level of the patients was highest at the onset of the clinical illness and then rapidly decreased. In contrast, a high level of circulating IgG antibody titers lasted for more than 4 years. In Japan, a non-endemic country, the prevalence of the IgG class antibody to HEV in healthy individuals was found to range from 1.9% to 14.1%, depending on the geographical area. Only one out of 900 (0.1%) serum samples was IgM-positive. The IgM class antibody to HEV was detected in 10.8% of non-A, non-B, and non-C acute hepatitis patients in northeast China, whereas none of the patients in Korea had the IgM antibody. The ELISA utilizing the VLPs is sensitive and specific in its detection of the IgM and IgG antibodies to HEV. The ELISA is therefore useful for diagnosing HEV infection and for seroepidemiological study of hepatitis E.
Adolescent↗
High concentrations of serum inhibin in pre-eclampsia.
OBJECTIVE: To evaluate maternal serum immunoreactive inhibin (ir-inhibin) concentrations in women with pre-eclampsia, and assess the correlation between serum ir-inhibin and HCG. METHODS: The subjects comprised 28 pregnant women with suspected intrauterine growth retardation (IUGR) during the third trimester. Serum concentrations of ir-inhibin and HCG were measured in 13 women with pre-eclampsia and 15 pregnant women as control subjects. Serum ir-inhibin was determined by a double antibody radioimmunoassay, and HCG by a solid-phase immunoradiometric assay. RESULTS: There were no significant differences in maternal characteristics between the pre-eclamptic group and control group. The pre-eclamptic group had significantly higher concentrations of serum ir-inhibin and HCG compared with the control group. The serum concentrations of ir-inhibin correlated positively with those of HCG. CONCLUSION: The pre-eclamptic patients displayed high serum levels of ir-inhibin and HCG, and this might reflect hyperplasia of trophoblastic cells.
Adult↗
Predicting incomplete uterine rupture with vaginal sonography during the late second trimester in women with prior cesarean.
OBJECTIVE: To evaluate the usefulness of serial transvaginal ultrasonographic measurement of the thickness of the lower uterine segment in the late second trimester for predicting the risk of intrapartum incomplete uterine rupture in women with previous cesarean delivery. METHODS: Serial transvaginal ultrasonography with full bladder was performed in 374 women without previous cesarean delivery (control group) and 348 women with previous cesarean delivery (cesarean group) from 19 to 39 weeks' gestation. The thickness of the lower uterine segment was measured in the longitudinal plane of the cervical canal. RESULTS: The thickness of the lower uterine segment decreased from 6.7 +/- 2.4 mm (mean +/- standard deviation [SD]) at 19 weeks' gestation to 3.0 +/- 0.7 mm at 39 weeks' gestation in the control group, but the thickness was more than 2.0 mm throughout this period in each control subject. In the cesarean group, the thickness decreased from 6.8 +/- 2.3 mm at 19 weeks' to 2.1 +/- 0.7 mm at 39 weeks' gestation and was significantly thinner than that of the control group after 27 weeks' gestation (P <.05). Eleven of 12 women (91%) with lower uterine segment less than the mean control - 1 SD in the late second trimester had a very thin lower uterine segment at cesarean delivery with fetal hair being visible through the amniotic membrane, ie, incomplete uterine rupture. In 17 of 23 women (74%) with lower uterine segment less than 2.0 mm in thickness within 1 week (4 +/- 3 days) before repeat cesarean delivery, intrapartum incomplete uterine rupture developed. CONCLUSION: Transvaginal ultrasonography is useful for measurement of the uterine wall after previous cesarean delivery.
Adult↗
Suppression of interferon-induced antiviral activity in cells expressing hepatitis C virus proteins.
To elucidate the mechanism of the persistent nature of hepatitis C virus (HCV) infection, we examined whether the expression of HCV proteins affect the antiviral activity of interferon (IFN). Antiviral activity of IFN in HepG2 cells expressing all HCV (type 1b) proteins was much lower than vector control (VC) HepG2 cells when encephalomyocarditis virus (EMCV) was used as a challenge virus. Lesser sensitivity to IFN was also observed in cells expressing NS3, NS4, and NS5 and in cells expressing only NS5A. In contrast, HepG2 cells expressing core, E1, E2, NS2, and NS3 proteins were equally sensitive to IFN as VC cells. We then tested the antiviral activity by IFN in two human amnion-derived FL cell lines expressing NS5A from two different clones, one with an intact sequence of IFN sensitivity-determining region (ISDR) and the other with a mutated ISDR sequence. They were almost equally insensitive to IFN treatment when EMCV was challenged. HCV thus has functional protein(s), possibly NS5A, to suppress IFN-induced antiviral activity and plays an important role in virus-cell interaction and regulation of viral replication.
Antiviral Agents↗
Membrane structure of the hepatitis B virus surface antigen particle.
Expression of S protein, an envelope protein of hepatitis B virus, in the absence of other viral proteins, leads to the secretion of hepatitis B virus surface antigen (HBsAg) particles that are formed by budding from the endoplasmic reticulum membranes. The HBsAg particles produced by mouse fibroblast cells show a unique lipid composition, with 1,2-diacyl glycerophosphocholine being the dominant component. The lipid organization of the HBsAg particles was studied by measuring electron spin resonance (ESR) using various spin-labeled fatty acids, and the results were compared with a parallel study on HVJ (Sendai virus) and vesicles reconstituted with total lipids of the HBsAg particles (HBs-lipid vesicles). HVJ and the HBs-lipid vesicles showed typical ESR spectra of lipids arranged in a lipid bilayer structure. In contrast, the ESR spectra obtained with the HBsAg particles showed that the movement of lipids in the particle is severely restricted and a typical immobilized signal characteristic of tight lipid-protein interactions was also evident. Phosphatidylcholine (PC) in the HBsAg particles was not exchangeable by a PC-specific exchange protein purified from bovine liver, while phospholipase A(2) from Naja naja vemon was able to hydrolyze all the PC in the particles. These analyses suggest that the lipids in the HBsAg particles are not organized in a typical lipid bilayer structure, but are located at the surface of the particles and are in a highly immobilized state. Based on these observations we propose a unique lipid assembly and membrane structure model for HBsAg particles.
Androgen-Binding Protein↗
Expression of recombinant capsid proteins of chitta virus, a genogroup II Norwalk virus, and development of an ELISA to detect the viral antigen.
The second open reading frame (ORF2) gene of the Chitta virus (CHV) was cloned to construct a recombinant baculovirus. The CHV ORF2 is predicted to encode a capsid protein of 535 amino acids (aa). CHV showed a high aa identity in the capsid region with genogroup II Norwalk virus (NV) (65-85%), but a low aa identity with genogroup I NV (44-46%). Phylogenetic analysis of the ORF2 gene demonstrated that CHV is genetically closely related to the Hawaii virus included in genogroup II NV. The recombinant capsid protein of CHV (rCHV) self-assembled to form empty virus-like particles (VLPs) when expressed in insect cells with the recombinant baculovirus. An enzyme-linked immunosorbent assay (ELISA) based on antisera to rCHV was developed to detect CHV antigen in stools. The antigen ELISA appeared to be highly specific to both rCHV and CHV-like strains. In addition, combined use of antigen ELISAs using antibodies against two antigenically distinct recombinant VLPs, the recombinant Chiba virus (rCV) and recombinant Seto virus (rSEV), enabled us to determine the genetic as well as antigenic relationship among these three viruses.
Animals↗