Yasuyuki Gomi

Comparison of the Complete DNA Sequences of the Oka Varicella Vaccine and Its Parental Virus

ABSTRACT The DNA sequences of the Oka varicella vaccine virus (V-Oka) and its parental virus (P-Oka) were completed. Comparison of the sequences revealed 42 base substitutions, which led to 20 amino acid conversions and length differences in tandem repeat regions (R1, R3, and R4) and in an origin of DNA replication. Amino acid substitutions existed in open reading frames (ORFs) 6, 9A, 10, 21, 31, 39, 50, 52, 55, 59, 62, and 64. Of these, 15 base substitutions, leading to eight amino acid substitutions, were in the gene 62 region alone. Further DNA sequence analysis showed that these substitutions were specific for V-Oka and were not present in nine clinical isolates. The immediate-early gene 62 product (IE62) of P-Oka had stronger transactivational activity than the mutant IE62 contained in V-Oka in 293 and CV-1 cells. An infectious center assay of a plaque-purified clone (S7-01) from the V-Oka with 8 amino acid substitutions in ORF 62 showed smaller plaque formation and less-efficient virus-spreading activity than did P-Oka in human embryonic lung cells. Another clone (S-13) with only five substitutions in ORF 62 spread slightly faster than S7-01 but not as effectively as P-Oka. Moreover, transient luciferase assay in 293 cells showed that transactivational activities of IE62s of S7-01 and S7-13 were lower than that of P-Oka. Based on these results, it appears that amino acid substitutions in ORF 62 are responsible for virus growth and spreading from infected to uninfected cells. Furthermore, the Oka vaccine virus was completely distinguishable from P-Oka and 54 clinical isolates by seven restriction-enzyme fragment length polymorphisms that detected differences in the DNA sequence.

Oka varicella vaccine is distinguishable from its parental virus in DNA sequence of open reading frame 62 and its transactivation activity

When the nucleotide sequences of the Oka vaccine and its parental varicella‐zoster virus (VZV) were compared in 6 open reading frames (ORFs), glycoprotein C (gC) and 5 transactivator genes, mutations were detected only in the immediate‐early gene 62. The vaccine virus contained a mixture of different sequences that had variations at 15 nucleotide positions, but only one sequence was found for the Oka parental virus gene 62. The Oka vaccine virus gene 62 could be distinguished from the parental virus gene using a simplified restriction‐enzyme fragment length polymorphism analysis, using NaeI and BssHII. This analysis was based on the sequence data obtained in this study. Studies of the regulatory activities of the ORF62 gene product (IE62) in a transient transfection assay indicated that IE62 of the parental virus had a stronger transactivational activity than that of the vaccine virus in activating immediate‐early, early, and late gene promoters. These data suggest that IE62 might play an important role in the attenuation of VZV. J. Med. Virol. 61:497–503, 2000.

Anti-influenza activity of marchantins, macrocyclic bisbibenzyls contained in liverworts.

The H1N1 influenza A virus of swine-origin caused pandemics throughout the world in 2009 and the highly pathogenic H5N1 avian influenza virus has also caused epidemics in Southeast Asia in recent years. The threat of influenza A thus remains a serious global health issue and novel drugs that target these viruses are highly desirable. Influenza A possesses an endonuclease within its RNA polymerase which comprises PA, PB1 and PB2 subunits. To identify potential new anti-influenza compounds in our current study, we screened 33 different types of phytochemicals using a PA endonuclease inhibition assay in vitro and an anti-influenza A virus assay. The marchantins are macrocyclic bisbibenzyls found in liverworts, and plagiochin A and perrottetin F are marchantin-related phytochemicals. We found from our screen that marchantin A, B, E, plagiochin A and perrottetin F inhibit influenza PA endonuclease activity in vitro. These compounds have a 3,4-dihydroxyphenethyl group in common, indicating the importance of this moiety for the inhibition of PA endonuclease. Docking simulations of marchantin E with PA endonuclease suggest a putative “fitting and chelating model” as the mechanism underlying PA endonuclease inhibition. The docking amino acids are well conserved between influenza A and B. In a cultured cell system, marchantin E was further found to inhibit the growth of both H3N2 and H1N1 influenza A viruses, and marchantin A, E and perrotein F showed inhibitory properties towards the growth of influenza B. These marchantins also decreased the viral infectivity titer, with marchantin E showing the strongest activity in this assay. We additionally identified a chemical group that is conserved among different anti-influenza chemicals including marchantins, green tea catechins and dihydroxy phenethylphenylphthalimides. Our present results indicate that marchantins are candidate anti-influenza drugs and demonstrate the utility of the PA endonuclease assay in the screening of phytochemicals for anti-influenza characteristics.

Measurement of Varicella-Zoster Virus (VZV)-Specific Cell-Mediated Immunity: Comparison between VZV Skin Test and Interferon-γ Enzyme-Linked Immunospot Assay

Cell-mediated immunity (CMI) is critical for the prevention and control of varicella-zoster virus (VZV)-related disease. To assess CMI to VZV, a varicella skin test and interferon-gamma enzyme-linked immunospot (ELISPOT) assay were both performed in healthy volunteers, and the results were compared. A total of 151 subjects were examined: 16 aged 20-29 years, 26 aged 30-39 years, 18 aged 40-49 years, 73 aged 50-59 years, and 18 aged 60-69 years. All were seropositive by a glycoprotein antigen-based enzyme-linked immunosorbent assay (gpELISA). Skin test reactivity was significantly correlated with the ELISPOT count, and both decreased with increasing age, indicating an age-dependent decline in CMI to VZV. In contrast, the antibody titer obtained by the gpELISA did not correlate with skin test reactivity. The results suggest that the skin test and ELISPOT assay are both reliable for assessing CMI to VZV and can easily be applied to screen individuals susceptible to the development of herpes zoster.

Expression of human herpesvirus 6B rep within infected cells and binding of its gene product to the TATA-binding protein in vitro and in vivo.

ABSTRACT We have characterized the human herpesvirus 6B (HHV-6B)rep gene, which is a homologue of the adeno-associated virus type 2 rep and is unique in the herpesvirus family. Three transcripts, 9.0, 5.0, and 2.7 kb (the major transcript), were detected by Northern blotting using an HHV-6B rep probe under late conditions. We investigated the expression kinetics of therep gene using cycloheximide (CHX) and phosphonoformic acid (PFA), which are inhibitors of protein synthesis and viral DNA synthesis, respectively. The 5.2-kb transcript was mainly detected in the absence of protein biosynthesis upon infection, and none of the 9.0-, 5.0-, and 2.7-kb transcripts detected under the late conditions were detected in the presence of CHX and PFA. Sequences obtained from a cDNA library showed that the 5.0- and 2.7-kb transcripts were spliced from two and three exons, respectively, and the 2.7-kb transcript was more abundant. Immunohistochemistry using an antibody raised against the HHV-6 rep gene product (REP) revealed that REP was mainly present in the nucleus of MT-4 cells within 24 h after infection with HHV-6B. Using pull-down assays, coimmunoprecipitation, and a mammalian two hybrid system, we showed that HHV-6 REP binds to a transcription factor, human TATA-binding protein, through its N-terminal region.

Immunogenicity and efficacy of two types of West Nile virus-like particles different in size and maturation as a second-generation vaccine candidate.

Virus-like particles (VLPs) of flaviviruses generated from the prM and E genes are a promising vaccine candidate. We have established cell clones continuously releasing VLPs of West Nile virus (WNV) in serum-free conditions. Two types of VLPs were distinguished by sedimenting analyses in sucrose density gradients. Fast sedimenting VLPs (F-VLPs) were large (40-50 nm) and composed of the E and processed mature M proteins, whereas slowly sedimenting VLPs (S-VLPs) were small (20-30 nm) particles consisting of the E and immature prM proteins. F-VLPs induced higher neutralizing antibody and anti-WNV IgG titers than S-VLPs. Furthermore, IgG2a was dominant over IgG1 by immunization with F-VLPs as with whole virion-derived antigens. Mice vaccinated with a low dose (3 ng) of F-VLPs showed higher protective efficacy (83% survivals) against WNV infection than S-VLP-immune mice (17% survivals). These results indicate that F-VLPs more closely resemble the virions and take a better immunogenic form than S-VLPs as WNV vaccine candidates.

Comparison of Virus Transcription during Lytic Infection of the Oka Parental and Vaccine Strains of Varicella-Zoster Virus

ABSTRACT The attenuated Oka vaccine (V-Oka) strain of varicella-zoster virus (VZV) effectively reduces disease produced by primary infection and virus reactivation. V-Oka was developed by propagation of the Oka parental (P-Oka) strain of VZV in guinea pig and human embryo fibroblasts. Complete DNA sequencing of both viruses has revealed 63 sites that differ between P-Oka and V-Oka, 37 of which are located within 21 unique open reading frames (ORFs). Of the ORFs that differ, ORF 62 contains the greatest number (10) of mutated sites. ORF 62 encodes IE 62, the major immediate-early transactivator of virus genes, and is essential for lytic virus replication. To determine whether a disproportionate number of mutations in ORF 62 might account for virus attenuation, we compared the global pattern of V-Oka gene expression to that of P-Oka. Transcription of ORFs 62, 65, 66, and 67 was suppressed, whereas ORF 41 was elevated in V-Oka-infected cells compared to P-Oka-infected cells (P < 0.01; z test). Suppression of ORF 62, 65, and 66 transcription was confirmed by quantitative dot blot and Western blot analyses. Transient-transfection assays to determine whether mutations within V-Oka-derived IE 62 affected its ability to transactivate VZV gene promoters revealed similar IE 62 transactivation of VZV gene 20, 21, 28, 29, 65, and 66 promoters in both P-Oka and V-Oka. Together, our results indicate that mutations in V-Oka IE 62 alone are unlikely to account for vaccine virus attenuation.

Comparison of DNA sequence and transactivation activity of open reading frame 62 of Oka varicella vaccine and its parental viruses

When nucleotide sequences of Oka vaccine and its parental viruses of varicella-zoster virus (VZV) were compared in 5 open reading frames (ORFs) including glycoprotein C (gC) and 4 immediate-early genes, mutations were detected only in gene 62 which is one of the immediate-early genes. Compared with its parental virus, the vaccine virus contained 15 nucleotide substitutions. With the differentiation method using the simplified restriction-enzyme fragment length polymorphism analysis by Nae I and Bss HII, which was established based on the sequence analysis data in this study, the Oka vaccine virus could be distinguished from its parental virus. Studies of the regulatory activities of the ORF62 gene product (IE62) in a transient assay indicate the IE62 of the parental virus had a stronger transactivational activity than that of the vaccine virus against immediate-early, early and late gene promoters. These data suggest that gene 62 might have an important role for attenuation of VZV. This is the first report in which many substitutions of nucleotides in gene 62 of Oka vaccine virus was found, compared with that of Oka parental virus.

Incidences of Herpes Zoster and Postherpetic Neuralgia in Japanese Adults Aged 50 Years and Older From a Community-based Prospective Cohort Study: The SHEZ Study

Background Many cross-sectional studies have examined the incidences of herpes zoster (HZ) and postherpetic neuralgia (PHN), but prospective studies in Japanese older adults are lacking. Therefore, we conducted a community-based prospective cohort study to determine the incidence in Japanese adults aged ≥50 years. Methods We recruited 12 522 participants from Shozu County, Kagawa Prefecture, between December 2008 and November 2009 and followed participants for 3 years. When a subject presented with symptoms suggestive of HZ, they were examined at collaborating medical institutions and cooperated with onset and recovery surveys (eg, measurement of varicella zoster virus-specific immunity and a pain survey). The hazard ratios (HRs) of HZ and PHN according to sex and age were analyzed by Cox regression analysis with a significance level of 5%. Results The incidence of HZ was 10.9/1000 person-years (men: 8.5/1000 person-years; women: 12.8/1000 person-years) and was significantly higher in women than in men (HR 1.5; 95% confidence interval, 1.2–1.8). The incidence of PHN was 2.1/1000 person-years (men: 1.7/1000 person-years; women: 2.4/1000 person-years), with no significant sex differences. A total of 19% of HZ cases progressed to PHN; no sex-specific difference in the proportion of PHN cases was observed. Conclusions We clarified the accurate incidences of HZ and PHN in a population of Japanese older adults. These incidences increased with age. HZ incidence was higher in women than in men, while PHN incidence did not differ markedly between the sexes.

Superior immunogenicity of a freeze-dried, cell culture-derived Japanese encephalitis vaccine (inactivated).

Japanese encephalitis is an infectious disease caused by the Japanese encephalitis virus, which is widespread throughout Asia. The worldwide incidence is 50,000 cases per year. There is no specific treatment available, but inactivated mouse brain-derived vaccine was used from the 1950s to prevent infection. However, quality control of mouse brain-derived vaccines is difficult, and therefore a new freeze-dried, cell culture-derived Japanese encephalitis vaccine (inactivated) (JEBIK V; development code: BK-VJE) was developed. In this paper, we report an analysis of neutralizing antibody titers in vaccinated subjects enrolled in clinical study of BK-VJE at various doses, and study of BK-VJE with the mouse brain-derived vaccine as a control. The results show that BK-VJE has superior immunogenicity compared to mouse brain-derived vaccine.