Publications (21)46.53 Total impact
- [Show abstract] [Hide abstract] ABSTRACT: Recently, several new strains of canine distemper virus (CDV) have been isolated in Japan. To investigate their pathogenesis in dogs, the Yanaka and Bunkyo-K strains were investigated by infecting dogs and determining clinical signs, amount of virus, and antibody responses. The Yanaka strain is avirulent and induced an antibody response. The Bunkyo-K strain induced typical CDV clinical signs in infected dogs and virulence was enhanced by brain passage. Molecular and phylogenetic analyses of H genes demonstrated the Bunkyo-K strains were of a different lineage from Asia-1 group including the Yanaka strain and Asia-2 group that contain recent Japanese isolates, which were recently identified as major prevalent strains worldwide but distinct from old vaccine strains. Based on these data, we tested the ability of the Yanaka strain for vaccination. Inoculation with the Yanaka strain efficiently induced CDV neutralizing antibodies with no clinical signs, and the protection effects against challenge with either old virulent strain or Bunkyo-K strain were equal or greater when compared with vaccination by an original vaccine strain. Thus, the Yanaka strain is a potential vaccine candidate against recent prevalent CDV strains.
- [Show abstract] [Hide abstract] ABSTRACT: Measles, caused by measles virus (MeV) infection, is the leading cause of death in children because of secondary infections attributable to MeV-induced immune suppression. Recently, we have shown that wild-type MeVs induce the suppression of protein synthesis in host cells (referred to as "shutoff") and that viral mRNAs are preferentially translated under shutoff conditions in infected cells. To determine the mechanism behind the preferential translation of viral mRNA, we focused on the 5' untranslated region (UTR) of nucleocapsid (N) mRNA. The La/SSB autoantigen (La) was found to specifically bind to an N-5'UTR probe. Recombinant La enhanced the translation of luciferase mRNA containing the N-5'UTR (N-fLuc), and RNA interference of La suppressed N-fLuc translation. Furthermore, recombinant MeV lacking the La-binding motif in the N-5'UTR displayed delayed viral protein synthesis and growth kinetics at an early phase of infection. These results suggest that La induced predominant translation of N mRNA via binding to its 5'UTR under shutoff conditions. This is the first report on a cellular factor that specifically regulates paramyxovirus mRNA translation.
- [Show abstract] [Hide abstract] ABSTRACT: The interaction of Nipah virus (NiV) nucleocapsid (N) protein with phosphoprotein (P) during nucleocapsid assembly is the essential process in the viral life cycle, since only the encapsidated RNA genome can be used for replication. To identify the region responsible for N-P interaction, we utilized fluorescent protein tags to visualize NiV N and P proteins in live cells and analyzed their cellular localization. N protein fused to monomeric enhanced cyan fluorescence protein (N-ECFP) exhibited a dotted pattern in transfected cells, while P protein fused to monomeric red fluorescent protein (P-mRFP) showed diffuse distribution. When the two proteins were coexpressed, P-mRFP colocalized with N-ECFP dots. N-ECFP mutants with serial amino acid deletions were generated to search for the region(s) responsible for this N-P colocalization. We found that, in addition to the 467- to 496-amino-acid (aa) region reported previously, aa 135 to 146 were responsible for the N-P colocalization. The residues crucial for N-P interaction were further investigated by introducing alanine substitutions into the untagged N protein. Alanine scanning in the region of aa 135 to 146 has revealed that there are distinct regions essential for the interaction of N-P and the function of N. This is the first study to visualize Nipah viral proteins in live cells and to assess the essential domain of N protein for the interaction with P protein.
- [Show abstract] [Hide abstract] ABSTRACT: Nipah virus (NiV) P gene encodes P protein and three accessory proteins (V, C and W). It has been reported that all four P gene products have IFN antagonist activity when the proteins were transiently expressed. However, the role of those accessory proteins in natural infection with NiV remains unknown. We generated recombinant NiVs lacking V, C or W protein, rNiV(V-), rNiV(C-), and rNiV(W-), respectively, to analyze the functions of these proteins in infected cells and the implications in in vivo pathogenicity. All the recombinants grew well in cell culture, although the maximum titers of rNiV(V-) and rNiV(C-) were lower than the other recombinants. The rNiV(V-), rNiV(C-) and rNiV(W-) suppressed the IFN response as well as the parental rNiV, thereby indicating that the lack of each accessory protein does not significantly affect the inhibition of IFN signaling in infected cells. In experimentally infected golden hamsters, rNiV(V-) and rNiV(C-) but not the rNiV(W-) virus showed a significant reduction in virulence. These results suggest that V and C proteins play key roles in NiV pathogenicity, and the roles are independent of their IFN-antagonist activity. This is the first report that identifies the molecular determinants of NiV in pathogenicity in vivo.
- [Show abstract] [Hide abstract] ABSTRACT: Comparative and mutational analysis of promoter regions of rinderpest virus was conducted. Minigenomic RNAs harboring the genomic and antigenomic promoter of the lapinized virulent strain (Lv) or an attenuated vaccine strain (RBOK) were constructed, and the expression of the reporter gene was examined. The activities of the antigenomic promoters of these strains were similar, whereas the activity of the genomic promoter (GP) of the RBOK strain was significantly higher than that of the Lv strain, regardless of cell type and the source of the N, P and L proteins. Increased replication (and/or encapsidation) activities were observed in the minigenomes that contained RBOK GP. Mutational analysis revealed that the nucleotides specific to the RBOK strain are responsible for the strong GP activity of the strain. It was also demonstrated that other virulent strains of RPV (Kabete O, Saudi/81 and Kuwait 82/1) have weaker GPs than that of the RBOK strain.
- [Show abstract] [Hide abstract] ABSTRACT: Enhanced green fluorescent protein (EGFP) is a useful marker protein which enables the tracing of virus infection. Recombinant viruses expressing EGFP are useful for the investigation of the underlying mechanism of viral infection in vitro and in vivo. Using EGFP-expressing recombinant Nipah virus (NiV) and canine distemper virus (CDV), we tested the susceptibility of a variety of cells to infection. Receptor usage in CDV infection was also investigated. © 2009 Humana Press, a part of Springer Science + Business Media, LLC.
- [Show abstract] [Hide abstract] ABSTRACT: The wide tissue tropism of the measles virus (MV) suggests that it involves ubiquitously expressed molecules. We have constructed a recombinant MV expressing the enhanced green fluorescent protein (EGFP) (rMV-EGFP) and demonstrated that the rMV-EGFP infected several cell types (HEK-293, HepG2, Hep3B, Huh7, and WRL68 cells) that do not express the human signalling lymphocyte activation molecule (SLAM), which is known as a cellular receptor for morbilliviruses. MV infection of HEK-293 and HepG2 cells was not inhibited in an infectivity-inhibition assay using an anti-SLAM monoclonal antibody, indicating that MV could infect cells without using SLAM. Soluble heparin (HP) inhibited the rMV-EGFP infectivity in SLAM-negative cell lines in a dose-dependent manner. Direct interaction between purified virions and HP was detected in a surface plasmon resonance assay. We also demonstrated that the hemagglutinin (H) protein, but not the fusion (F) protein is responsible for the interaction between the virions and HP. Taken together, our results suggest that HP-like glycosaminoglycans bind to the H protein of MV and play a key role in the infection of SLAM-negative cells.
- [Show abstract] [Hide abstract] ABSTRACT: Ten wild masked palm civets infected with canine distemper virus (CDV), captured in Japan from 2005 to 2007, were histopathologically and phylogenetically analyzed. Phylogenetic analysis based on the amino acid sequences of the H protein of two CDV isolates from masked palm civets revealed that the two isolates were classified into the clade of recent isolates in Japan. Histopathologically marked lesions of virus encephalitis were present in the brain, whereas gastrointestinal lesions were absent or at a mild degree. The distribution of the lesions resembles that of recent CDV cases in dogs. Therefore, recent CDV infections in masked palm civets could be caused by recently prevalent CDV in dogs. The possibility of the masked palm civet as a spreader of CDV among wildlife is also discussed.
- [Show abstract] [Hide abstract] ABSTRACT: We report the first identification of phosphorylation sites of the nucleoprotein (N) of the family Paramyxoviridae. The N protein is known to be the most abundant protein in infected cells; it constructs the N-RNA complex (nucleocapsid) and supports transcription and replication of viral genomic RNA. To determine the role of phosphorylation of the N protein, we expressed the N protein of the HL strain of measles virus (MV) in mammalian cells and purified the nucleocapsid. After separation of the C-terminal region from the core region, phosphorylated amino acids were assayed using MALDI-TOF/TOF and ESI-Q-TOF MS analyses. Two amino acids, S479 and S510, were shown to be phosphorylated by both methods of analysis. Metabolic labeling of the N protein with (32)P demonstrated that these two sites are the major phosphorylated sites within the MV-N protein. In transcriptional analysis using negative-strand minigenomic RNA containing the ORF of the luciferase gene, mutants of each phosphorylation site showed approximately 80% reduction in luciferase activity compared with the wild-type N, suggesting that the phosphorylation of N protein is important in the activation of the transcription of viral mRNA and/or replication of the genome in vivo.
- [Show abstract] [Hide abstract] ABSTRACT: We constructed recombinant viruses expressing enhanced green fluorescent protein (EGFP) or firefly luciferase from cDNA clones of the canine distemper virus (CDV) (a Japanese field isolate, Yanaka strain). Using these viruses, we examined susceptibilities of different cell lines to CDV infection. The results revealed that the recombinant CDVs can infect a broad range of cell lines. Infectivity inhibition assay using a monoclonal antibody specific to the human SLAM molecule indicated that the infection of B95a cells with these recombinant CDVs is mainly mediated by SLAM but the infection of 293 cell lines with CDV is not, implying the presence of one or more alternative receptors for CDV in non-lymphoid tissue. Infection of 293 cells with the recombinant CDV was inhibited by soluble heparin, and the recombinant virus bound to immobilized heparin. Both F and H proteins of CDV could bind to immobilized heparin. These results suggest that heparin-like molecules are involved in CDV infection.
- [Show abstract] [Hide abstract] ABSTRACT: Seven strains of canine parvovirus (CPV) were isolated from affected dogs in Japan between 1999 and 2000, and their VP2 genes were genetically analyzed. Comparison of the predicted amino acid sequences of VP2 suggested that three field isolates corresponded to CPV type 2a, while the other four to CPV type 2b. The phylogenetic tree constructed from the VP2 genes showed that the newly isolated strains are classified into the cluster consisting of recent Japanese and Taiwanese field isolates, which are distinct from Vietnamese isolates, United States Isolates, or classical CPV type 2. These results suggest that the CPV transmission occurred between Japan and Taiwan in 1990s, and the offspring are still circulating in both countries.
- [Show abstract] [Hide abstract] ABSTRACT: Forty Caspian seals were surveyed seroepidemiologically between 1993 and 1998 around the times of mass mortality that occurred in 1997 in the Caspian Sea and seven Baikal seals were also surveyed in 1998. Virus neutralizing tests and ELISA clearly suggested that distemper virus epidemic was caused in Caspian seals before the spring of 1997 and that CDV infection continued to occur in Lake Baikal in recent years.
- [Show abstract] [Hide abstract] ABSTRACT: The nucleotide sequence of the matrixprotein (M) gene of the lapinized rinderpest virus (RPV-L) was determined. The full-length cDNA of the RPV-L M gene is composed of 1460 base pairs and is supposed to contain an open reading frame of 1005 nucleotides encoding on M protein of 335 amino acids. The homology of the predicted amino acid among congeneric morbilliviruses such as RPV Kabete 'O' strain (wild strain of RPV), RPV RBOK strain (vaccine strain of RPV for cattle), measles virus (MV), and canine distemper virus (CDV), is approximately 94%, 93%, 87% and 77%, respectively. In the present study, all coding regions of the RPV-L strain have been determined.
- [Show abstract] [Hide abstract] ABSTRACT: We constructed two recombinant feline herpesvirus type 1 (FHV-1) expressing the envelope (Env) protein of feline immunodeficiency virus (FIV). One recombinant, designated dlTK-env, has the whole FIV env gene inserted at a thymidine kinase (TK) deletion site. The second recombinant, designated dlTK(gCp)-env, has a cassette containing a partial FIV env gene fused with the signal sequence of the gC protein of FHV-1 (under the control of the gC promoter) inserted at the same site. Growth kinetics of both the recombinants in Crandell feline kidney (CRFK) cells were similar to that of the parent strain of FHV-1. By indirect immunofluorescence assays and immunoblot analyses, we confirmed the expression of the FIV Env protein in CRFK cells infected with both recombinants. Enzyme-linked immunosorbent assays showed that the maximum Env expression level achieved by dlTK(gCp)-env was more than four times higher than that observed for dlTK-env. Flow cytometric analyses revealed that the Env protein produced by both recombinants was efficiently expressed on the cell surface. The dlTK(gCp)-env reported here may thus be a promising candidate for a live recombinant vaccine to protect against FIV infection.
- [Show abstract] [Hide abstract] ABSTRACT: We previously reported the attenuation of thymidine kinase (TK) deficient mutant (C7301dlTK) of feline herpesvirus type 1 (FHV-1) in cats and the construction of a recombinant FHV-1 (C7301dlTK-Cap) inserted a precursor capsid gene of feline calcivirus (FCV) into the TK deletion locus of the C7301dlTK. In this study, we constructed a further improved recombinant FHV-1 (dlTK(gCp)-Cap) carrying a putative FHV-1 gC promoter sequence upstream of the FCV precursor capsid gene of the C7301dlTK-Cap. Growth kinetics of the dlTK(gCp)-Cap in cell cultures was similar to those of C7301dlTK and C7301dlTK-Cap. A strong expression of FCV immunogenic antigen by dlTK(gCp)-Cap was confirmed by indirect immunofluorescence and enzyme-linked immunosorbent assays. In addition, one vaccination with dlTK(gCp)-Cap protected cats more effective against subsequent virulent FCV challenge than that with C7301dlTK-Cap.
- [Show abstract] [Hide abstract] ABSTRACT: The YP11mu strain of a plaque-selected canine herpesvirus (CHV) encoded a smaller molecular weight (MW) of gD than those of other strains including YP2 strain (Xuan et al., 1990). When nucleotide sequence of the mutated gD of YP11mu strain (gD(YP11mu)) was compared with that of gDs of other CHV strains, gD(YP11mu) lacked 12 nucleotides encoding 4 amino acids, NKTI, including one predicted potential N-linked glycosylation site and no other change was found in other regions. When the gD(YP11mu) and gD of YP2 strain (gD(YP2)) expressed in COS-7 and insect (Spodoptera frugiperda; Sf9) cells were compared each other, both gDs reacted with a panel of monoclonal antibodies (MAbs) against CHV gD by indirect immunofluorescence analysis and the gD(YP11mu) possessed an MW of approximately 47-51 and 39-44 kDa in COS-7 and Sf9 cells, respectively, which were smaller than the expressed gD(YP2) (approximately 51-55 and 41-46 kDa, respectively) by immunoblot analysis. After treatment with tunicamycin, the MW of both gDs in Sf9 cells became approximately 37 kDa. When hemagglutination (HA) test using canine red blood cells (RBC) were carried out, lysates of Sf9 cells expressing CHV gDs agglutinated canine RBC. Serum from mice inoculated with lysates of Sf9 cells expressing the gDs possessed a high titer of virus-neutralizing (VN) activities against CHV. These results indicated that the deletion of 4 amino acids possessing approximately 4 kDa of glyco-chain from gD of CHV in mammalian cells does not affect HA activity and VN antibody-inducing activity and that this deletion of gD(YP11mu) might be a good selective marker for development of recombinant viruses as a live vaccine.
- [Show abstract] [Hide abstract] ABSTRACT: Feline herpesvirus type 1 (FHV-1) possesses a very narrow host range, but the mechanism of its infection has not yet been analyzed. Heparan sulfate on the cell surface serves as a receptor for several herpesviruses. In this study, we determined that infection of FHV-1 is inhibited by addition of soluble heparin in cells cultures. Using heparin-affinity column, it was shown that FHV-1 gC is a major heparin-binding protein, and FHV-1 gB weakly binds to heparin, but FHV-1 gD does not. Furthermore, the FHV-1 gC expressed in insect cells can also bind to heparin despite of being immature glycosylation. Our results suggested that FHV-1 gC can bind to heparin as observed in other herpesviruses and that glycosylation of the gC does not affect its heparin-binding activity. In addition, mice immunized with the gC expressed in insect cells produced complement-dependent virus-neutralizing antibody.
- [Show abstract] [Hide abstract] ABSTRACT: Glycoprotein D (gD) of canine herpesvirus (CHV) YP2 strain was expressed in COS-7 and insect (Spodoptera frugiperda; Sf9) cells. The gDs expressed in COS-7 and Sf9 cells reacted with a panel of monoclonal antibodies (MAbs) against CHV gD (hemagglutinin) and an MAb 25C9 against feline herpesvirus type 1 (FHV-1) gD by indirect immunofluorescence assay, and possessed a molecular weight (MW) of approximately 51-55 and 41-46 kilodalton (kDa), respectively, when examined by immunoblot analysis. After treatment with tunicamycin, the MW of the gD expressed in Sf9 cells became approximately 37 kDa. By hemadsorption (HAD) tests using canine or feline red blood cells (RBC), COS-7 cells expressing CHV gD adsorbed only canine RBC, but not feline RBC, whereas control COS-7 cells expressing FHV-1 gD adsorbed feline RBC, but not canine RBC. By hemagglutination (HA) tests, lysates of Sf9 cells expressing CHV gD agglutinated canine RBC, but not feline RBC. These HA and HAD activities were inhibited by HA-inhibition MAbs against CHV gD. Control lysates of Sf9 cells expressing FHV-1 gD agglutinated only feline RBC. Serum from mice inoculated with lysates of Sf9 cells expressing CHV gD possessed a high titer of virus-neutralizing activities against CHV infection. These results indicated that CHV gD is structurally similar to FHV-1 gD, but is functionally different from FHV-1 gD.
- [Show abstract] [Hide abstract] ABSTRACT: The feline herpesvirus type 1 (FHV-1) gene encoding glycoprotein C (gC) has been sequenced and identified based on its genomic location and comparative analysis to other alphaherpesvirus gCs, and the expressed gC protein was also identified by using specific monoclonal antibodies. The FHV-1 gC gene was located within a 7.0 kbp EcoRI fragment, and was 1602 bp in length. The amino acid sequence deduced from the nucleotide sequence was predicted to encode a membrane glycoprotein containing a characteristic N-terminal hydrophobic signal sequence, nine potentialN -linked glycosylation sites, and C-terminal transmembrane and cytoplasmic domains. The FHV-1 gC was expressed in COS-7 cells. When flowcytometric analysis was carried out, the gC expressed in COS-7 cells reacted with a panel of monoclonal antibodies against gp113. By immunoprecipitation analysis, the gC expressed in COS-7 cells possessed molecular masses of 125–;150 kilodalto n, and was similar in size to that in FHV-1-infected CRFK cells.
- [Show abstract] [Hide abstract] ABSTRACT: We analyzed the mechanism for feline herpesvirus type 1 (FHV-1) immediate early (IE) gene expression. We demonstrated that (i) the transcription initiation site of the FHV-1 IE transcript lies in the putative FHV-1 ICP4 binding site, (ii) the FHV-1 IE transcript is spliced to remove 906 bp intron from the leader region, (iii) cis-acting elements of the FHV-1 IE promoter map both down- and upstream of the transcription initiation site, and (iv) a deletion of a 58 bp sequence which includes the putative FHV-1 ICP4 binding site in the IE promoter resulted in significant induction of promoter activity by a FHV-1 IE gene product although the FHV-1 IE gene product slightly stimulates the FHV-1 IE promoter, indicating that the FHV-1 IE gene product down-regulates its own promoter via the region.
The University of Tokyo
- • Laboratory Animal Research Center
- • Faculty and Graduate School of Agriculture and Life Sceince