Sequence analysis of VP2 gene of canine parvovirus isolated from domestic dogs in Japan in 1999 and 2000.

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.

Characterization of monoclonal antibodies directed against the canine distemper virus nucleocapsid protein.

We have established four monoclonal antibodies (MAbs) against the nucleocapsid protein (NP) of canine distemper virus (CDV). A competitive binding assay has revealed that the MAbs are directed against two antigenic domains. An immunofluorescence assay using a series of deletion clones of the NP and an immunoprecipitation assay using the NP have revealed that two of the MAbs recognize the C-terminal region of the NP while the other two recognize the tertiary structure of the N-terminal domain. These MAbs reacted with all eight strains of CDV used in this study, but showed different reactivities against measles virus and rinderpest virus.

Measles virus infection induces interleukin-8 release in human pulmonary epithelial cells.

Measles virus (MV) infection primarily targets epithelial cells of the respiratory tract, which have the potential to synthesize a variety of cytokines. In this report, we studied the effect of MV infection on the production of interleukin (IL)-8 by the pulmonary epithelial cells. A549 cells, a lower airway epithelial cell line, produced IL-8 after MV inoculation in a dose- and time-dependent manner. The IL-8 production was little affected by UV-inactivation of MV and scarcely suppressed by cycloheximide treatment. These results indicated that MV particle binding to and/or incorporation into cells stimulated IL-8 expression in A549 cells.

Efficacy of Recombinant Canine Distemper Virus Expressing Leishmania Antigen against Leishmania Challenge in Dogs.

Canine distemper virus (CDV) vaccination confers long-term protection against CDV reinfection. To investigate the utility of CDV as a polyvalent vaccine vector for Leishmania, we generated recombinant CDVs, based on an avirulent Yanaka strain, that expressed Leishmania antigens: LACK, TSA, or LmSTI1 (rCDV-LACK, rCDV-TSA, and rCDV-LmSTI1, respectively). Dogs immunized with rCDV-LACK were protected against challenge with lethal doses of virulent CDV, in the same way as the parental Yanaka strain. To evaluate the protective effects of the recombinant CDVs against cutaneous leishmaniasis in dogs, dogs were immunized with one recombinant CDV or a cocktail of three recombinant CDVs, before intradermal challenge (in the ears) with infective-stage promastigotes of Leishmania major. Unvaccinated dogs showed increased nodules with ulcer formation after 3 weeks, whereas dogs immunized with rCDV-LACK showed markedly smaller nodules without ulceration. Although the rCDV-TSA- and rCDV-LmSTI1-immunized dogs showed little protection against L. major, the cocktail of three recombinant CDVs more effectively suppressed the progression of nodule formation than immunization with rCDV-LACK alone. These results indicate that recombinant CDV is suitable for use as a polyvalent live attenuated vaccine for protection against both CDV and L. major infections in dogs.

Phylogenetic analysis of the hemagglutinin (H) gene of canine distemper viruses isolated from wild masked palm civets (Paguma larvata).

Hemagglutinin (H) gene of two CDV isolates, the Haku93 and Haku00 strains, from masked palm civets was molecularly analyzed. H genes of both two CDVs contained one open reading frame encoding 607 amino acids. Nucleotide and predicted amino acid sequences of H gene of the CDV Haku93 and Haku00 revealed high similarity to those of recent field isolates such as the Yanaka and Tanu96, while they showed limited identity to those of old vaccine strains. Potential N-linked glycosylation sites in both Haku93 and Haku00 were identical to other recent CDV isolates. Phylogenetic analysis revealed that the CDV strains derived from masked palm civets were classified into the group of recent Japanese CDV isolates.

Prevalence of canine distemper virus, feline immunodeficiency virus and feline leukemia virus in captive African lions (Panthera leo) in Japan.

Sero-prevalences of canine distemper virus (CDV), feline immunodeficiency virus (FIV) and feline leukemia virus (FeLV) were evaluated in 20 captive lions in two Japanese zoos. Anti-CDV antibody was detected in 13 of 20 lions. We could pursue antibody responses against CDV in three lions back to 1996. Sera collected in 1996 were negative for anti-CDV antibody, therefore, all of them showed sero-conversion in 2000. This result suggested that the epidemic of CDV infection in this zoo might have happened between 1996 and 2000. The lions were also examined for FIV and FeLV infections. We had no evidence for FeLV infection but eight lions were sero-positive for anti-FIV antibody.

Cytotoxic T-lymphocyte activity specific for hemagglutinin (H) protein of canine distemper virus in dogs.

Cytotoxic T-lymphocyte (CTL) responses to hemagglutinin (H) protein of canine distemper virus (CDV) were evaluated in dogs using the replication-deficient adenovirus protein expression system. Skin fibroblasts were isolated from two dogs and were infected with recombinant adenovirus bearing the CDV-H gene (Ade-CDVH). CTL assay was performed using fibroblasts expressing CDV-H protein as target cells and peripheral blood lymphocytes (PBL) collected from the same dogs one week after immunization of CDV as effector cells. Specific cytotoxic activity was observed against autologous but not heterologous fibroblasts expressing CDV-H protein. These results indicate that the CTL epitope(s) were localized in the H protein.

Characterization of two recent Japanese field isolates of canine distemper virus and examination of the avirulent strain utility as an attenuated vaccine.

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.

Measles virus induces cell-type specific changes in gene expression.

Measles virus (MV) causes various responses including the induction of immune responses, transient immunosuppression and establishment of long-lasting immunity. To obtain a comprehensive view of the effects of MV infection on target cells, DNA microarray analyses of two different cell-types were performed. An epithelial (293SLAM; a 293 cell line stably expressing SLAM) and lymphoid (COBL-a) cell line were inoculated with purified wild-type MV. Microarray analyses revealed significant differences in the regulation of cellular gene expression between these two different cells. In 293SLAM cells, upregulation of genes involved in the antiviral response was rapidly induced; in the later stages of infection, this was followed by regulation of many genes across a broad range of functional categories. On the other hand, in COBL-a cells, only a limited set of gene expression profiles was modulated after MV infection. Since it was reported that V protein of MV inhibited the IFN signaling pathway, we performed a microarray analysis using V knockout MV to evaluate V protein's effect on cellular gene expression. The V knockout MV displayed a similar profile to that of parental MV. In particular, in COBL-a cells infected with the virus, no alteration of cellular gene expression, including IFN signaling, was observed. Furthermore, IFN signaling analyzed in vitro was completely suppressed by MV infection in the COBL-a cells. These results reveal that MV induces different cellular responses in a cell-type specific manner. Microarray analyses will provide us useful information about potential mechanisms of MV pathogenesis.

A novel monolayer cell line derived from human umbilical cord blood cells shows high sensitivity to measles virus.

Measles virus (MeV) research is largely dependent on the B95a cell line, which is derived from marmoset B lymphocytes. As this cell line is persistently infected with Epstein-Barr virus (EBV), a novel cell line, COBL-a, was established from human umbilical cord blood. COBL-a cells have a significant advantage over B95a cells because they are of human origin, are free from EBV and have higher sensitivity to wild-type MeV. Thus, COBL-a cells should prove very valuable for both epidemiological and basic studies of MeV.

Host range and receptor utilization of canine distemper virus analyzed by recombinant viruses: Involvement of heparin-like molecule in CDV infection.

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.

Morbillivirus nucleoprotein possesses a novel nuclear localization signal and a CRM1-independent nuclear export signal.

Morbilliviruses, which belong to the Mononegavirales, replicate its RNA genome in the cytoplasm of the host cell. However, they also form characteristic intranuclear inclusion bodies, consisting of nucleoprotein (N), in infected cells. To analyze the mechanisms of nucleocytoplasmic transport of N protein, we characterized the nuclear localization (NLS) and nuclear export (NES) signals of canine distemper virus (CDV) N protein by deletion mutation and alanine substitution of the protein. The NLS has a novel leucine/isoleucine-rich motif (TGILISIL) at positions 70-77, whereas the NES is composed of a leucine-rich motif (LLRSLTLF) at positions 4-11. The NLS and NES of the N proteins of other morbilliviruses, that is, measles virus (MV) and rinderpest virus (RPV), were also analyzed. The NLS of CDV-N protein is conserved at the same position in MV-N protein, whereas the NES of MV-N protein is located in the C-terminal region. The NES of RPV-N protein is also located at the same position as CDV-N protein, whereas the NLS motif is present not only at the same locus as CDV-N protein but also at other sites. Interestingly, the nuclear export of all these N proteins appears to proceed via a CRM1-independent pathway.

Rinderpest virus phosphoprotein gene is a major determinant of species-specific pathogenicity.

We previously demonstrated that the rinderpest virus (RPV) hemagglutinin (H) protein plays an important role in determining host range but that other viral proteins are clearly required for full RPV pathogenicity to be manifest in different species. To examine the effects of the RPV nucleocapsid (N) protein and phosphoprotein (P) genes on RPV cross-species pathogenicity, we constructed two new recombinant viruses in which the H and P or the H, N, and P genes of the cattle-derived RPV RBOK vaccine were replaced with those from the rabbit-adapted RPV-Lv strain, which is highly pathogenic in rabbits. The viruses rescued were designated recombinant RPV-lapPH (rRPV-lapPH) and rRPV-lapNPH, respectively. Rabbits inoculated with RPV-Lv become feverish and show leukopenia and a decrease in body weight gain, while clinical signs of infection are never observed in rabbits inoculated with RPV-RBOK or with rRPV-lapH. However, rabbits inoculated with either rRPV-lapPH or rRPV-lapNPH became pyrexic and showed leukopenia. Further, histopathological lesions and high virus titers were clearly observed in the lymphoid tissues from animals infected with rRPV-lapPH or rRPV-lapNPH, although they were not observed in rabbits infected with RPV-RBOK or rRPV-lapH. The clinical, virological, and histopathological signs in rabbits infected with the two new recombinant viruses did not differ significantly; therefore, the RPV P gene was considered to be a key determinant of cross-species pathogenicity.

Identification and transcriptional analysis of the homologues of the herpes simplex virus type 1 UL30 to UL40 genes in the genome of nononcogenic Marek's disease virus serotype 2.

Studies on Marek's disease virus serotype 2 (MDV2) are important for understanding the natural nononcogenic phenotype of MDV. This study reports a 27,535 bp nucleotide sequence of part of the MDV2 genome located in the central unique long (U(L)) region. The analysis revealed 11 complete ORFs with high amino acid sequence identities to the products of other alphaherpesviruses. The MDV2 ORFs were arranged collinearly with the prototype sequence of herpes simplex virus type 1, ranging from the UL30 to UL40 genes. Sequences that were particularly well conserved among alphaherpesviruses were the putative functional domain of the DNA polymerase (UL30) and the ribonucleotide reductase large and small subunits (UL39 and UL40). On the other hand, in contrast to oncogenic MDV1, MDV2 did not contain the conserved proline-repeat region in the UL36 homologue. All the genes identified were confirmed to be transcribed as 3'-coterminal mRNAs and/or unique transcripts in virus-infected cells.