Tsuyoshi Gemma

Molecular and phylogenetic analyses of the haemagglutinin (H) proteins of field isolates of canine distemper virus from naturally infected dogs.

We isolated three strains of canine distemper virus (CDV)--the Ueno, Hamamatsu, and Yanaka strains--from dogs in Japan and analysed the molecular properties of their haemagglutinin (H) proteins. Immunoprecipitation of all three strains with a monoclonal antibody revealed H proteins with molecular masses of 84 kDa, which differs from the molecular mass (78 kDa) of the H protein of the Onderstepoort vaccine strain. However, after tunicamycin treatment immunoprecipitation identified H proteins of identical molecular mass (68 kDa) for all three field isolates and the vaccine strain. Sequence analysis showed nine potential sites for asparagine-linked glycosylation in the H proteins of the new isolates, in contrast to four in the H protein of the Onderstepoort strain. Thus, variation in glycosylation of the H proteins of the isolates and the vaccine strain may cause differences in antigenicity of the viruses. Sequences of the H genes showed that the new Japanese isolates have 99% identity with each other, 95% with other European and American isolates (from seals, a German dog, a ferret and large felids) and 90% with the vaccine strain. Phylogenetically, the new Japanese isolates form one cluster which is separate from recent European or American isolates, all of which are distinct from vaccine strains.

Immunohistochemical analysis of the lymphoid organs of dogs naturally infected with canine distemper virus

The pathogenesis of acute canine distemper in three naturally infected dogs was investigated. The lymphoid organs showed atrophy without secondary follicles. The distribution of canine distemper virus (CDV) antigens was examined immunohistochemically with monoclonal antibodies specific for canine Thy-1, immunoglobulin (Ig) M, CD4, CD8, CD21 and CD45RB, and anti-measles virus nucleocapsid protein serum. The viral antigens were located in the T-cell-dependent areas and in the follicles of lymphoid organs; they were observed mainly in the Thy-1, or CD4-positive cells, but also in the CD8-, CD21-, or IgM-positive cells. The results indicated that Thy-1-positive and CD4-positive T cells serve as major target cells for CDV during the acute stage of infection.

Histopathological features of canine distemper recently observed in Japan

Eleven dogs with canine distemper (CD) from the Chubu region of Japan and the Tokyo area were examined. Clinically, respiratory and neurological signs were present in all animals. Histopathologically, all showed characteristic CD lesions of bronchopneumonia and demyelinating encephalitis. However, some differences in gastrointestinal abnormalities were observed. Three out of four dogs from the Chubu region had severe diarrhoea and gastroenteritis, associated with numerous eosinophilic inclusion bodies in the mucosal epithelia. The remaining dog from this area showed vomiting, but not diarrhoea, and also had a number of intraepithelial inclusion bodies in the gastric and intestinal mucosa. In contrast, the seven dogs from the Tokyo area showed neither gastrointestinal symptoms nor intraepithelial inclusions in the stomach or intestine. Immunohistochemical examination for CD virus antigen, however, revealed that these seven dogs had immunoreactive products in the mucosal epithelia, suggesting that the epithelial cells had either a low level of infection with CD virus or were infected with a less cytopathogenic virus. These findings suggest that the dogs in this study were probably affected by two distinct types of CD, in terms of epitheliotropism and cytopathogenic effects on the gastrointestinal tissues.

Expression and identification of the feline herpesvirus type 1 glycoprotein B (gp143/108)

The gene for feline herpesvirus type 1 (FHV-1) glycoprotein B (gB) has been cloned into an expression vector, pRVSVneo, containing the long terminal repeat of Rous sarcoma virus and polyadenylation signal of SV40. This expression vector containing FHV-1 gB gene, pRVSVgBneo, was transfected into Crandell feline kidney (CRFK) cells which are susceptible to FHV-1 infection. By indirect immunofluorescence analysis, the expressed gB was recognized with a panel of monoclonal antibodies (MAbs) against FHV-1 gp143/108. Immunoprecipitation analysis using a MAb 34H12 showed that molecular weights of the gB were 143 and 108 kDa under non-denaturing conditions that 108, 70, 64, and 58 kDa under denaturing conditions. The molecular weights were similar to those of the gB expressed in FHV-1-infected CRFK cells. In addition, when plasmid DNAs were injected into mice to obtain gB-monospecific serum, the pooled serum from mice inoculated with pRVSVgBneo, but not with pRVSVgDneo or pRVSVneo, recognized the FHV-1 gB polypeptides.

Screening and identification of T helper 1 and linear immunodominant antibody-binding epitopes in the spike 2 domain and the nucleocapsid protein of feline infectious peritonitis virus

Abstract The antibody-dependent enhancement (ADE) of feline infectious peritonitis virus (FIPV) infection has been recognized in experimentally infected cats, and cellular immunity is considered to play an important role in preventing the onset of feline infectious peritonitis (FIP). In the present study, we synthesized eighty-one kinds of peptides derived from the spike (S)2 domain of type I FIPV KU-2 strain, the S2 domain of type II FIPV 79-1146 strain, and the nucleocapcid (N) protein of FIPV KU-2 strain. To detect the T helper (Th)1 epitope, peripheral blood mononuclear cells (PBMCs) obtained from FIPV-infected cats were cultured with each peptide, and Th1-type immune responses were measured using feline interferon (fIFN)-γ production as an index. To detect the linear immunodominant antibody-binding epitope, we investigated the reactivity of plasma collected from FIPV-infected cats against each peptide by ELISA. Four and 2 peptides containing Th1 epitopes were identified in the heptad repeat (HR)1 and inter-helical (IH) regions of the S2 domain of type I FIPV, respectively, and these were located on the N-terminal side of the regions. In the S2 domain of type II FIPV, 2, 3, and 2 peptides containing Th1 epitopes were identified in the HR1, IH, and HR2 regions, respectively, and these were mainly located on the C-terminal side of the regions. In the S2 domain of type I FIPV, 3 and 7 peptides containing linear immunodominant antibody-binding epitopes were identified in the IH and HR2 regions, respectively. In the S2 domain of type II FIPV, 4 peptides containing linear immunodominant antibody-binding epitopes were identified in the HR2 region. The Th1 epitopes in the S2 domain of type I and II FIPV were located in different regions, but the linear immunodominant antibody-binding epitopes were mostly located in the HR2 region. Eight peptides containing Th1 epitopes were identified in N protein, and 3 peptides derived from residues 81 to 100 and 137 to 164 showed strong inductivity of fIFN-γ production in PBMCs isolated from type I FIPV- and type II FIPV-infected non-FIP cats. In N protein, 4 peptides containing linear immunodominant antibody-binding epitopes were identified, and 2 peptides derived from residues 345 to 372 showed strong reactivity with plasma of type I FIPV- and type II FIPV-infected cats. The Th1 and linear immunodominant antibody-binding epitopes were located at different positions in both the S2 domain and N protein. Our results may provide important information for the development of peptide-based vaccine against FIPV infection.

The biological characterization of field isolates of canine distemper virus from Japan

Eight isolates of canine distemper virus (CDV) were obtained from seven dogs suffering from distemper by co-cultivation of their mononuclear cells with a marmoset B lymphoblastoid cell line, B95a. Six of the seven dogs had received one or more vaccinations. All of the isolates readily proliferated in B95a cells, but were not completely neutralized by anti-CDV canine plasma, which had high neutralizing activity against the Onderstepoort laboratory strain of CDV. Furthermore, different reactivities of monoclonal antibodies (MAbs) against CDV were observed between the field isolates and laboratory or vaccine strains of CDV in immunofluorescence studies. Immunoprecipitation analysis using MAbs detected the haemagglutinin protein of each new field isolate as 69K, 75K and 155K forms, and the fusion protein as 64K and 65K forms; the corresponding proteins of the Onderstepoort strain were detected as 75K and 61K proteins respectively. It is apparent from these results that the new field isolates of CDV have very different antigenic properties from the Onderstepoort vaccine strain.

Identification of cytosine-phosphorothioate-guanine oligodeoxynucleotide sequences that induce interferon-γ production in feline immune cells

Unmethylated CpG‐ODN are known to enhance Th1‐type immune response. However, optimal sequences of CpG‐ODN for activating Th1‐type immune cells vary among species. It is necessary to identify the effective CpG‐ODN sequences in each species. In the present study, in order to identify the sequences of CpG‐ODN that produce fIFN‐γ in cats, 14 kinds of ODN were synthesized and examined regarding their ability to induce fIFN‐γ in feline PBMC and splenocytes. It was shown that some CpG‐ODN significantly induced fIFN‐γ production in splenocytes, but not in PBMC. We found that three kinds of CpG‐ODN (no. 2, 5′‐ggTGCATCGATGCAGggggG‐3′; no. 5, 5′‐ggTGCGTCGACGCAGggggG‐3′; no. 10, 5′‐ggTGCTACGTAGCAGggggG‐3′) specifically and significantly induced fIFN‐γ production in feline splenocytes. The reverse sequences, GpC‐ODN, do not cause significant fIFN‐γ production. The fIFN‐γ production inductivity of a mixture of CpG‐ODN nos. 2, 5 and 10 was higher than those of individual CpG‐ODN. When the CpG‐ODN mixture was encapsulated in an MCL and administrated to cats, the number of fIFN‐γ+ cells in PBMC significantly increased. CpG‐ODN nos. 2, 5 and 10 should be useful to elicit a Th1‐type immune response as a vaccine adjuvant in cats.