Kenji Motokawa

Comparison of the Amino Acid Sequence and Phylogenetic Analysis of the Peplomer, Integral Membrane and Nucleocapsid Proteins of Feline, Canine and Porcine Coronaviruses

Complete nucleotide sequences were determined by cDNA cloning of peplomer (S), integral membrane (M) and nucleocapsid (N) genes of feline infectious peritonitis virus (FIPV) type I strain KU‐2, UCD1 and Black, and feline enteric coronavirus (FECV) type II strain 79–1683. Only M and N genes were analyzed in strain KU‐2 and strain 79–1683, which still had unknown nucleotide sequences. Deduced amino acid sequences of S, M and N proteins were compared in a total of 7 strains of coronaviruses, which included FIPV type II strain 79–1146, canine coronavirus (CCV) strain Insavc‐1 and transmissible gastroenteritis virus of swine (TGEV) strain Purdue. Comparison of deduced amino acid sequences of M and N proteins revealed that both M and N proteins had an identity of at least 90% between FIPV type I and type II. The phylogenetic tree of the M and N protein‐deduced amino acid sequences showed that FIPV type I and type II form a group with FECV type II, and that these viruses were evolutionarily distant from CCV and TGEV. On the other hand, when the S protein‐deduced amino acid sequences was compared, identity of only about 45% was found between FIPV type I and type II. The phylogenetic tree of the S protein‐deduced amino acid sequences indicated that three strains of FIPV type I form a group, and that it is a very long distance from the FIPV type II, FECV type II, CCV and TGEV groups.

Molecular cloning and sequence determination of the peplomer protein gene of feline infectious peritonitis virus type I

SummarycDNA clones spanning the entire region of the peplomer (S) gene of feline infectious peritonitis virus (FIPV) type I strain KU-2 were obtained and their complete nucleotide sequences were determined. A long open reading frame (ORF) encoding 1464 amino acid residues was found in the gene, which was 12 residues longer than the ORF of the FIPV type II strain 79–1146. The sequences of FIPV type I and mainly FIPV type II were compared. The homologies at the N- (amino acid residues 1–693) and C- (residues 694–1464) terminal halves were 29.8 and 60.7%, respectively. This was much lower than that between FIPV type II and other antigenically related coronaviruses, such as transmissible gastroenteritis virus of swine and canine coronavirus. This supported the serological relatedness of the viruses and confirmed that the peplomer protein of FIPV type I has distinct structural features that differ from those of antigenically related viruses.

Comparative study of the cell tropism of feline immunodeficiency virus isolates of subtypes A, B and D classified on the basis of the env gene V3-V5 sequence.

Feline immunodeficiency virus (FIV) isolates have been classified into subtypes A, B, C and D based on the env gene V3-V5 sequence. The cell tropism of seven new Japanese isolates and a Petaluma (prototype) isolate of FIV, which classified into subtypes A, B and D, for feline lymphoblastoid and feline fibroblastoid cell lines was compared. FeT-1 (CD4+/-, CD8-, AND CD9+2) and Kumi-1 (CD4+2, CD8- and CD9+2) cells were used as the interleukin-2 (IL-2)-dependent feline T-lymphocyte cell lines and FeT-J (CD4+, CD8+/- and CD9+2) and 3201 (CD4+2, CD8+ and CD9-) cells were used as the IL-2- independent feline T-lymphocyte cell lines. The feline fibroblastoid cell lines used were Crandell feline kidney (CrFK) and fewf-4 (both CD4-, CD8- and CD9+2) cells. All FIV isolates replicated in all lymphoblastoid cell lines used. All isolates showed the greatest cytopathogenicity for Kumi-1 cells. All isolates replicated even in the CD9-negative 3201 cells. More isolates caused persistent infection in IL-2-independent cell lines than in IL-2-dependent cell lines. The number of subtype B isolates that established persistent infection was limited, only one of four strains. Only the subtype A isolates replicated in CrFK cells, whereas none of the isolates replicated in fewf-4 cells, which have similar cell surface markers to CrFK cells. The subtype A viruses (CrFK/Petaluma, CrFK/Sendai-1) growing in CrFK cells showed greater cytopathogenicity for lymphoblastoid cell lines than did those (FL-4/Petaluma, Kumi-1/Sendai-1) growing in a lymphoblastoid cell line.

Genetic subtyping and epidemiological study of feline immunodeficiency virus by nested polymerase chain reaction-restriction fragment length polymorphism analysis of the gag gene

Genetic subtyping of feline immunodeficiency virus (FIV) was carried out by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analysis. A 329-base pair fragment in the FIV gag gene was amplified by nested PCR, then digested with restriction enzymes, HindIII, PvuII and BamHI. Using these restriction enzymes, FIV isolates belonging to subtypes A, B and D, which had been classified on the basis of the env gene V3-V5 sequence, could be discriminated. Genetic subtypes of FIV prevalent in Japan were investigated using the gag-nested PCR-RFLP analysis. Of 88 FIV-infected cats, PCR products of 70 cats showed a subtype B RFLP pattern (digestion only with PvuII), those of nine cats had a subtype D RFLP pattern (digestion only with BamHI), and those of seven cats had a subtype A RFLP pattern (digestion only with HindIII). The PCR products of the remaining two cats had subtype A and B RFLP patterns (digestion with both HindIII and PvuII). The digestion pattern of the gag-nested PCR-RFLP analysis was unchanged after in vivo passages of the virus. These results suggest that the gag-nested PCR-RFLP analysis is useful as a simple method for FIV genetic subtyping.

Effect of dual-subtype vaccine against feline immunodeficiency virus infection

Abstract Dual-subtype feline immunodeficiency virus (FIV) vaccine, consisting of inaactivated cells infected with subtypes A (Petaluma strain) and D (Shizuoka strain), was developed and tested for its vaccine efficacy against FIV infection in specific pathogen free (SPF) cats. Animals were monitored for proviral DNA by FIV-specific PCR and for FIV-specific antibody profiles by ELISA and virus-neutralization assays. In addition, blood from challenged cats was inoculated into naive SPF cats to confirm the viral status of the vaccinated cats. All cats immunized with Petaluma vaccine alone were protected against homologous Petaluma challenge, but only one of four cats was protected against heterologous Shizuoka challenge. More importantly, all cats immunized with the dual-subtype vaccine were protected against both Petaluma and Shizuoka challenges. These results suggest that a multi-subtype vaccine approach may provide the broad-spectrum immunity necessary for vaccine protection against strains from different subtypes.

Vaccine efficacy of a cell lysate with recombinant baculovirus-expressed feline infectious peritonitis (FIP) virus nucleocapsid protein against progression of FIP.

Abstract The Type II feline infectious peritonitis virus (FIPV) infection of feline macrophages is enhanced by a monoclonal antibody (MAb) to the S protein of FIPV. This antibody-dependent enhancement (ADE) activity increased with the MAb that showed a neutralizing activity with feline kidney cells, suggesting that there was a distinct correlation between ADE activity and the neutralizing activity. The close association between enhancing and neutralizing epitopes is an obstacle to developing a vaccine containing only neutralizing epitopes without enhancing epitopes. In this study, we immunized cats with cell lysate with recombinant baculovirus-expressed N protein of the Type I FIPV strain KU-2 with an adjuvant and investigated its preventive effect on the progression of FIP. Cats immunized with this vaccine produced antibodies against FIPV virion-derived N protein but did not produce virus-neutralizing antibodies. A delayed type hypersensitivity skin response to N protein was observed in these vaccinated cats, showing that cell mediated immunity against the FIPV antigen was induced. When these vaccinated cats were challenged with a high dose of heterologous FIPV, the survival rate was 75% (6/8), while the survival rate in the control group immunized with SF-9 cell-derived antigen was 12.5% (1/8). This study showed that immunization with the cell lysate with baculovirus-expressed N protein was effective in preventing the progression of FIP without inducing ADE of FIPV infection in cats.

CD8+ T cells from feline immunodeficiency virus (FIV) infected cats suppress exogenous FIV replication of their peripheral blood mononuclear cells in vitro

Summary. Feline immunodeficiency virus (FIV) isolates from domestic cats have been classified into five subtypes, designated A, B, C, D and E. Although many FIV-infected cats may have frequent contact with multiple strains of FIV, they usually become infected with a single FIV subtype. In the present study, we demonstrate that peripheral blood mononuclear cells (PBMC) of FIV infected cats were resistant to exogenous FIV (second virus) replication in vitro and that the resistance of these PBMC was mediated by CD8+ T cells. In cats with a low anti-FIV activity of CD8+ T cells, the proviral DNA of the second virus inoculated into PBMC was detected intracellularly, and both the second and the originally infecting strain (original virus) were produced in the culture supernatant. In contrast, in cats with a high anti-FIV activity of CD8+ T cells, both the proviral DNA of the second virus and the original virus were detected in PBMC intracellularly, but neither virus was produced in the culture supernatant. However, when PBMCs from these cats were depleted of CD8+ T cells, the RNA of both viruses was detected in the culture supernatant. These results suggest that CD8+ T cells inhibit the late phase of FIV replication after viral integration. Moreover, the inhibition was also effective against FIV strains of different subtypes from that of the original strain. It appears that the CD8+ T cell-mediated immune response plays important roles in the maintenance of an asymptomatic state in FIV-infected cats and their resistance to superinfection.

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.

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.

Characterization of T helper (Th)1- and Th2-type immune responses caused by baculovirus-expressed protein derived from the S2 domain of feline infectious peritonitis virus, and exploration of the Th1 and Th2 epitopes in a mouse model

Feline infectious peritonitis virus (FIPV) may cause a lethal infection in cats. Antibody‐dependent enhancement (ADE) of FIPV infection has been recognized, and cellular immunity is considered to play an important role in preventing the onset of feline infectious peritonitis. In the present study, whether or not the T helper (Th)1 epitope was present in the spike (S)2 domain was investigated, the ADE epitope being thought to be absent from this domain. Three kinds of protein derived from the C‐terminal S2 domain of S protein of the FIPV KU‐2 strain were developed using a baculovirus expression system. These expressed proteins were the pre‐coil region which is the N‐terminal side of the putative fusion protein (FP), the region from FP to the heptad repeat (HR)2 (FP‐HR2) region, and the inter‐helical region which is sandwiched between HR1 and HR2. The ability of three baculovirus‐expressed proteins to induce Th1‐ and Th2‐type immune responses was investigated in a mouse model. It was shown that FP‐HR2 protein induced marked Th1‐ and Th2‐type immune responses. Furthermore, 30 peptides derived from the FP‐HR2 region were synthesized. Five and 16 peptides which included the Th1 and Th2 epitopes, respectively, were identified. Of these, four peptides which included both Th1 and Th2 epitopes were identified. These findings suggest that the identification of Th1 epitopes in the S2 domain of FIPV has important implications in the cat.