Hajime Kusuhara

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.

Molecular characterization and pathogenicity of a genogroup GVI feline norovirus

Abstract Norovirus (NoV) has been classified into 6 genogroups, GI-GVI. In the present study, we identified novel feline NoV (FNoV) M49-1 strain. The C-terminal of RNA-dependent RNA polymerase of the FNoV M49-1 strain was highly homologous with GIV FNoV and GIV lion norovirus, whereas VP1 was highly homologous with GVI canine NoV (CNoV). Based on the results of the Simplot analysis, the FNoV M49-1 strain may have been produced by recombination between GIV.2 FNoV and GVI.1 CNoV. In addition, specific pathogen-free cats inoculated with FNoV gene-positive-fecal samples developed diarrhea symptoms, and the viral gene was detected in their feces and blood.

Ability of CD8+ T Cell Anti-Feline Immunodeficiency Virus Activity Correlated with Peripheral CD4+ T Cell Counts and Plasma Viremia

In the host defense mechanism against feline immunodeficiency virus (FIV) infection, CD8+ T cells specifically attack virus‐infected cells and suppress the replication of the virus in a non‐cytolytic manner by secreting soluble factors. In this study, we measured CD8+ T cell anti‐FIV activity in 30 FIV‐infected cats. We investigated its relationship with the number of peripheral blood lymphocytes, particularly the CD4+ T cell and CD8+ T cell counts, and the relationship between anti‐FIV activity and the number of T cells of CD8α+βlo and CD8α+β− phenotypes. A clearly significant correlation was observed between anti‐FIV activity and the number of CD4+ T cells. A weaker anti‐FIV activity was associated with a greater decrease in the number of CD4+ T cells. However, there was no significant correlation between anti‐FIV activity and the number of B or CD8+ T cells. Compared with SPF cats, FIV‐infected cats had significantly higher CD8α+βlo T cell and CD8α+β− T cell counts, but, no significant correlation was observed between these cell counts and anti‐FIV activity. This anti‐FIV activity significantly correlated with plasma viremia, which was detected in cats with a weak anti‐FIV activity. These results suggest that the anti‐FIV activity of CD8+ T cells plays an important role in plasma viremia and the maintenance of CD4+ T cells in the body. It is unlikely that CD8α+βlo or CD8α+β− T cells appearing after FIV infection represent a phenotype of CD8+ cells with anti‐FIV activity.

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.

Viral shedding and clinical status of feline-norovirus-infected cats after reinfection with the same strain

Norovirus (NoV) infection is the most common cause of acute gastroenteritis in humans of all ages worldwide. When cats are experimentally infected with feline norovirus (FNoV), they develop symptoms of acute gastroenteritis. Therefore, FNoV infection may serve as an animal model for the disease caused by human norovirus infection. In this study, we examined whether FNoV of cats infected with genogroup GVI are protected from reinfection with the same strain. The blood anti-FNoV IgG level was inversely correlated with the viral load in stool samples and the clinical score of FNoV-infected cats, but complete prevention of reinfection was not observed. These findings were similar to the results of a reinfection experiment with NoV in human volunteers.