Tomomi Takano

A "possible" involvement of TNF-alpha in apoptosis induction in peripheral blood lymphocytes of cats with feline infectious peritonitis.

Abstract Feline infectious peritonitis (FIP) cats show a decrease in peripheral blood lymphocyte counts, and a particularly marked decrease in T cells including CD4+ and CD8+ cells. In this study, we showed that lymphopenia observed in FIP cats was due to apoptosis, and that the ascitic fluid, plasma, and culture supernatant of peritoneal exudate cells (adherent cells with macrophage morphology, or PEC) from FIP cats readily induced apoptosis in specific pathogen-free cat peripheral blood mononuclear cells, particularly CD8+ cells. In addition, TNF-alpha released from macrophages and TNF-receptor (TNFR) 1 and TNFR2 mRNA expression in lymphocytes were closely involved in this apoptosis induction. In particular, in CD8+ cells cultured in the presence of the PEC culture supernatant, the expression levels of TNFR1 and TNFR2 mRNA were increased, indicating that CD8+ cells are more susceptible to apoptosis induction by TNF-alpha than other lymphocyte subsets, particularly B cells (CD21+ cells). The results of this study suggest that TNF-alpha, produced by virus-infected macrophages, is responsible for induction of apoptosis in uninfected T cells, primarily CD8+ T cells.

TNF-alpha, produced by feline infectious peritonitis virus (FIPV)-infected macrophages, upregulates expression of type II FIPV receptor feline aminopeptidase N in feline macrophages

Abstract The pathogenicity of feline infectious peritonitis virus (FIPV) is known to depend on macrophage tropism, and this macrophage infection is enhanced by mediation via anti-S antibody (antibody-dependent enhancement, ADE). In this study, we found that TNF-alpha production was increased with viral replication in macrophages inoculated with a mixture of FIPV and anti-S antibody, and demonstrated that this culture supernatant had feline PBMC apoptosis-inducing activity. We also demonstrated that the expression level of the FIPV virus receptor, feline aminopeptidase N (fAPN), was increased in macrophages of FIP cats. For upregulation of TNF-alpha and fAPN in macrophages, viral replication in macrophages is necessary, and their expressions were increased by ADE of FIPV infection. It was demonstrated that a heat-resistant fAPN-inducing factor was present in the culture supernatant of FIPV-infected macrophages, and this factor was TNF-alpha: fAPN expression was upregulated in recombinant feline TNF-alpha-treated macrophages, and FIPV infectivity was increased in these macrophages. These findings suggested that FIPV replication in macrophages increases TNF-alpha production in macrophages, and the produced TNF-alpha acts and upregulates fAPN expression, increasing FIPV sensitivity.

Neutrophil survival factors (TNF-alpha, GM-CSF, and G-CSF) produced by macrophages in cats infected with feline infectious peritonitis virus contribute to the pathogenesis of granulomatous lesions.

Feline infectious peritonitis (FIP) is a feline coronavirus (FCoV)-induced fatal disease of domestic and wild cats. The infiltration of neutrophils into granulomatous lesions is unusual for a viral disease, but it is a typical finding of FIP. This study aimed to investigate the reason for the lesions containing neutrophils in cats with FIP. Neutrophils of cats with FIP were cultured, and changes in the cell survival rate were assessed. In addition, the presence or absence of neutrophil survival factors was investigated in specimens collected from cats with FIP. Furthermore, it was investigated whether macrophages, one of the target cells of FIPV infection, produce neutrophil survival factors (TNF-alpha, GM-CSF, and G-CSF). We showed that virus-infected macrophages overproduce neutrophil survival factors, and these factors act on neutrophils and up-regulate their survival. These observations suggest that sustained production of neutrophil survival factors by macrophages during FCoV infection is sufficient for neutrophil survival and contributes to development of granulomatous lesions.

Analysis of the mechanism of antibody-dependent enhancement of feline infectious peritonitis virus infection: aminopeptidase N is not important and a process of acidification of the endosome is necessary.

Infection of the monocyte/macrophage lineage with feline infectious peritonitis virus (FIPV) is enhanced in the presence of anti-FIPV antibodies (antibody-dependent enhancement or ADE). We investigated the following unclear points concerning ADE of FIPV infection: (i) involvement of the virus receptor, feline aminopeptidase N (fAPN), in ADE activity in FIPV infection; (ii) necessity of acidification of the endosome in cellular invasion of FIPV. Virus receptor-blocking experiments using anti-fAPN antibodies at 4 or 37 degrees C and experiments using fAPN-negative U937 cells revealed that fAPN is not involved in ADE of FIPV infection. Experiments using lysosomotropic agents clarified that acidification of the endosome is necessary for cellular invasion by FIPV, regardless of the presence or absence of antibodies. These findings may be very important for understanding the mechanism of ADE of FIPV infection.

Effect of chloroquine on feline infectious peritonitis virus infection in vitro and in vivo.

Abstract Feline infectious peritonitis (FIP) is a feline coronavirus-induced fatal disease in domestic and wild cats. Several studies have investigated potential treatments for FIP. However, there have been no reports on agents that have exhibited a therapeutic effect. Recently, chloroquine has been reported to antiviral effect. We investigated whether chloroquine can be used to treat FIP in vitro and in vivo. It was demonstrated that chloroquine has inhibitory effect against the replication of FIPV and anti-inflammatory effect in vitro. In vivo study using cats with experimentally induced FIP, the clinical score of chloroquine-treatment groups were better than in chloroquine-untreated group. However, alanine aminotransferase levels increased in the chloroquine-treated groups. It will be necessary to further investigate the possibility of FIP treatment with a combination of chloroquine and other agents.

B-cell activation in cats with feline infectious peritonitis (FIP) by FIP-virus-induced B-cell differentiation/survival factors

It has been suggested that antibody overproduction plays a role in the pathogenesis of feline infectious peritonitis (FIP). However, only a few studies on the B-cell activation mechanism after FIP virus (FIPV) infection have been reported. The present study shows that: (1) the ratio of peripheral blood sIg+ CD21− B-cells was higher in cats with FIP than in SPF cats, (2) the albumin-to-globulin ratio has negative correlation with the ratio of peripheral blood sIg+ CD21− B-cell, (3) cells strongly expressing mRNA of the plasma cell master gene, B-lymphocyte-induced maturation protein 1 (Blimp-1), were increased in peripheral blood in cats with FIP, (4) mRNA expression of B-cell differentiation/survival factors, IL-6, CD40 ligand, and B-cell-activating factor belonging to the tumor necrosis factor family (BAFF), was enhanced in macrophages in cats with FIP, and (5) mRNAs of these B-cell differentiation/survival factors were overexpressed in antibody-dependent enhancement (ADE)-induced macrophages. These data suggest that virus-infected macrophages overproduce B-cell differentiation/survival factors, and these factors act on B-cells and promote B-cell differentiation into plasma cells in FIPV-infected cats.

Mutation of neutralizing/antibody-dependent enhancing epitope on spike protein and 7b gene of feline infectious peritonitis virus: influences of viral replication in monocytes/macrophages and virulence in cats.

Abstract We previously prepared neutralizing monoclonal antibody (MAb)-resistant (mar) mutant viruses using a laboratory strain feline infectious peritonitis virus (FIPV) 79-1146 (Kida et al., 1999). Mar mutant viruses are mutated several amino acids of the neutralizing epitope of Spike protein, compared with the parent strain, FIPV 79-1146. We clarified that MAb used to prepare mar mutant viruses also lost its activity to enhance homologous mar mutant viruses, strongly suggesting that neutralizing and antibody-dependent enhancing epitopes are present in the same region in the strain FIPV 79-1146. We also discovered that amino acid mutation in the neutralizing epitope reduced viral replication in monocytes/macrophages. We also demonstrated that the mutation or deletion of two nucleotides in 7b gene abrogate the virulence of strain FIPV 79-1146.

Vascular endothelial growth factor (VEGF), produced by feline infectious peritonitis (FIP) virus-infected monocytes and macrophages, induces vascular permeability and effusion in cats with FIP

Abstract Feline infectious peritonitis virus (FIPV) causes a fatal disease called FIP in Felidae. The effusion in body cavity is commonly associated with FIP. However, the exact mechanism of accumulation of effusion remains unclear. We investigated vascular endothelial growth factor (VEGF) to examine the relationship between VEGF levels and the amounts of effusion in cats with FIP. Furthermore, we examined VEGF production in FIPV-infected monocytes/macrophages, and we used feline vascular endothelial cells to examine vascular permeability induced by the culture supernatant of FIPV-infected macrophages. In cats with FIP, the production of effusion was related with increasing plasma VEGF levels. In FIPV-infected monocytes/macrophages, the production of VEGF was associated with proliferation of virus. Furthermore, the culture supernatant of FIPV-infected macrophages induced hyperpermeability of feline vascular endothelial cells. It was suggested that vascular permeability factors, including VEGF, produced by FIPV-infected monocytes/macrophages might increase the vascular permeability and the amounts of effusion in cats with FIP.

Detection of canine astrovirus in dogs with diarrhea in Japan

Canine astrovirus (CAstV) is the causative agent of gastroenteritis in dogs. We collected rectal swabs from dogs with or without diarrhea symptoms in Japan and examined the feces for the presence of CAstV by RT-PCR with primers based on a conserved region of the ORF1b gene. The ORF1b gene of CAstV was not detected in the 42 dogs without clinical illness but was present in three pups out of the 31 dogs with diarrhea symptoms. Based on the full-length capsid protein, the CAstV KU-D4-12 strain that we detected in this study shared high homology with the novel virulent CAstV VM-2011 strain.

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.