Tomoyoshi Doki

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.

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.

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.

Generation, characterization and therapeutic potential of anti-feline TNF-alpha MAbs for feline infectious peritonitis

Abstract Feline infectious peritonitis (FIP) is a lethal infectious disease affecting domestic and wild cats. Several reports suggested that TNF-alpha is related to the progression of FIP. Thus, the administration of a feline TNF-alpha-neutralizing antibody to cats with FIP may reduce the disease progression. In this study, we have prepared nine monoclonal antibodies (MAbs) that recognize feline TNF-alpha. All MAbs neutralized recombinant TNF-alpha. The 50% inhibitory concentrations (IC50) of the MAbs for the cytotoxicity of recombinant TNF-alpha were 5–684ng/ml. MAb 2–4 exhibited high neutralizing activity against natural TNF-alpha derived from FIPV-infected macrophages, and was confirmed to inhibit the following feline TNF-alpha-induced conditions in vitro: (i) an increase in the survival rate of neutrophils from cats with FIP, (ii) aminopeptidase N (APN) mRNA expression in macrophages, and (iii) apoptosis of a feline T-lymphocyte cell line.

Differential effect of cholesterol on type I and II feline coronavirus infection.

Feline infectious peritonitis (FIP) is a fatal disease of domestic and wild felidae that is caused by feline coronavirus (FCoV). FCoV has been classified into types I and II. Since type I FCoV infection is dominant in the field, it is necessary to develop antiviral agents and vaccines against type I FCoV infection. However, few studies have been conducted on type I FCoV. Here, we compare the effects of cholesterol on types I and II FCoV infections. When cells were treated methyl-β-cyclodextrin (MβCD) and inoculated with type I FCoV, the infection rate decreased significantly, and the addition of exogenous cholesterol to MβCD-treated cells resulted in the recovery of the infectivity of type I FCoV. Furthermore, exogenous cholesterol increased the infectivity of type I FCoV. In contrast, the addition of MβCD and exogenous cholesterol had little effect on the efficiency of type II FCoV infection. These results strongly suggest that the dependence of infection by types I and II FCoV on cholesterol differs.

Differential effects of viroporin inhibitors against feline infectious peritonitis virus serotypes I and II

Feline infectious peritonitis virus (FIP virus: FIPV), a feline coronavirus of the family Coronaviridae, causes a fatal disease called FIP in wild and domestic cat species. The genome of coronaviruses encodes a hydrophobic transmembrane protein, the envelope (E) protein. The E protein possesses ion channel activity. Viral proteins with ion channel activity are collectively termed “viroporins”. Hexamethylene amiloride (HMA), a viroporin inhibitor, can inhibit the ion channel activity of the E protein and replication of several coronaviruses. However, it is not clear whether HMA and other viroporin inhibitors affect replication of FIPV. We examined the effect of HMA and other viroporin inhibitors (DIDS [4,4′-disothiocyano-2,2′-stilbenedisulphonic acid] and amantadine) on infection by FIPV serotypes I and II. HMA treatment drastically decreased the titers of FIPV serotype I strains Black and KU-2 in a dose-dependent manner, but it only slightly decreased the titer of FIPV serotype II strain 79-1146. In contrast, DIDS treatment decreased the titer of FIPV serotype II strain 79-1146 in dose-dependent manner, but it only slightly decreased the titers of FIPV serotype I strains Black and KU-2. We investigated whether there is a difference in ion channel activity of the E protein between viral serotypes using E. coli cells expressing the E protein of FIPV serotypes I and II. No difference was observed, suggesting that a viroporin other than the E protein influences the differences in the actions of HMA and DIDS on FIPV serotypes I and II.

Evaluation of protective efficacy of the synthetic peptide vaccine containing the T-helper 1 epitope with CpG oligodeoxynucleotide against feline infectious peritonitis virus infection in cats.

BACKGROUND Feline infectious peritonitis (FIP) is a feline coronavirus-induced fatal disease in domestic and wild cats. Cellular immunity is considered to play an important role in the prevention of FIP. Thus, induction of the cellular immune response is essential in vaccines against FIP virus (FIPV) infection. METHODS We immunized cats with peptides containing T-helper (Th)1 epitopes derived from the nucleocapsid (N) protein of the type I FIPV KU-2 strain (NP7 and NP8) with feline CpG-oligodeoxynucleotides (fCpG-ODNs) as a vaccine adjuvant. RESULTS Prevention against type II FIPV 79-1146 strain-induced FIP was slightly better in specific pathogen-free cats treated with NP7 and NP8 with fCpG-ODNs. However, immune tolerance was suggested to be induced by the high dose and frequency of NP7 and NP8 with fCpG-ODNs. CONCLUSIONS Further investigations on the combination and concentrations of the peptides and fCpG-ODNs, dose, frequency and route of administration are needed.

Therapeutic effect of anti-feline TNF-alpha monoclonal antibody for feline infectious peritonitis.

Abstract Feline infectious peritonitis virus (FIPV) replication in macrophages/monocytes induced tumor necrosis factor (TNF)-alpha production, and that the TNF-alpha produced was involved in aggravating the pathology of FIP. We previously reported the preparation of a feline TNF-alpha (fTNF-alpha)-neutralizing mouse monoclonal antibody (anti-fTNF-alpha mAb). This anti-fTNF-alpha mAb 2–4 was confirmed to inhibit the following fTNF-alpha-induced conditions in vitro. In the present study, we investigated whether mAb 2–4 improved the FIP symptoms and survival rate of experimentally FIPV-inoculated SPF cats. Progression to FIP was prevented in 2 out of 3 cats treated with mAb 2–4, whereas all 3 cats developed FIP in the placebo control group. Plasma alpha1-glycoprotein and vascular endothelial growth factor levels were improved by the administration of mAb 2–4, and the peripheral lymphocyte count also recovered. These results strongly suggested that the anti-fTNF-alpha antibody is effective for the treatment of FIP.

Identification of the peptide derived from S1 domain that inhibits type I and type II feline infectious peritonitis virus infection

Abstract Feline infectious peritonitis virus (FIPV) can cause a lethal disease in cats, feline infectious peritonitis (FIP). A therapeutic drug that is effective against FIP has not yet been developed. Peptides based on viral protein amino acid sequences have recently been attracting attention as new antiviral drugs. In the present study, we synthesized 30 overlapping peptides based on the amino acid sequence of the S1 domain of the type I FIPV strain KU-2 S protein, and investigated their inhibitory effects on FIPV infection. To evaluate the inhibitory effects on type I FIPV infection of these peptides, we investigated a method to increase the infection efficiency of poorly replicative type I FIPV. The efficiency of type I FIPV infection was increased by diluting the virus with medium containing a polycation. Of the 30 peptides, I-S1-8 (S461-S480), I-S1-9 (S471-S490), I-S1-10 (S481-S500), I-S1-16 (S541-S560), and I-S1-22 (S601-S620) significantly decreased the infectivity of FIPV strain KU-2 while I-S1-9 and I-S1-16 exhibited marked inhibitory effects on FIPV infection. The inhibitory effects on FIPV infection of these 2 peptides on other type I and type II FIPV strains, feline herpesvirus (FHV), and feline calicivirus (FCV) were also examined. These 2 peptides specifically inhibited type I and type II FIPV, but did FHV or FCV infection. In conclusion, the possibility of peptides derived from the S protein of type I FIPV strain KU-2 as anti-FIPV agents effective not only for type I, but also type II FIPV was demonstrated in vitro.

Use of recombinant nucleocapsid proteins for serological diagnosis of Feline coronavirus infection by three immunochromatographic tests.

Abstract Three types of immunochromatographic assays (ICAs) were designed to detect anti-feline coronavirus (FCoV) antibodies. Recombinant FCoV nucleocapsid protein (rNP) was used as a conjugate or test line in all 3 ICA kits (CJIgG/TNP, CJNP/TNP, and CJNP/TPA). All three ICA kits were capable of detecting anti-FCoV antibodies; however, non-specific positive reactions of anti-FCoV antibody-negative plasma samples with the test line were observed in 2 ICA kits (CJIgG/TNP and CJNP/TNP), in which rNP was used as the test line. On the other hand, the specific detection of anti-FCoV antibodies was possible in all plasma, serum, whole blood, and ascitic fluid samples using the ICA kit with protein A blotted as the test line (CJNP/TPA). In addition, the specificity and sensitivity of ICA (CJNP/TPA) were equivalent to those of the reference ELISA. The development of simple antibody test methods using the principle of ICA (CJNP/TPA) for other coronavirus and feline viral infections is expected in the future.