Therapeutic effect of an anti-human-TNF-alpha antibody and itraconazole on feline infectious peritonitis.

Feline infectious peritonitis (FIP) is a fatal disease in wild and domestic cat species. Although several drugs are expected to be useful as treatments for FIP, no drugs are available in clinical practice. In this study, we evaluated the therapeutic effect of combined use of adalimumab (an anti-human-TNF-alpha monoclonal antibody, ADA) and itraconazole (ICZ), which are presently available to veterinarians. The neutralizing activity of ADA against fTNF-alpha-induced cytotoxicity was measured in WEHI-164 cells. Ten specific pathogen-free (SPF) cats were inoculated intraperitoneally with type I FIPV KU-2. To the cats that developed FIP, ADA (10 mg/animal) was administered twice between day 0 and day 4 after the start of treatment. ICZ (50 mg/head, SID) was orally administered daily from day 0 after the start of treatment. ADA demonstrated dose-dependent neutralizing activity against rfTNF-alpha. In an animal experiment, 2 of 3 cats showed improvements in FIP clinical symptoms and blood chemistry test results, an increase in the peripheral blood lymphocyte count, and a decrease in the plasma alpha 1-AGP level were observed after the beginning of treatment. One of the cats failed to respond to treatment and was euthanized, although the viral gene level in ascites temporarily decreased after the start of treatment. ADA was found to have neutralizing activity against rfTNF-alpha. The combined use of ADA and ICZ showed a therapeutic effect for experimentally induced FIP. We consider these drugs to be a treatment option until effective anti-FIPV drugs become available.

Antiviral activity of itraconazole against type I feline coronavirus infection.

Feline coronaviruses (FCoVs) are the causative agents of severe systemic disease (feline infectious peritonitis: FIP) in domestic and wild cats. FCoVs have been classified into serotypes I and II. Type I FCoV is the dominant serotype (approximately 70-90%) worldwide. Therefore, it is necessary to provide antiviral agents for type I FCoV infection. In this study, we demonstrated that itraconazole (ICZ), practically used for fungal infections in cats, inhibits the type I FCoV infection. ICZ also exhibited antiviral effect in cells after viral infection, suggesting that ICZ could potentially be used as a therapeutic.

Novel single-stranded, circular DNA virus identified in cats in Japan.

We detected a novel feline stool-associated circular DNA virus (FeSCV) in fecal samples from cats with diarrhea using consensus primers matching those of circovirus and cyclovirus. FeSCV is a circular DNA virus containing a genome with a total length of 2,046 nt encoding 2 open reading frames. Phylogenetic analyses indicated that FeSCV is classified into a clade different from that of circovirus and cyclovirus. Since the FeSCVs detected in several cats in the same household had genetically similar genomes, these viruses are most likely derived from the same origin.

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.

Antibody-dependent enhancement of serotype II feline enteric coronavirus infection in primary feline monocytes.

Feline coronavirus (FCoV) has been classified into two biotypes: avirulent feline coronavirus (feline enteric coronavirus: FECV) and virulent feline coronavirus (feline infectious peritonitis virus: FIPV). In FIPV infection, antibody-dependent enhancement (ADE) has been reported and was shown to be associated with severe clinical disease. On the other hand, the potential role of ADE in FECV infection has not been examined. In this study, using laboratory strains of serotype II FIPV WSU 79-1146 (FIPV 79-1146) and serotype II FECV WSU 79-1683 (FECV 79-1683), we investigated the relationship between ADE and induction of inflammatory cytokines, which are pathogenesis-related factors, for each strain. As with ADE of FIPV 79-1146 infection, a monoclonal antibody against the spike protein of FCoV (mAb 6-4-2) enhanced FECV 79-1683 replication in U937 cells and primary feline monocytes. However, the ADE activity of FECV 79-1683 was lower than that of FIPV 79-1146. Moreover, mRNA levels of inflammatory cytokines (TNF-α, IL-1ß, and IL-6) significantly increased with ADE of FIPV 79-1146 infection in primary feline monocytes, but FECV 79-1683 did not demonstrate an increase in these levels. In conclusion, infection of monocytes by FECV was enhanced by antibodies, but the efficiency of infection was lower than that of FIPV.

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-ß-cyclodextrin (MßCD) and inoculated with type I FCoV, the infection rate decreased significantly, and the addition of exogenous cholesterol to MßCD-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 MßCD 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.

Genetic characterization of feline bocavirus detected in cats in Japan.

Feline bocavirus (FBoV) has been classified into three genotypes (FBoV1-FBoV3). FBoVs are mainly detected in feces. In the present study, we collected rectal swabs from cats in Japan and examined the samples for the presence of FBoV. The FBoV infection rate was 9.9 % in 101 cats. No significant association was observed between FBoV infection and clinical symptoms. Based on the full-length NS1 protein, the three strains of FBoVs detected in the present study shared high homologies with the genotype 2 FBoV POR1 strain. This is the first study to report FBoV in Japan.

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.

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.

Both Feline Coronavirus Serotypes 1 and 2 Infected Domestic Cats Develop Cross-Reactive Antibodies to SARS-CoV-2 Receptor Binding Domain: Its Implication to Pan-CoV Vaccine Development.

The current study was initiated when our specific-pathogen-free laboratory toms developed unexpectedly high levels of cross-reactive antibodies to human SARS-CoV-2 (SCoV2) receptor binding domain (RBD) upon mating with feline coronavirus (FCoV)-positive queens. Multi-sequence alignment analyses of SCoV2 Wuhan RBD and four strains each from FCoV serotypes 1 and 2 (FCoV1 and FCoV2) demonstrated an amino acid sequence identity of 11.5% and a similarity of 31.8% with FCoV1 RBD (12.2% identity and 36.5% similarity for FCoV2 RBD). The sera from toms and queens cross-reacted with SCoV2 RBD and reacted with FCoV1 RBD and FCoV2 spike-2, nucleocapsid, and membrane proteins, but not with FCoV2 RBD. Thus, the queens and toms were infected with FCoV1. Additionally, the plasma from six FCoV2-inoculated cats reacted with FCoV2 and SCoV2 RBDs, but not with FCoV1 RBD. Hence, the sera from both FCoV1-infected cats and FCoV2-infected cats developed cross-reactive antibodies to SCoV2 RBD. Furthermore, eight group-housed laboratory cats had a range of serum cross-reactivity to SCoV2 RBD even 15 months later. Such cross-reactivity was also observed in FCoV1-positive group-housed pet cats. The SCoV2 RBD at a high non-toxic dose and FCoV2 RBD at a 60-400-fold lower dose blocked the in vitro FCoV2 infection, demonstrating their close structural conformations essential as vaccine immunogens. Remarkably, such cross-reactivity was also detected by the peripheral blood mononuclear cells of FCoV1-infected cats. The broad cross-reactivity between human and feline RBDs provides essential insights into developing a pan-CoV vaccine.

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.

In Vivo Antiviral Effects of U18666A Against Type I Feline Infectious Peritonitis Virus.

Background: The cationic amphiphilic drug U18666A inhibits the proliferation of type I FIPV in vitro. In this study, we evaluated the in vivo antiviral effects of U18666A by administering it to SPF cats challenged with type I FIPV. Methods: Ten SPF cats were randomly assigned to two experimental groups. FIPV KU-2 were inoculated intraperitoneally to cats. The control group was administered PBS, and the U18666A-treated group was administered U18666A subcutaneously at 2.5 mg/kg on day 0, and 1.25 mg/kg on days 2 and 4 after viral inoculation. Results: Two of the five control cats administered PBS alone developed FIP. Four of the five cats administered U18666A developed no signs of FIP. One cat that temporarily developed fever, had no other clinical symptoms, and no gross lesion was noted on an autopsy after the end of the experiment. The FIPV gene was detected intermittently in feces and saliva regardless of the development of FIP or administration of U18666A. Conclusions: When U18666A was administered to cats experimentally infected with type I FIPV, the development of FIP might be suppressed compared with the control group. However, the number of animals with FIP is too low to establish anti-viral effect of U18666A in cats.

Antiviral Effects of Hydroxychloroquine and Type I Interferon on In Vitro Fatal Feline Coronavirus Infection.

Feline infectious peritonitis (FIP) is a viral disease with a high morbidity and mortality by the FIP virus (FIPV, virulent feline coronavirus). Several antiviral drugs for FIP have been identified, but many of these are expensive and not available in veterinary medicine. Hydroxychloroquine (HCQ) is a drug approved by several countries to treat malaria and immune-mediated diseases in humans, and its antiviral effects on other viral infections (e.g., SARS-CoV-2, dengue virus) have been confirmed. We investigated whether HCQ in association with interferon-ω (IFN-ω) is effective for FIPV in vitro. A total of 100 µM of HCQ significantly inhibited the replication of types I and II FIPV. Interestingly, the combination of 100 µM of HCQ and 104 U/mL of recombinant feline IFN-ω (rfIFN-ω, veterinary registered drug) increased its antiviral activity against type I FIPV infection. Our study suggested that HCQ and rfIFN-ω are applicable for treatment of FIP. Further clinical studies are needed to verify the combination of HCQ and rIFN-ω will be effective and safe treatment for cats with FIP.

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.

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.

Pathogenesis of oral type I feline infectious peritonitis virus (FIPV) infection: Antibody-dependent enhancement infection of cats with type I FIPV via the oral route.

Feline infectious peritonitis virus (FIPV) causes a severe, immune-mediated disease called FIP in domestic and wild cats. It is unclear whether FIP transmits from cat to cat through the oral route of FIPV infection, and the reason for this includes that FIP is caused by oral inoculation with some FIPV strains (e.g., type II FIPV WSU 79-1146), but is not caused by other FIPV (e.g., type I FIPV KU-2 strain: FIPV-I KU-2). In this study, when cats passively immunized with anti-FIPV-I KU-2 antibodies were orally inoculated with FIPV-I KU-2, FIP was caused at a 50% probability, i.e., FIPV not causing FIP through oral infection caused FIP by inducing antibody-dependent enhancement. Many strains of type I FIPV do not cause FIP by inoculation through the oral route in cats. Based on the findings of this study, type I FIPV which orally infected cats may cause FIP depending on the condition.

Antibody-dependent enhancement occurs upon re-infection with the identical serotype virus in feline infectious peritonitis virus infection.

Feline infectious peritonitis virus (FIPV) is classified into serotype I and serotype II according to the amino acid sequence of its spike(S) protein. Antibody dependent enhancement (ADE) of macrophage infection occurs in the presence of antibodies to FIPV S protein, and a close relationship between ADE and neutralizing epitopes has been reported. The importance of differences in FIPV serotype on the induction of ADE remains unclear. In this study, we investigated whether the same or different serotype of FIPV induces ADE in cats. Specific pathogen-free cats were passively immunized with anti-type I or II FIPV antibodies, and we investigated the induction of ADE following subcutaneous inoculation with type I FIPV. Inoculation using FIPV serotype I enhanced the onset of FIP in cats passively immunized with FIPV serotype I-specific antibodies but not in those passively immunized with antibodies to FIPV serotype II. These data suggest that re-infection with the same serotype induces ADE in cats infected with FIPV.

Differential induction of type I interferon by type I and type II feline coronaviruses in vitro.

Feline infectious peritonitis (FIP) is a feline coronavirus (FCoV)-induced fatal disease in wild and domestic cats. There are two FCoV serotypes. Both type I and II FCoV can replicate in Felis catus whole fetus (fcwf)-4 cells, but the replicability of type I FCoV in feline cell lines is lower than that of type II FCoV, the reason for which is unclear. Inhibition of IFNß production by non-structural and structural proteins, excluding spike protein has been reported in many coronavirus infections. In this study, we investigated whether IFNß is involved in the difference in replicability in feline cell lines between types I and II FCoV. When fcwf-4 cells were infected with FCoV, the virus titer of type II FCoV in the culture supernatant was higher than that of type I FIPV. When the IFNß expression level in FCoV-infected fcwf-4 cells was semi-quantitatively analyzed, infection with type I FIPV, excluding type I FIPV UCD-1, highly induced IFNß expression. In contrast, induction of IFNß by type II FCoV infection was significantly lower than that by type I FIPV. In addition, when fcwf-4 cells were adsorbed by FIPV and then stimulated with Poly(I:C), type II FCoV infection inhibited Poly(I:C)-induced IFNß gene expression. Also, the proliferation of type I FIPV was enhanced by a IFN inhibitor. These findings clarified that, unlike type I FIPV, type II FCoV strongly inhibits IFNß expression in infected cells. It was also suggested that the IFNß-inducing ability is different among type I FIPV strains.

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

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.

Serological differentiation of FIV-infected cats from dual-subtype feline immunodeficiency virus vaccine (Fel-O-Vax FIV) inoculated cats.

Feline immunodeficiency virus (FIV) vaccine, Fel-O-Vax FIV, was released for sale in the US in 2002. The antibodies of vaccinated cats interfere with serological assays by currently available FIV diagnostic kits. In this study, we investigated whether it is possible to distinguish serologically cats vaccinated with Fel-O-Vax FIV from cats experimentally or naturally infected with FIV. A total of 153 sera taken from 97 cats were used as serum samples. Enzyme linked immunosorbent assay (ELISA) was performed using whole FIV antigen and formalin treated whole FIV antigen, recombinant-gag (r-gag) antigen, and transmembrane (TM) peptide. Statistical analysis was performed using ELISA optical density (O.D.) values obtained with each antigen as variables. Except for the ELISA O.D. values obtained with r-gag antigen, a significant difference in ELISA O.D. values was observed between the vaccinated and the infected groups. However, it was not possible to distinguish both groups unequivocally. Using discriminant analysis, it was possible to distinguish the two groups with an accuracy of 97.1% with two discriminating variables (ELISA O.D. values obtained with formalin treated whole FIV antigen, and TM peptide), 97.8% with three discriminating variables (ELISA O.D. values obtained with whole FIV antigen, formalin treated whole FIV antigen, and TM peptide). Therefore, it was considered possible to distinguish cats vaccinated with Fel-O-Vax FIV from FIV-infected cats by ELISA using two types of antigens including formalin treated whole FIV antigen and TM peptide, or three types of antigens including formalin treated whole FIV antigen, TM peptide and whole FIV antigen.