Feline Infectious Peritonitis Virus Infection Research Articles

Feline infectious peritonitis virus ORF7a is a virulence factor involved in inflammatory pathology in cats

A hyperinflammatory response is a prominent feature of feline infectious peritonitis (FIP), but the mechanisms behind the feline infectious peritonitis virus (FIPV)-induced cytokine storm in the host have not been clarified. Studies have shown that coronaviruses encode accessory proteins that are involved in viral replication and associated with viral virulence, the inflammatory response and immune regulation. Here, we found that FIPV ORF7a gene plays a key role in viral infection and host proinflammatory responses. The recombinant FIPV strains lacking ORF7a (rQS-79Δ7a) exhibit low replication rates in macrophages and do not induce dramatic upregulation of inflammatory factors. Furthermore, through animal experiments, we found that the rQS-79Δ7a strain is nonpathogenic and do not cause symptoms of FIP in cats. Unexpectedly, after three vaccinations with rQS-79Δ7a strain, humoral and cellular immunity was increased and provided protection against virulent strains in cats, and the protection rate reaches 40%. Importantly, our results demonstrated that ORF7a is a key virulence factor that exacerbates FIPV infection and inflammatory responses. Besides, our findings will provide novel implications for future development of live attenuated FIPV vaccines.

A Combined Method Based on the FIPV N Monoclonal Antibody Immunofluorescence Assay and RT-nPCR Method for the Rapid Diagnosis of FIP-Suspected Ascites

Feline infectious peritonitis (FIP), which is caused by feline infectious peritonitis virus (FIPV), is a fatal and immunologically mediated infectious disease among cats. At present, due to the atypical clinical symptoms and clinicopathological changes, the clinical diagnosis of FIP is still difficult. The gold standard method for the differential diagnosis of FIP is immunohistochemistry (IHC) which is time-consuming and requires specialized personnel and equipment. Therefore, a rapid and accurate clinical diagnostic method for FIPV infection is still urgently needed. In this study, based on the etiological investigation of FIPV in parts of southern China, we attempted to explore a new rapid and highly sensitive method for clinical diagnosis. The results of the etiological investigation showed that the N gene of the FIPV BS8 strain had the highest homology with other strains. Based on this, a specific FIPV BS8 N protein monoclonal antibody was successfully prepared by expression of the recombinant proteins, immunization of mice, fusion and selection of hybridoma cell lines, and screening and purification of monoclonal antibodies. Furthermore, we carried out a time-saving combination method including indirect immunofluorescence assay (IFA) and nested reverse transcription polymerase chain reaction (RT-nPCR) to examine FIP-suspected clinical samples. These results were 100% consistent with IHC. The results revealed that the combined method could be a rapid and accurate application in the diagnosis of suspected FIPV infection within 24 hours. In conclusion, the combination of IFA and RT-nPCR was shown to be a fast and reliable method for clinical FIPV diagnosis. This study will provide insight into the exploitation of FIPV N antibodies for the clinical diagnosis of FIP-suspected ascites samples.

First study on in vitro antiviral and virucidal effects of flavonoids against feline infectious peritonitis virus at the early stage of infection.

Feline infectious peritonitis (FIP), one of the most important infectious diseases in cats is caused by FIP virus (FIPV), a mutated variant of feline coronavirus. Feline infectious peritonitis has a negative impact on feline health, with extremely high mortality in clinical FIP-infected cats, particularly young cats. There are no approved drugs for FIP treatment, and therapeutic possibilities for FIP treatment are limited. This study aimed to utilize nature-derived bioactive flavonoids with antiviral properties to inhibit FIPV infection in Crandell-Rees feline kidney (CRFK) cells. The cytotoxicity of 16 flavonoids was evaluated on CRFK cells using a colorimetric method (MTS) assay. Viral kinetics of FIPV at 50 tissue culture infectious dose (TCID50)/well was determined during the first 24-h post-infection (HPI). Antiviral activity was evaluated based on the replication steps of the virus life cycle, including pre-compound, attachment, penetration, post-viral entry, and virucidal assays. The antiviral efficacy of flavonoids against FIPV was determined based on positive FIPV-infected cells with the immunoperoxidase monolayer assay and viral load quantification using reverse transcription-quantitative polymerase chain reaction. Two flavonoids, namely, isoginkgetin and luteolin, inhibited FIPV replication during post-viral entry in a dose-dependent manner, with 50% maximal effective concentrations = 4.77 ± 0.09 and 36.28 ± 0.03 μM, respectively. Based on viral kinetics, both flavonoids could inhibit FIPV replication at the early stage of infection at 0-6-HPI for isoginkgetin and 2-6-HPI for luteolin using a time-of-addition assay. Isoginkgetin exerted a direct virucidal effect that reduced the viral titers by 2 and 1.89 log10 TCID50/mL at 60 and 120 min, respectively. Isoginkgetin interfered with FIPV replication during both post-viral infection and virucidal experiments on CRFK cells, whereas luteolin inhibited the virus after infection. These results demonstrate the potential of herbal medicine for treating FIP.

Adaptive Mutation in the Main Protease Cleavage Site of Feline Coronavirus Renders the Virus More Resistant to Main Protease Inhibitors.

The rapid global emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused serious health problems, highlighting the urgent need for antiviral drugs. The viral main protease (Mpro) plays an important role in viral replication and thus remains the target of choice for the prevention or treatment of several viral diseases due to high sequence and structural conservation. Prolonged use of viral protease inhibitors can lead to the development of mutants resistant to those inhibitors and to many of the available antiviral drugs. Here, we used feline infectious peritonitis virus (FIPV) as a model to investigate its development of resistance under pressure from the Mpro inhibitor GC376. Passage of wild-type (WT) FIPV in the presence of GC376 selected for a mutation in the nsp12 region where Mpro cleaves the substrate between nsp12 and nsp13. This mutation confers up to 3-fold resistance to GC376 and nirmatrelvir, as determined by EC50 assay. In vitro biochemical and cellular experiments confirmed that FIPV adapts to the stress of GC376 by mutating the nsp12 and nsp13 hydrolysis site to facilitate cleavage by Mpro and release to mediate replication and transcription. Finally, we demonstrate that GC376 cannot treat FIP-resistant mutants that cause FIP in animals. Taken together, these results suggest that Mpro affects the replication of coronaviruses (CoVs) and the drug resistance to GC376 by regulating the amount of RdRp from a distant site. These findings provide further support for the use of an antiviral drug combination as a broad-spectrum therapy to protect against contemporary and emerging CoVs. IMPORTANCE CoVs cause serious human infections, and antiviral drugs are currently approved to treat these infections. The development of protease-targeting therapeutics for CoV infection is hindered by resistance mutations. Therefore, we should pay attention to its resistance to antiviral drugs. Here, we identified possible mutations that lead to relapse after clinical treatment of FIP. One amino acid substitution in the nsp12 polymerase at the Mpro cleavage site provided low-level resistance to GC376 after selection exposure to the GC376 parental nucleoside. Resistance mutations enhanced FIPV viral fitness in vitro and attenuated the therapeutic effect of GC376 in an animal model of FIPV infection. Our research explains the evolutionary characteristics of coronaviruses under antiviral drugs, which is helpful for a more comprehensive understanding of the molecular basis of virus resistance and provides important basic data for the effective prevention and control of CoVs.

Expression of Toll-like receptors 3, 7, 9 and cytokines in feline infectious peritonitis virus-infected CRFK cells and feline peripheral monocytes.

BackgroundThe role of Toll-like receptors (TLRs) in a feline infectious peritonitis virus (FIPV) infection is not completely understood.ObjectivesThis study examined the expression of TLR3, TLR7, TLR9, tumor necrosis factor-alpha (TNF-α), interferon (IFN)-β, and interleukin (IL)-10 upon an FIPV infection in Crandell-Reese feline kidney (CRFK) cells and feline monocytes.MethodsCRFK cells and monocytes from feline coronavirus (FCoV)-seronegative cats and FCoV-seropositive cats were infected with type II FIPV-79-1146. At four, 12, and 24 hours post-infection (hpi), the expression of TLR3, TLR7, TLR9, TNF-α, IFN-β, and IL-10, and the viral load were measured using reverse transcription quantitative polymerase chain reaction. Viral protein production was confirmed using immunofluorescence.ResultsFIPV-infected CRFK showed the upregulation of TLR9, TNF-α, and IFN-β expression between 4 and 24 hpi. Uninfected monocytes from FCoV-seropositive cats showed lower TLR3 and TLR9 expression but higher TLR7 expression compared to uninfected monocytes from FCoV-seronegative cats. FIPV-infected monocytes from FCoV-seropositive cats downregulated TLR7 and TNF-α expression between 4 and 24 hpi, and 4 and 12 hpi, respectively. IFN-β was upregulated early in FIPV-infected monocytes from FCoV-seropositive cats, with a significant difference observed at 12 hpi compared to FCoV-seronegative cats. The viral load in the CRFK and FIPV-infected monocytes in both cohorts of cats was similar over time.ConclusionTLR7 may be the key TLR involved in evading the innate response against inhibiting TNF-α production. Distinct TLR expression profiles between FCoV-seronegative and FCoV-seropositive cats were observed. The associated TLR that plays a role in the induction of IFN-β needs to be explored further.

Structural-based virtual screening and in vitro assays for small molecules inhibiting the feline coronavirus 3CL protease as a surrogate platform for coronaviruses

Feline infectious peritonitis (FIP) which is caused by feline infectious peritonitis virus (FIPV), a variant of feline coronavirus (FCoV), is a member of family Coronaviridae, together with severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and SARS-CoV-2. So far, neither effective vaccines nor approved antiviral therapeutics are currently available for the treatment of FIPV infection. Both human and animal CoVs shares similar functional proteins, particularly the 3CL protease (3CLpro), which plays the pivotal role on viral replication. We investigated the potential drug-liked compounds and their inhibitory interaction on the 3CLpro active sites of CoVs by the structural-bases virtual screening. Fluorescence resonance energy transfer (FRET) assay revealed that three out of twenty-eight compounds could hamper FIPV 3CLpro activities with IC50 of 3.57 ± 0.36 μM to 25.90 ± 1.40 μM, and Ki values of 2.04 ± 0.08 to 15.21 ± 1.76 μM, respectively. Evaluation of antiviral activity using cell-based assay showed that NSC629301 and NSC71097 could strongly inhibit the cytopathic effect and also reduced replication of FIPV in CRFK cells in all examined conditions with the low range of EC50 (6.11 ± 1.90 to 7.75 ± 0.48 μM and 1.99 ± 0.30 to 4.03 ± 0.60 μM, respectively), less than those of ribavirin and lopinavir. Analysis of FIPV 3CLpro-ligand interaction demonstrated that the selected compounds reacted to the crucial residues (His41 and Cys144) of catalytic dyad. Our investigations provide a fundamental knowledge for the further development of antiviral agents and increase the number of anti-CoV agent pools for feline coronavirus and other related CoVs.

In Vitro Evaluation of Curcumin-Encapsulated Chitosan Nanoparticles against Feline Infectious Peritonitis Virus and Pharmacokinetics Study in Cats.

Feline infectious peritonitis (FIP) is an important feline viral disease, causing an overridden inflammatory response that results in a high mortality rate, primarily in young cats. Curcumin is notable for its biological activities against various viral diseases; however, its poor bioavailability has hindered its potential in therapeutic application. In this study, curcumin was encapsulated in chitosan nanoparticles to improve its bioavailability. Curcumin-encapsulated chitosan (Cur-CS) nanoparticles were synthesised based on the ionic gelation technique and were spherical and cuboidal in shape, with an average particle size of 330 nm and +42 mV in zeta potential. The nanoparticles exerted lower toxicity in Crandell-Rees feline kidney (CrFK) cells and enhanced antiviral activities with a selective index (SI) value three times higher than that of curcumin. Feline-specific bead-based multiplex immunoassay and qPCR were used to examine their modulatory effects on proinflammatory cytokines, including tumour necrosis factor (TNF)α, interleukin- (IL-) 6, and IL-1β. There were significant decrements in IL-1β, IL-6, and TNFα expression in both curcumin and Cur-CS nanoparticles. Based on the multiplex immunoassay, curcumin and the Cur-CS nanoparticles could lower the immune-related proteins in FIP virus (FIPV) infection. The single- and multiple-dose pharmacokinetics profiles of curcumin and the Cur-CS nanoparticles were determined by high-performance liquid chromatography (HPLC). Oral delivery of the Cur-CS nanoparticles to cats showed enhanced bioavailability with a maximum plasma concentration (Cmax) value of 621.5 ng/mL. Incorporating chitosan nanoparticles to deliver curcumin improved the oral bioavailability and antiviral effects of curcumin against FIPV infection. This study provides evidence for the potential of Cur-CS nanoparticles as a supplementary treatment of FIP.

Cellular Metabolic Profiling of CrFK Cells Infected with Feline Infectious Peritonitis Virus Using Phenotype Microarrays.

Feline infectious peritonitis (FIP) is a fatal feline immune-mediated disease caused by feline infectious peritonitis virus (FIPV). Little is known about the biological pathways associated in FIP pathogenesis. This is the first study aiming to determine the phenotypic characteristics on the cellular level in relation to specific metabolic pathways of importance to FIP pathogenesis. Methods: The internalization of type II FIPV WSU 79-1146 in Crandell-Rees Feline Kidney (CrFK) cells was visualized using a fluorescence microscope, and optimization prior to phenotype microarray (PM) study was performed. Then, four types of Biolog Phenotype MicroArray™ plates (PM-M1 to PM-M4) precoated with different carbon and nitrogen sources were used to determine the metabolic profiles in FIPV-infected cells. Results: The utilization of palatinose was significantly low in FIPV-infected cells; however, there were significant increases in utilizing melibionic acid, L-glutamine, L-glutamic acid and alanyl-glutamine (Ala-Gln) compared to non-infected cells. Conclusion: This study has provided the first insights into the metabolic profiling of a feline coronavirus infection in vitro using PMs and deduced that glutamine metabolism is one of the essential metabolic pathways for FIPV infection and replication. Further studies are necessary to develop strategies to target the glutamine metabolic pathway in FIPV infection.

Epidemiological investigation of feline infectious peritonitis in cats living in Harbin, Northeast China from 2017 to 2019 using a combination of an EvaGreen-based real-time RT-PCR and serum chemistry assays

Feline infectious peritonitis (FIP) is caused by the FIP virus (FIPV), a highly virulent mutant form of feline coronavirus (FCoV). This disease is one of the most important infectious diseases in cats, and it is associated with high mortality, particularly among younger cats. In this study, we isolated a wild-type FIPV HRB-17 epidemic strain from the blood sample of household pet cat exhibiting the characteristic wet-form FIP symptoms, which has been confirmed further by animal infection. Further, we developed an EvaGreen-based real-time RT-PCR assay for the accurate detection of FCoV based on the amplification of the highly conserved FIPV N gene. Then, using a combination of the real-time RT-PCR approach and a serum chemistry assay, we performed an epidemiological survey of FIPV infection in cats living in Harbin City, Northeast China. The results indicated that the EvaGreen-based real-time RT-PCR assay can be used for screening FCoV infection in the affected cats at an analytical detection limit of 8.2 × 101 viral genome copies/μL, but could not effectively distinguish FIPVs from FECVs. Additionally, the results of the epidemiological survey investigating feline blood samples (n = 1523) collected between July 2017 to July 2019 revealed an FIPV prevalence of approximately 12% (189/1523). Maybe, the prevalence would be less than 12% due to the real-time RT-PCR assay could not accurately differentiate FIPV and FECV. Nevertheless, it still highlighted the severity of the FIP epidemic in cats and reiterated the urgent need to develop effective anti-FIP therapeutic agents and anti-FIPV vaccines. As pet cats are household animals, risk communication and continuous region-extended surveillance cat programs are recommended.

Characterization of antiviral T cell responses during primary and secondary challenge of laboratory cats with feline infectious peritonitis virus (FIPV)

BackgroundFeline infectious peritonitis (FIP) is considered highly fatal in its naturally occurring form, although up to 36% of cats resist disease after experimental infection, suggesting that cats in nature may also resist development of FIP in the face of infection with FIP virus (FIPV). Previous experimental FIPV infection studies suggested a role for cell-mediated immunity in resistance to development of FIP. This experimental FIPV infection study in specific pathogen free (SPF) kittens describes longitudinal antiviral T cell responses and clinical outcomes ranging from rapid progression, slow progression, and resistance to disease.ResultsDifferences in disease outcome provided an opportunity to investigate the role of T cell immunity to FIP determined by T cell subset proliferation after stimulation with different viral antigens. Reduced total white blood cell (WBC), lymphocyte and T cell counts in blood were observed during primary acute infection for all experimental groups including cats that survived without clinical FIP. Antiviral T cell responses during early primary infection were also similar between cats that developed FIP and cats remaining healthy. Recovery of antiviral T cell responses during the later phase of acute infection was observed in a subset of cats that survived longer or resisted disease compared to cats showing rapid disease progression. More robust T cell responses at terminal time points were observed in lymph nodes compared to blood in cats that developed FIP. Cats that survived primary infection were challenged a second time to pathogenic FIPV and tested for antiviral T cell responses over a four week period. Nine of ten rechallenged cats did not develop FIP or T cell depletion and all cats demonstrated antiviral T cell responses at multiple time points after rechallenge.ConclusionsIn summary, definitive adaptive T cell responses predictive of disease outcome were not detected during the early phase of primary FIPV infection. However emergence of antiviral T cell responses after a second exposure to FIPV, implicated cellular immunity in the control of FIPV infection and disease progression. Virus host interactions during very early stages of FIPV infection warrant further investigation to elucidate host resistance to FIP.

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.

Nanoparticulate vacuolar ATPase blocker exhibits potent host-targeted antiviral activity against feline coronavirus

Feline infectious peritonitis (FIP), caused by a mutated feline coronavirus, is one of the most serious and fatal viral diseases in cats. The disease remains incurable, and there is no effective vaccine available. In light of the pathogenic mechanism of feline coronavirus that relies on endosomal acidification for cytoplasmic entry, a novel vacuolar ATPase blocker, diphyllin, and its nanoformulation are herein investigated for their antiviral activity against the type II feline infectious peritonitis virus (FIPV). Experimental results show that diphyllin dose-dependently inhibits endosomal acidification in fcwf-4 cells, alters the cellular susceptibility to FIPV, and inhibits the downstream virus replication. In addition, diphyllin delivered by polymeric nanoparticles consisting of poly(ethylene glycol)-block-poly(lactide-co-glycolide) (PEG-PLGA) further demonstrates an improved safety profile and enhanced inhibitory activity against FIPV. In an in vitro model of antibody-dependent enhancement of FIPV infection, diphyllin nanoparticles showed a prominent antiviral effect against the feline coronavirus. In addition, the diphyllin nanoparticles were well tolerated in mice following high-dose intravenous administration. This study highlights the therapeutic potential of diphyllin and its nanoformulation for the treatment of FIP.

Natural resistance to experimental feline infectious peritonitis virus infection is decreased rather than increased by positive genetic selection

A previous study demonstrated the existence of a natural resistance to feline infectious peritonitis virus (FIPV) among 36% of randomly bred laboratory cats. A genome wide association study (GWAS) on this population suggested that resistance was polygenic but failed to identify any strong specific associations. In order to enhance the power of GWAS or whole genome sequencing to identify strong genetic associations, a decision was made to positively select for resistance over three generations. The inbreeding experiment began with a genetically related parental (P) population consisting of three toms and four queens identified from among the survivors of the earlier study and belonging to a closely related subgroup (B). The subsequent effects of inbreeding were measured using 42 genome-wide STR markers. P generation cats produced 57 first filial (F1) kittens, only five of which (9.0%) demonstrated a natural resistance to FIPV infection. One of these five F1 survivors was then used to produce six F1/P-backcrosses kittens, only one of which proved resistant to FIP. Six of eight of the F1 and F1/P survivors succumbed to a secondary exposure 4–12 months later. Therefore, survival after both primary and secondary infection was decreased rather than increased by positive selection for resistance. The common genetic factor associated with this diminished resistance was a loss of heterozygosity.

Development of a mouse-feline chimeric antibody against feline tumor necrosis factor-alpha.

Feline infectious peritonitis (FIP) is a fatal inflammatory disease caused by FIP virus infection. Feline tumor necrosis factor (fTNF)-alpha is closely involved in the aggravation of FIP pathology. We previously described the preparation of neutralizing mouse anti-fTNF-alpha monoclonal antibody (mAb 2–4) and clarified its role in the clinical condition of cats with FIP using in vitro systems. However, administration of mouse mAb 2–4 to cat may lead to a production of feline anti-mouse antibodies. In the present study, we prepared a mouse-feline chimeric mAb (chimeric mAb 2–4) by fusing the variable region of mouse mAb 2–4 to the constant region of feline antibody. The chimeric mAb 2–4 was confirmed to have fTNF-alpha neutralization activity. Purified mouse mAb 2–4 and chimeric mAb 2–4 were repeatedly administered to cats, and the changes in the ability to induce feline anti-mouse antibody response were investigated. In the serum of cats treated with mouse mAb 2–4, feline anti-mouse antibody production was induced, and the fTNF-alpha neutralization effect of mouse mAb 2–4 was reduced. In contrast, in cats treated with chimeric mAb 2–4, the feline anti-mouse antibody response was decreased compared to that of mouse mAb 2–4-treated cats.

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

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.

The influence of age and genetics on natural resistance to experimentally induced feline infectious peritonitis

Naturally occurring feline infectious peritonitis (FIP) is usually fatal, giving the impression that immunity to the FIP virus (FIPV) is extremely poor. This impression may be incorrect, because not all cats experimentally exposed to FIPV develop FIP. There is also a belief that the incidence of FIP may be affected by a number of host, virus, and environmental cofactors. However, the contribution of these cofactors to immunity and disease incidence has not been determined. The present study followed 111 random-bred specific pathogen free (SPF) cats that were obtained from a single research breeding colony and experimentally infected with FIPV. The cats were from several studies conducted over the past 5 years, and as a result, some of them had prior exposure to feline enteric coronavirus (FECV) or avirulent FIPVs. The cats were housed under optimized conditions of nutrition, husbandry, and quarantine to eliminate most of the cofactors implicated in FIPV infection outcome and were uniformly challenge exposed to the same field strain of serotype 1 FIPV. Forty of the 111 (36%) cats survived their initial challenge exposure to a Type I cat-passaged field strains of FIPV. Six of these 40 survivors succumbed to FIP to a second or third challenge exposure, suggesting that immunity was not always sustained. Exposure to non-FIP-inducing feline coronaviruses prior to challenge with virulent FIPV did not significantly affect FIP incidence but did accelerate the disease course in some cats. There were no significant differences in FIP incidence between males and females, but resistance increased significantly between 6 months and 1 or more years of age. Genetic testing was done on 107 of the 111 infected cats. Multidimensional scaling (MDS) segregated the 107 cats into three distinct families based primarily on a common sire(s), and resistant and susceptible cats were equally distributed within each family. Genome-wide association studies (GWAS) on 73 cats that died of FIP after one or more exposures (cases) and 34 cats that survived (controls) demonstrated four significant associations after 100k permutations. When these same cats were analyzed using a sib-pair transmission test, three of the four associations were confirmed although not with genome-wide significance. GWAS was then done on three different age groups of cases to take into account age-related resistance, and different associations were observed. The only common and strong association identified between the various GWAS case configurations was for the 34.7–45.8Mb region of chromosome A3. No obvious candidate genes were present in this region.

In VitroAntiviral Activity of Circular Triple Helix Forming Oligonucleotide RNA towards Feline Infectious Peritonitis Virus Replication

Feline Infectious Peritonitis (FIP) is a severe fatal immune-augmented disease in cat population. It is caused by FIP virus (FIPV), a virulent mutant strain of Feline Enteric Coronavirus (FECV). Current treatments and prophylactics are not effective. The in vitro antiviral properties of five circular Triple-Helix Forming Oligonucleotide (TFO) RNAs (TFO1 to TFO5), which target the different regions of virulent feline coronavirus (FCoV) strain FIPV WSU 79-1146 genome, were tested in FIPV-infected Crandell-Rees Feline Kidney (CRFK) cells. RT-qPCR results showed that the circular TFO RNAs, except TFO2, inhibit FIPV replication, where the viral genome copy numbers decreased significantly by 5-fold log10 from 1014 in the virus-inoculated cells to 109 in the circular TFO RNAs-transfected cells. Furthermore, the binding of the circular TFO RNA with the targeted viral genome segment was also confirmed using electrophoretic mobility shift assay. The strength of binding kinetics between the TFO RNAs and their target regions was demonstrated by NanoITC assay. In conclusion, the circular TFOs have the potential to be further developed as antiviral agents against FIPV infection.

Suppression of NK cells and regulatory T lymphocytes in cats naturally infected with feline infectious peritonitis virus

A strong cell-mediated immunity (CMI) is thought to be indispensable for protection against infection with feline infectious peritonitis virus (FIPV) in cats. In this study, the role of natural killer (NK) cells and regulatory T cells (Tregs), central players in the innate and adaptive CMI respectively, was examined during natural FIPV infection. When quantified, both NK cells and Tregs were drastically depleted from the peripheral blood, mesenteric lymph node (LN) and spleen in FIP cats. In contrast, mesentery and kidney from FIP cats did not show any difference when compared to healthy non-infected control animals. In addition, other regulatory lymphocytes (CD4+CD25−Foxp3+ and CD3+CD8+Foxp3+) were found to be depleted from blood and LN as well. Phenotypic analysis of blood-derived NK cells in FIP cats revealed an upregulation of activation markers (CD16 and CD25) and migration markers (CD11b and CD62L) while LN-derived NK cells showed upregulation of only CD16 and CD62L. LN-derived NK cells from FIPV-infected cats were also significantly less cytotoxic when compared with healthy cats. This study reveals for the first time that FIPV infection is associated with severe suppression of NK cells and Tregs, which is reflected by cell depletion and lowered cell functionality (only NK cells). This will un-doubtfully lead to a reduced capacity of the innate immune system (NK cells) to battle FIPV infection and a decreased capacity (Tregs) to suppress the immunopathology typical for FIP. However, these results will also open possibilities for new therapies targeting specifically NK cells and Tregs to enhance their numbers and/or functionality during FIPV infection.

Intriguing interplay between feline infectious peritonitis virus and its receptors during entry in primary feline monocytes

Two potential receptors have been described for the feline infectious peritonitis virus (FIPV): feline aminopeptidase N (fAPN) and feline dendritic cell-specific intercellular adhesion molecule grabbing non-integrin (fDC-SIGN). In cell lines, fAPN serves as a receptor for serotype II, but not for serotype I FIPV. The role of fAPN in infection of in vivo target cells, monocytes, is not yet confirmed. Both serotype I and II FIPVs use fDC-SIGN for infection of monocyte-derived cells but how is not known. In this study, the role of fAPN and fDC-SIGN was studied at different stages in FIPV infection of monocytes. First, the effects of blocking the potential receptor(s) were studied for the processes of attachment and infection. Secondly, the level of co-localization of FIPV and the receptors was determined. It was found that FIPV I binding and infection were not affected by blocking fAPN while blocking fDC-SIGN reduced FIPV I binding to 36% and practically completely inhibited infection. Accordingly, 66% of bound FIPV I particles co-localized with fDC-SIGN. Blocking fAPN reduced FIPV II binding by 53% and infection by 80%. Further, 60% of bound FIPV II co-localized with fAPN. fDC-SIGN was not involved in FIPV II binding but infection was reduced with 64% when fDC-SIGN was blocked. In conclusion, FIPV I infection of monocytes depends on fDC-SIGN. Most FIPV I particles already interact with fDC-SIGN at the plasma membrane. For FIPV II, both fAPN and fDC-SIGN are involved in infection with only fAPN playing a receptor role at the plasma membrane.

Development of monoclonal antibodies (MAbs) to feline interferon (fIFN)- γ as tools to evaluate cellular immune responses to feline infectious peritonitis virus (FIPV)

Feline infectious peritonitis virus (FIPV) can cause a lethal disease in cats, feline infectious peritonitis (FIP). The antibody-dependent enhancement (ADE) of FIPV infection has been recognised in experimentally infected cats, and cellular immunity is considered to play an important role in preventing the onset of FIP. To evaluate the importance of cellular immunity for FIPV infection, monoclonal antibodies (MAbs) against feline interferon (fIFN)-γ were first created to establish fIFN-γ detection systems using the MAbs. Six anti-fIFN-γ MAbs were created. Then, the difference in epitope which those MAbs recognise was demonstrated by competitive enzyme-linked immunosorbent assay (ELISA) and IFN-γ neutralisation tests. Detection systems for fIFN-γ (sandwich ELISA, ELISpot assay, and two-colour flow cytometry) were established using anti-fIFN-γ MAbs that recognise different epitopes. In all tests, fIFN-γ production from peripheral blood mononuclear cells (PBMCs) obtained from cats experimentally infected with an FIPV isolate that did not develop the disease was significantly increased by heat-inactivated FIPV stimulation in comparison with medium alone. Especially, CD8+fIFN-γ+ cells, but not CD4+fIFN-γ+ cells, were increased. In contrast, fIFN-γ production from PBMCs isolated from cats that had developed FIP and specific pathogen-free (SPF) cats was not increased by heat-inactivated FIPV stimulation. These results suggest that cellular immunity plays an important role in preventing the development of FIP. Measurement of fIFN-γ production with the anti-fIFN-γ MAbs created in this study appeared to be useful in evaluating cellular immunity in cats.