Establishment of Full-Length cDNA Clones and an Efficient Oral Infection Model for Feline Coronavirus in Cats.

Feline infectious peritonitis virus (FIPV) is the etiologic agent of feline infectious peritonitis (FIP) and causes fatal disease in cats of almost all ages. Currently, there are no clinically approved drugs or effective vaccines for FIP. Furthermore, the pathogenesis of FIP is still not fully understood. There is an urgent need for an effective infection model of feline infectious peritonitis induced by FIPV. Here, we constructed a field type I FIPV full-length cDNA clone, pBAC-QS, corresponding to the isolated FIPV QS. By replacing the FIPV QS spike gene with the commercially available type II FIPV 79-1146 (79-1146_CA) spike gene, we established and rescued a recombinant virus, designated rQS-79. Moreover, we constructed 79-1146_CA infectious full-length cDNA pBAC-79-1146_CA, corresponding to recombinant feline coronavirus (FCoV) 79-1146_CA (r79-1146_CA). In animal experiments with 1- to 2-year-old adult cats orally infected with the recombinant virus, rQS-79 induced typical FIP signs and 100% mortality. In contrast to cats infected with rQS-79, cats infected with 79-1146_CA did not show obvious signs. Furthermore, by rechallenging rQS-79 in surviving cats previously infected with 79-1146_CA, we found that there was no protection against rQS-79 with different titers of neutralizing antibodies. However, high titers of neutralizing antibodies may help prolong the cat survival time. Overall, we report the first reverse genetics of virulent recombinant FCoV (causing 100% mortality in adult cats) and attenuated FCoV (causing no mortality in adult cats), which will be powerful tools to study pathogenesis, antiviral drugs, and vaccines for FCoV. IMPORTANCE Tissue- or cell culture-adapted feline infectious peritonitis virus (FIPV) usually loses pathogenicity. To develop a highly virulent FIPV, we constructed a field isolate type I FIPV full-length clone with the spike gene replaced by the 79-1146 spike gene, corresponding to a virus named rQS-79, which induces high mortality in adult cats. rQS-79 represents the first described reverse genetics system for highly pathogenic FCoV. By further constructing the cell culture-adapted FCoV 79-1146_CA, we obtained infectious clones of virulent and attenuated FCoV. By in vitro and in vivo experiments, we established a model that can serve to study the pathogenic mechanisms of FIPV. Importantly, the wild-type FIPV replicase skeleton of serotype I will greatly facilitate the screening of antiviral drugs, both in vivo and in vitro.

FELINE CORONAVIRUS AND FELINE INFECTIOUS PERITONITIS IN NONDOMESTIC FELID SPECIES.

Feline coronavirus (FCoV) is reported worldwide and known to cause disease in domestic and nondomestic felid species. Although FCoV often results in mild to inapparent disease, a small subset of cats succumb to the fatal, systemic disease feline infectious peritonitis (FIP). An outbreak of FIP in Cheetahs (Acinonyx jubatus) in a zoological collection demonstrated the devastating effect of FCoV introduction into a naïve group of animals. In addition to cheetahs, FIP has been described in European wildcats (Felis silvestris), a tiger (Panthera tigris), a mountain lion (Puma concolor), and lion (Panthera leo). This paper reviews the reported cases of FIP in nondomestic felid species and highlights the surveys of FCoV in populations of nondomestic felids.

A retrospective study of clinical and laboratory features and treatment on cats highly suspected of feline infectious peritonitis in Wuhan, China.

Feline infectious peritonitis (FIP) is a systemic, potentially fatal viral disease. The objectives of this study were to review clinical and laboratory features and treatment of cats highly suspected of FIP in Wuhan, China. The clinical records of 127 cats highly suspected of FIP were reviewed for history, clinical signs, physical findings, and diagnostic test results. Sex, neutering status, breed, age, and month of onset of disease were compared with the characteristics of the clinic population. Age and neutering status were significantly correlated with FIP-suspicion. Sex, breed and onset month were not associated with FIP. There were many more FIP-suspected cases in cats in young cats or male intact cats. Effusion was observed in 85.8% of the FIP-suspected cats. Increased serum amyloid A (SAA) and lymphopenia were common laboratory abnormalities in the FIP cases. Furthermore, 91.7% of the cats highly suspected of FIP had an albumin/globulin (A/G) ratio < 0.6, while 85.3% had an A/G ratio < 0.5. The mortality rate for FIP-suspected cats was 67%, and six submitted cases were confirmed by FIP-specific immunohistochemistry. Of the 30 cats treated with GS-441524 and/or GC376, 29 were clinically cured. The study highlights the diverse range of clinical manifestations by clinicians in diagnosing this potentially fatal disease. A/G ratio and SAA were of higher diagnostic value. GS-441524 and GC376 were efficient for the treatment of FIP-suspected cats.

Mutation analysis of the spike protein in Italian feline infectious peritonitis virus and feline enteric coronavirus sequences.

Feline coronavirus (FCoV) exists as two different genotypes, FCoV type I and II, each including two biotypes, feline enteric coronavirus (FECV) and feline infectious peritonitis virus (FIPV), the latter being a virulent variant originating from the former virus. Recently, two amino acid substitutions, M1058L and S1060A, within the spike protein have been associated to the FECV/FIPV virulence change. In this study, we have analysed the frequency of detection of such mutations in FIPV and FECV strains circulating in Italian cats and obtained information about their evolutionary relationships with reference isolates. A total of 40 FCoV strains, including 19 strains from effusions or tissue samples of FIP cats and 21 strains from faecal samples of non-FIP cats, were analysed. Mutation M1058L was detected in 16/18 FCoV-I and 1/1 FCoV-II strains associated with FIP, while change S1060A was presented by two FIPV strains. By phylogenetic analysis, FCoV sequences clustered according to the genotype but not according to the biotype, with FECV/FIPV strains recovered from the same animal being closely related. Further studies are needed to better define the genetic signatures associated with the FECV/FIPV virulence shift.

Assay validation and determination of in vitro binding of mefloquine to plasma proteins from clinically normal and FIP-affected cats.

The antimalarial agent mefloquine is currently being investigated for its potential to inhibit feline coronavirus and feline calicivirus infections. A simple, high pressure liquid chromatography assay was developed to detect mefloquine plasma concentrations in feline plasma. The assay's lower limit of quantification was 250 ng/mL. The mean ± standard deviation intra- and inter-day precision expressed as coefficients of variation were 6.83 ± 1.75 and 5.33 ± 1.37%, respectively, whereas intra- and inter-day accuracy expressed as a percentage of the bias were 11.40 ± 3.73 and 10.59 ± 3.88%, respectively. Accordingly, this validated assay should prove valuable for future in vivo clinical trials of mefloquine as an antiviral agent against feline coronavirus and feline calicivirus. However, the proportion of mefloquine binding to feline plasma proteins has not been reported. The proportion of drug bound to plasma protein binding is an important concept when developing drug dosing regimens. As cats with feline infectious peritonitis (FIP) demonstrate altered concentrations of plasma proteins, the proportion of mefloquine binding to plasma proteins in both clinically normal cats and FIP-affected cats was also investigated. An in vitro method using rapid equilibrium dialysis demonstrated that mefloquine was highly plasma protein bound in both populations (on average > 99%).

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.

A Tale of Two Viruses: The Distinct Spike Glycoproteins of Feline Coronaviruses.

Feline coronavirus (FCoV) is a complex viral agent that causes a variety of clinical manifestations in cats, commonly known as feline infectious peritonitis (FIP). It is recognized that FCoV can occur in two different serotypes. However, differences in the S protein are much more than serological or antigenic variants, resulting in the effective presence of two distinct viruses. Here, we review the distinct differences in the S proteins of these viruses, which are likely to translate into distinct biological outcomes. We introduce a new concept related to the non-taxonomical classification and differentiation among FCoVs by analyzing and comparing the genetic, structural, and functional characteristics of FCoV and the FCoV S protein among the two serotypes and FCoV biotypes. Based on our analysis, we suggest that our understanding of FIP needs to consider whether the presence of these two distinct viruses has implications in clinical settings.

Feline Infectious Peritonitis as a Systemic Inflammatory Disease: Contribution of Liver and Heart to the Pathogenesis.

Feline infectious peritonitis (FIP) is a fatal immune-mediated disease of cats, induced by feline coronavirus (FCoV). A combination of as yet poorly understood host and viral factors combine to cause a minority of FCoV-infected cats to develop FIP. Clinicopathological features include fever, vasculitis, and serositis, with or without effusions; all of which indicate a pro-inflammatory state with cytokine release. As a result, primary immune organs, as well as circulating leukocytes, have thus far been of most interest in previous studies to determine the likely sources of these cytokines. Results have suggested that these tissues alone may not be sufficient to induce the observed inflammation. The current study therefore focussed on the liver and heart, organs with a demonstrated ability to produce cytokines and therefore with huge potential to exacerbate inflammatory processes. The IL-12:IL-10 ratio, a marker of the immune system's inflammatory balance, was skewed towards the pro-inflammatory IL-12 in the liver of cats with FIP. Both organs were found to upregulate mRNA expression of the inflammatory triad of cytokines IL-1ß, IL-6, and TNF-α in FIP. This amplifying step may be one of the missing links in the pathogenesis of this enigmatic disease.

Establishment of a Virulent Full-Length cDNA Clone for Type I Feline Coronavirus Strain C3663.

Feline infectious peritonitis (FIP) is one of the most important infectious diseases in cats and is caused by feline coronavirus (FCoV). Tissue culture-adapted type I FCoV shows reduced FIP induction in experimental infections, which complicates the understanding of FIP pathogenesis caused by type I FCoV. We previously found that the type I FCoV strain C3663 efficiently induces FIP in specific-pathogen-free cats through the naturally infectious route. In this study, we employed a bacterial artificial chromosome-based reverse genetics system to gain more insights into FIP caused by the C3633 strain. We successfully generated recombinant virus (rC3663) from Fcwf-4 cells transfected with infectious cDNA that showed growth kinetics similar to those shown by the parental virus. Next, we constructed a reporter C3663 virus carrying the nanoluciferase (Nluc) gene to measure viral replication with high sensitivity. The inhibitory effects of different compounds against rC3663-Nluc could be measured within 24 h postinfection. Furthermore, we found that A72 cells derived from canine fibroblasts permitted FCoV replication without apparent cytopathic effects. Thus, our reporter virus is useful for uncovering the infectivity of type I FCoV in different cell lines, including canine-derived cells. Surprisingly, we uncovered aberrant viral RNA transcription of rC3663 in A72 cells. Overall, we succeeded in obtaining infectious cDNA clones derived from type I FCoV that retained its virulence. Our recombinant FCoVs are powerful tools for increasing our understanding of the viral life cycle and pathogenesis of FIP-inducing type I FCoV.IMPORTANCE Feline coronavirus (FCoV) is one of the most significant coronaviruses, because this virus induces feline infectious peritonitis (FIP), which is a lethal disease in cats. Tissue culture-adapted type I FCoV often loses pathogenicity, which complicates research on type I FCoV-induced feline infectious peritonitis (FIP). Since we previously found that type I FCoV strain C3663 efficiently induces FIP in specific-pathogen-free cats, we established a reverse genetics system for the C3663 strain to obtain recombinant viruses in the present study. By using a reporter C3663 virus, we were able to examine the inhibitory effect of 68 compounds on C3663 replication in Fcwf-4 cells and infectivity in a canine-derived cell line. Interestingly, one canine cell line, A72, permitted FCoV replication but with low efficiency and aberrant viral gene expression.

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.

Reverse Genetics for Type I Feline Coronavirus Field Isolate To Study the Molecular Pathogenesis of Feline Infectious Peritonitis.

Feline infectious peritonitis (FIP), one of the most important lethal infections of cats, is caused by feline infectious peritonitis virus (FIPV), the high-virulence biotype of feline coronaviruses (FCoVs). FIPVs are suggested to emerge from feline enteric coronaviruses (FECVs) by acquiring mutations in specific genes in the course of persistent infections. Although numerous studies identified mutations predicted to be responsible for the FECV-FIPV biotype switch, the presumed roles of specific genetic changes in FIP pathogenesis have not been confirmed experimentally. Reverse genetics systems established previously for serotype I and the less common serotype II FCoVs were based on cell culture-adapted FIPV strains which, however, were shown to be unsuitable for FIP pathogenesis studies in vivo To date, systems to produce and manipulate recombinant serotype I field viruses have not been developed, mainly because these viruses cannot be grown in vitro Here, we report the first reverse genetics system based on a serotype I FECV field isolate that is suitable to produce high-titer stocks of recombinant FECVs. We demonstrate that these recombinant viruses cause productive persistent infections in cats that are similar to what is observed in natural infections. The system provides an excellent tool for studying FCoVs that do not grow in standard cell culture systems and will greatly facilitate studies into the molecular pathogenesis of FIP. Importantly, the system could also be adapted for studies of other RNA viruses with large genomes whose production and characterization in vivo are currently hampered by the lack of in vitro propagation systems.IMPORTANCE The availability of recombinant serotype I FCoV field isolates that are amenable to genetic manipulation is key to studying the molecular pathogenesis of FIP, especially since previous studies using cell culture-adapted FIPVs had proven unsuccessful. To our knowledge, we report the first serotype I FECV field isolate-based reverse genetics system that allows the production of high-titer recombinant virus stocks that can be used for subsequent in vivo studies in cats. The system represents a milestone in FCoV research. It provides an essential tool for studying the molecular pathogenesis of FIP and, more specifically, the functions of specific gene products in causing a fundamentally different progression of disease following acquisition of specific mutations. The system developed in this study will also be useful for studying other coronaviruses or more distantly related RNA viruses with large genomes for which suitable in vitro culture systems are not available.

Differential susceptibility of macrophages to serotype II feline coronaviruses correlates with differences in the viral spike protein.

The ability to infect and replicate in monocytes/macrophages is a critically distinguishing feature between the two feline coronavirus (FCoV) pathotypes: feline enteric coronavirus (FECV; low-virulent) and feline infectious peritonitis virus (FIPV; lethal). Previously, by comparing serotype II strains FIPV 79-1146 and FECV 79-1683 and recombinant chimeric forms thereof in cultured feline bone marrow macrophages, we mapped this difference to the C-terminal part of the viral spike (S) protein (S2). In view of the later identified diagnostic difference in this very part of the S protein of serotype I FCoV pathotypes, the present study aimed to further define the contribution of the earlier observed ten amino acids difference to the serotype II virus phenotype in macrophages. Using targeted RNA recombination as a reverse genetics system we introduced the mutations singly and in combinations into the S gene and evaluated their effects on the infection characteristics of the mutant viruses in macrophages. While some of the single mutations had a significant effect, none of them fully reverted the infection phenotype. Only by combining five specific mutations the infections mediated by the FIPV and FECV spike proteins could be fully blocked or potentiated, respectively. Hence, the differential macrophage infection phenotype is caused by the cooperative effect of five mutations, which occur in five functionally different domains of the spike fusion subunit S2. The significance of these observations will be discussed, taking into account also some questions related to the identity of the virus strains used.

Mutation of the S and 3c genes in genomes of feline coronaviruses.

Feline coronavirus (FCoV) is classified into two biotypes based on its pathogenicity in cats: a feline enteric coronavirus of low pathogenicity and a highly virulent feline infectious peritonitis virus. It has been suspected that FCoV alters its biotype via mutations in the viral genome. The S and 3c genes of FCoV have been considered the candidates for viral pathogenicity conversion. In the present study, FCoVs were analyzed for the frequency and location of mutations in the S and 3c genes from faecal samples of cats in an animal shelter and the faeces, effusions, and tissues of cats that were referred to veterinary hospitals. Our results indicated that approximately 95% FCoVs in faeces did not carry mutations in the two genes. However, 80% FCoVs in effusion samples exhibited mutations in the S and 3c genes with remainder displaying a mutation in the S or 3c gene. It was also suggested that mutational analysis of the 3c gene could be useful for studying the horizontal transmission of FCoVs in multi-cat environments.

Feline coronavirus: Insights into viral pathogenesis based on the spike protein structure and function.

Feline coronavirus (FCoV) is an etiological agent that causes a benign enteric illness and the fatal systemic disease feline infectious peritonitis (FIP). The FCoV spike (S) protein is considered the viral regulator for binding and entry to the cell. This protein is also involved in FCoV tropism and virulence, as well as in the switch from enteric disease to FIP. This regulation is carried out by spike's major functions: receptor binding and virus-cell membrane fusion. In this review, we address important aspects in FCoV genetics, replication and pathogenesis, focusing on the role of S. To better understand this, FCoV S protein models were constructed, based on the human coronavirus NL63 (HCoV-NL63) S structure. We describe the specific structural characteristics of the FCoV S, in comparison with other coronavirus spikes. We also revise the biochemical events needed for FCoV S activation and its relation to the structural features of the protein.

Feline Coronaviruses: Pathogenesis of Feline Infectious Peritonitis.

Feline infectious peritonitis (FIP) belongs to the few animal virus diseases in which, in the course of a generally harmless persistent infection, a virus acquires a small number of mutations that fundamentally change its pathogenicity, invariably resulting in a fatal outcome. The causative agent of this deadly disease, feline infectious peritonitis virus (FIPV), arises from feline enteric coronavirus (FECV). The review summarizes our current knowledge of the genome and proteome of feline coronaviruses (FCoVs), focusing on the viral surface (spike) protein S and the five accessory proteins. We also review the current classification of FCoVs into distinct serotypes and biotypes, cellular receptors of FCoVs and their presumed role in viral virulence, and discuss other aspects of FIPV-induced pathogenesis. Our current knowledge of genetic differences between FECVs and FIPVs has been mainly based on comparative sequence analyses that revealed "discriminatory" mutations that are present in FIPVs but not in FECVs. Most of these mutations result in amino acid substitutions in the S protein and these may have a critical role in the switch from FECV to FIPV. In most cases, the precise roles of these mutations in the molecular pathogenesis of FIP have not been tested experimentally in the natural host, mainly due to the lack of suitable experimental tools including genetically engineered virus mutants. We discuss the recent progress in the development of FCoV reverse genetics systems suitable to generate recombinant field viruses containing appropriate mutations for in vivo studies.

Feline Coronavirus 3c Protein: A Candidate for a Virulence Marker?

Feline infectious peritonitis virus (FIPV) is highly virulent and responsible for the highly fatal disease feline infectious peritonitis (FIP), whereas feline enteric coronavirus (FECV) is widespread among the feline population and typically causes asymptomatic infections. Some candidates for genetic markers capable of differentiating these two pathotypes of a unique virus (feline coronavirus) have been proposed by several studies. In the present survey, in order to search for markers that can differentiate FECV and FIPV, several clones of the 3a-c, E, and M genes were sequenced from samples obtained from cats with or without FIP. All genes showed genetic diversity and suggested the presence of FCoV mutant spectrum capable of producing a virulent pathotype in an individual-specific way. In addition, all the feline coronavirus FIPV strains demonstrated a truncated 3c protein, and the 3c gene was the only observed pathotypic marker for FCoVs, showing that 3c gene is a candidate marker for the distinction between the two pathotypes when the mutant spectrum is taken into account.

Glial response in the central nervous system of cats with feline infectious peritonitis.

OBJECTIVES: The aim of the study was to evaluate central nervous system (CNS) lesions in non-effusive and effusive cases of feline infectious peritonitis (FIP) regarding aspects related to astrocytic and microglial reactions. METHODS: Five necropsied cats that were naturally infected with FIP virus, confirmed by reverse transcriptase polymerase chain reaction and immunohistochemistry, with different intensities of CNS lesions, were studied. Brain and cerebellum were evaluated by light microscopy and immunohistochemistry for glial fibrillary acidic protein (GFAP) and vimentin to assess astrocytic morphology, and lectin histochemistry for Ricinus communis agglutinin-I (RCA-I) to detect microglia was performed to evaluate the glial response in the CNS of cats with FIP. RESULTS: An important astrocytic response in many areas of the CNS of all cats, including the periventricular areas of lateral ventricles and fourth ventricle, the molecular layer of the cerebellum and cerebral cortex, was visualized. This astrocytic reactivity was associated with areas of granulomatous or pyogranulomatous vasculitis/perivasculitis in most cases, and it was characterized by multifocal to coalescing astrocytosis and astrogliosis with an increase in the expression of intermediate filaments, such as GFAP. However, astrocytes exhibited strong vimentin expression in neuroparenchyma with severe inflammatory and necrotic changes, but GFAP expression was mild or absent in these cases. A microglial response was present only in severe lesions, and RCA-I expression was detected primarily in gitter cells and resting microglia. CONCLUSIONS AND RELEVANCE: The present study indicates a strong astrocytic response, including the presence of many less differentiated vimentin-positive astrocytes and gitter cells positive for RCA-1 in severe lesions in the CNS of cats with FIP.

Tackling feline infectious peritonitis via reverse genetics.

Feline infectious peritonitis (FIP) is caused by feline coronaviruses (FCoVs) and represents one of the most important lethal infectious diseases of cats. To date, there is no efficacious prevention and treatment, and our limited knowledge on FIP pathogenesis is mainly based on analysis of experiments with field isolates. In a recent study, we reported a promising approach to study FIP pathogenesis using reverse genetics. We generated a set of recombinant FCoVs and investigated their pathogenicity in vivo. The set included the type I FCoV strain Black, a type I FCoV strain Black with restored accessory gene 7b, two chimeric type I/type II FCoVs and the highly pathogenic type II FCoV strain 79-1146. All recombinant FCoVs and the reference strain isolates were found to establish productive infections in cats. While none of the type I FCoVs and chimeric FCoVs induced FIP, the recombinant type II FCoV strain 79-1146 was as pathogenic as the parental isolate. Interestingly, an intact ORF 3c was confirmed to be restored in all viruses (re)isolated from FIP-diseased animals.

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.

Emergence of pathogenic coronaviruses in cats by homologous recombination between feline and canine coronaviruses.

Type II feline coronavirus (FCoV) emerged via double recombination between type I FCoV and type II canine coronavirus (CCoV). In this study, two type I FCoVs, three type II FCoVs and ten type II CCoVs were genetically compared. The results showed that three Japanese type II FCoVs, M91-267, KUK-H/L and Tokyo/cat/130627, also emerged by homologous recombination between type I FCoV and type II CCoV and their parent viruses were genetically different from one another. In addition, the 3'-terminal recombination sites of M91-267, KUK-H/L and Tokyo/cat/130627 were different from one another within the genes encoding membrane and spike proteins, and the 5'-terminal recombination sites were also located at different regions of ORF1. These results indicate that at least three Japanese type II FCoVs emerged independently. Sera from a cat experimentally infected with type I FCoV was unable to neutralize type II CCoV infection, indicating that cats persistently infected with type I FCoV may be superinfected with type II CCoV. Our previous study reported that few Japanese cats have antibody against type II FCoV. All of these observations suggest that type II FCoV emerged inside the cat body and is unable to readily spread among cats, indicating that these recombination events for emergence of pathogenic coronaviruses occur frequently.