Pathogenic characteristics of persistent feline enteric coronavirus infection in cats

Feline coronaviruses (FCoV) comprise two biotypes: feline enteric coronaviruses (FECV) and feline infectious peritonitis viruses (FIPV). FECV is associated with asymptomatic persistent enteric infections, while FIPV causes feline infectious peritonitis (FIP), a usually fatal systemic disease in domestic cats and some wild Felidae. FIPV arises from FECV by mutation. FCoV also occur in two serotypes, I and II, of which the serotype I viruses are by far the most prevalent in the field. Yet, most of our knowledge about FCoV infections relates to serotype II viruses, particularly about the FIPV, mainly because type I viruses grow poorly in cell culture. Hence, the aim of the present work was the detailed study of the epidemiologically most relevant viruses, the avirulent serotype I viruses. Kittens were inoculated oronasally with different doses of two independent FECV field strains, UCD and RM. Persistent infection could be reproducibly established. The patterns of clinical symptoms, faecal virus shedding and seroconversion were monitored for up to 10 weeks revealing subtle but reproducible differences between the two viruses. Faecal virus, i.e. genomic RNA, was detected during persistent FECV infection only in the large intestine, downstream of the appendix, and could occasionally be observed also in the blood. The implications of our results, particularly our insights into the persistently infected state, are discussed.     

An enteric coronavirus infection of cats and its relationship to feline infectious peritonitis

An enteric coronavirus that is antigenically closely related to feline infectious peritonitis virus (FIPV) is ubiquitous in the cat population. This virus has been designated feline enteric coronavirus to differentiate it from FIPV. The virus is shed in the feces by many seropositive cats; in catteries it is a cause of inapparent to mildly severe enteritis in kittens 6 to 12 weeks of age. The virus may produce a more severe enteritis in young specific-pathogen-free kittens. Feline enteric coronavirus selectively infects the apical columnar epithelium of the intestinal villi, from the caudal part of the duodenum to the cecum. In severe infections, there are sloughing of the tips of the villi and villous atrophy. Many cats recovering from the disease remain carriers of the virus. Recovered cats, observed for 3 to 24 months, remained healthy and did not develop peritonitis, pleuritis, or granulomatous disease. The relationship of feline enteric coronavirus and FIPV was studied. Although the viruses were antigenically similar, they were distinctly different in their pathogenicities. The enteric coronavirus did not cause feline infectious peritonitis in coronavirus antibody-negative cats inoculated orally or intraperitoneally nor in coronavirus antibody-positive cats inoculated intraperitoneally or intratracheally. Serologic tests, using FIPV, canine coronavirus, and transmissible gastroenteritis virus of swine as substrate antigens in fluorescent antibody procedures may not accurately identify FIPV infection. These tests do not appear to distinguish between FIPV and this feline enteric coronavirus.     

Pathogenesis of feline gastric chlamydial infection

Studies were conducted to determine whether the gastric chlamydiae that have been observed recently in cats are of pathologic significance. Chlamydiae were isolated in mouse L cell cultures from the homogenized pooled gastric mucosa of 3 cats that had been identified, by histopathologic examination, to have gastric chlamydiosis. Ten specific-pathogen-free kittens were exposed by aerosol and oral inoculation to the harvested feline gastric chlamydiae cell-culture media. In general, the clinical signs and lesions were conjunctivitis, rhinitis, and mild gastritis. The clinical signs and lesions were most severe in 2 chlamydia-infected kittens that had received methylprednisolone acetate (50 mg/kg of body weight). Chlamydiae were demonstrated in epithelial cells of conjunctival and nasal smears in 10 of 10 infected kittens from postexposure days 7 through 35. In addition, chlamydiae were isolated in L cell cultures from a variety of antemortem and postmortem specimens from infected kittens. The present study provided evidence that feline gastric chlamydiae, under appropriate conditions, were capable of inducing, in cats, clinical signs and lesions similar to those induced by the feline pneumonitis agent.     

Perspectives on the epizootiology of feline enteric coronavirus and the pathogenesis of feline infectious peritonitis.

This review presents some current thoughts regarding the epizootiology of the feline coronaviruses; feline infectious peritonitis virus (FIPV) and feline coronavirus (FECV) with primary emphasis on the pathogenesis of these viruses in nature. Although the mechanism(s) whereby FIPV causes disease are still incompletely understood, there have been significant contributions to the literature over the past decade which provide a framework upon which plausible explanations can be postulated. Two concepts are presented which attempt to clarify the pathogenesis of FIPV and at the same time may serve as an impetus for further research. The first involves the hypothesis, originally promulgated by Pedersen in 1981, that FIPV is derived from FECV during virus replication in the gastrointestinal tract. The second involves a unique mechanism of the mucosal immune system referred to as oral tolerance, which under normal conditions promotes the production of secretory immunity and suppresses the production of systemic immunity. In the case of FIPV infection, we propose that oral tolerance is important in the control of the virus at the gastrointestinal tract level. Once oral tolerance is disrupted, FIPV is capable of systemic spread resulting in immune-mediated vasculitis and death. Thus, it may be that clinical forms of FIP are due to a combination of two events, the first being the generation of FIPV from FECV, and the second being the capacity of FIPV to circumvent oral tolerance.     

Experimental infection of young rabbits with a rabbit enteric coronavirus.

The clinical signs and lesions caused by the rabbit enteric coronavirus (RECV) were studied in young rabbits orally inoculated with a suspension containing RECV particles. The inoculated animals were observed daily for evidence of diarrhea. Fecal samples and specimens from the small intestine and from the gut associated lymphoid tissue (GALT) were collected from 2 h to 29 days postinoculation (PI) and processed for immune electron microscopy (IEM) and light microscopy. Coronavirus particles were detected in the cecal contents of most inoculated animals from 6 h to 29 days PI. Lesions were first observed 6 h PI and were characterized by a loss of the brush border of mature enterocytes located at the tips of intestinal villi and by necrosis of these cells. At 48 h PI, short intestinal villi and hypertrophic crypts were noted. In the GALT, complete necrosis of the M cells as well as necrosis of the enterocytes lining the villi above the lymphoid follicules with hypertrophy of the corresponding crypts were observed in all the animals. Five inoculated rabbits had diarrhea three days PI. The presence of RECV particles in the feces of the sick animals and the microscopic lesions observed in the small intestine suggested that the virus was responsible for the clinical signs. A few inoculated rabbits remained free of diarrhea. Fecal material collected at postmortem examination contained RECV particles. The results suggest that the virus could also produce a subclinical infection.     

Interferon-gamma in the serum and effusions of cats with feline coronavirus infection

The aim of this study was to quantify and compare interferon-γ (IFN-γ) concentrations in the serum of clinically normal cats infected with feline coronavirus (FCoV) with its concentration in the sera and effusions of cats with feline infectious peritonitis (FIP), a disease associated with infection with a mutated form of FCoV.Clinically normal FCoV-infected cats living in catteries with a high prevalence of FIP had the highest serum IFN-γ concentrations. The serum concentration of IFN-γ was not significantly different in cats with FIP compared with clinically normal FCoV-infected animals living in catteries with a low prevalence of the disease. Moreover, the concentration of IFN-γ was significantly higher in the effusions than in the serum of cats with FIP, probably due to IFN-γ production within lesions. These findings support the hypothesis that there is a strong, ‘systemic’ cell mediated immune response in clinically normal, FCoV-infected cats and that a similar process, albeit at a tissue level, is involved in the pathogenesis of FIP.     

Quarantine protects Falkland Islands (Malvinas) cats from feline coronavirus infection

Feline coronavirus (FCoV) causes feline infectious peritonitis (FIP). Since 2002, when 20 cats on the Falkland Islands were found to be FCoV seronegative, only seronegative cats could be imported. Between 2005-2007, 95 pet and 10 feral cats tested negative by indirect immunofluorescence antibody (IFA) analysis using two strains of type II FCoV, two transmissible gastroenteritis virus assays, an enzyme-linked immunosorbent assay and rapid immunomigration test. Twenty-four samples (23%) showed non-specific fluorescence, mostly attributable to anti-nuclear antibodies (ANA). The reason for ANA was unclear: reactive samples were negative for Erhlichia canis antibodies; seven were feline immunodeficiency virus positive, but 15 were negative. It was not possible to determine retrospectively whether the cats had autoimmune disease, hyperthyroidism treatment, or recent vaccination which may also cause ANA. The FCoV/ FIP-free status of the Falkland Islands cats should be maintained by FCoV testing incoming cats. However, ANA can complicate interpretation of IFA tests.     

Polymorphisms in the feline TNFA and CD209 genes are associated with the outcome of feline coronavirus infection

Feline infectious peritonitis (FIP), caused by feline coronavirus (FCoV) infection, is a highly lethal disease without effective therapy and prevention. With an immune-mediated disease entity, host genetic variant was suggested to influence the occurrence of FIP. This study aimed at evaluating cytokine-associated single nucleotide polymorphisms (SNPs), i.e., tumor necrosis factor alpha (TNF-α), receptor-associated SNPs, i.e., C-type lectin DC-SIGN (CD209), and the five FIP-associated SNPs identified from Birman cats of USA and Denmark origins and their associations with the outcome of FCoV infection in 71 FIP cats and 93 FCoV infected non-FIP cats in a genetically more diverse cat populations. A promoter variant, fTNFA - 421 T, was found to be a disease-resistance allele. One SNP was identified in the extracellular domain (ECD) of fCD209 at position +1900, a G to A substitution, and the A allele was associated with FIP susceptibility. Three SNPs located in the introns of fCD209, at positions +2276, +2392, and +2713, were identified to be associated with the outcome of FCoV infection, with statistical relevance. In contrast, among the five Birman FIP cat-associated SNPs, no genotype or allele showed significant differences between our FIP and non-FIP groups. As disease resistance is multifactorial and several other host genes could involve in the development of FIP, the five genetic traits identified in this study should facilitate in the future breeding of the disease-resistant animal to reduce the occurrence of cats succumbing to FIP.

Electronic supplementary material

The online version of this article (doi:10.1186/s13567-014-0123-6) contains supplementary material, which is available to authorized users.     

Long-term impact on a closed household of pet cats of natural infection with feline coronavirus, feline leukaemia virus and feline immunodeficiency virus

A closed household of 26 cats in which feline coronavirus (FCoV), feline leukaemia virus (FeLV) and feline immunodeficiency virus (FIV) were endemic was observed for 10 years. Each cat was seropositive for FCoV on at least one occasion and the infection was maintained by reinfection. After 10 years, three of six surviving cats were still seropositive. Only one cat, which was also infected with FIV, developed feline infectious peritonitis (FIP). Rising anti-FCoV antibody titres did not indicate that the cat would develop FIP. The FeLV infection was self-limiting because all seven of the initially viraemic cats died within five years and the remainder were immune. However, FeLV had the greatest impact on mortality. Nine cats were initially FIV-positive and six more cats became infected during the course of the study, without evidence of having been bitten. The FIV infection did not adversely affect the cats' life expectancy.     

Shedding of enteric coronavirus in adult cattle

Electron microscopic examination of fecal specimens from adult dairy cows indicated seasonal coronavirus shedding. Fifty-two cows from a 300 cow herd were monitored for shedding of coronavirus. Approximately 50% to 60% of the cows monitored shed coronavirus during the winter months (November to March) of the first year of the study. During 2 subsequent years of monitoring the same cows, 20% to 30% of the cows shed coronavirus during the winter months. Virus shedding was not detected during the summer months (July to September). Half of the cows monitored were vaccinated with a modified-live rotavirus-coronavirus-Escherichia coli combination vaccine; however, vaccination did not influence seasonal shedding of coronavirus, as compared with shedding in the nonvaccinated cows. In nonvaccinated cows that calved in the winter months, the incidence of coronavirus shedding increased from 20% to 30% during the last 2 months of gestation to 65% to 70% at parturition. In vaccinated cows, the incidence of shedding did not increase at parturition.     

Detection of antigenic heterogeneity in feline coronavirus nucleocapsid in feline pyogranulomatous meningoencephalitis

A new monoclonal antibody (mAb), CCV2-2, was compared with the widely used FIPV3-70 mAb, both directed against canine coronavirus (CCoV), as a diagnostic and research tool. Western blot showed that both anti-CCoV mAbs only reacted with a protein of 50 kD, a weight consistent with the feline coronavirus (FCoV) viral nucleocapsid. A competitive inhibition enzyme-linked immunosorbent assay showed that the 2 recognized epitopes are distinct. Preincubation of CCV2-2 mAb with FCoV antigen suppressed the immunostaining. Formalin-fixed, paraffin-embedded sections from brains of 15 cats with the dry form of feline infectious peritonitis (FIP) were examined by immunohistochemistry. Immunohistochemistry was performed with both anti-CCoV mAbs, either on consecutive or on the same sections. A myeloid-histiocytic marker, MAC 387, was also used to identify FIP virus-infected cells. In all regions where MAC 387-positive cells were present, positive staining with the CCV2-2 mAb was systematically detected, except at some levels in 1 cat. In contrast, none or only a few cells were positive for the FIPV3-70 mAb. Double immunostaining showed macrophages that were immunopositive for either CCV2-2 alone or alternatively for CCV2-2 and FIPV3-70 mAbs. This reveals the coexistence of 2 cohorts of phagocytes whose FIP viral contents differed by the presence or absence of the FIPV3-70-recognized epitope. These findings provide evidence for antigenic heterogeneity in coronavirus nucleocapsid protein in FIP lesions, a result that is in line with molecular observations. In addition, we provide for the first time morphologic depiction of viral variants distribution in these lesions.     

Common virus infections in cats, before and after being placed in shelters, with emphasis on feline enteric coronavirus

The purpose of this study was to determine the origin and subsequent spread of feline calicivirus (FCV), feline herpesvirus (FHV), and feline enteric coronavirus (FECV) in cats relinquished to shelters. FCV was isolated from the oral fauces of 11% of healthy cats upon entry, and isolation rates were highest for kittens (33%). FHV shedding was very low (4%) at the time of entry and occurred mainly in juveniles. FECV shedding was also common among newly relinquished cats (33%), especially older kittens and juveniles (90%). The subsequent spread of all three viruses was rapid and efficient in the shelter environment. Fifteen percent of cats were shedding FCV, 52% FHV, and 60% FECV after 1 week. More detailed studies were done with FECV shedding, which could be accurately quantitated. The amounts of FECV shed by infected cats ranged from 10(2)to 10(16)particles/swab of feces. FECV shedding was several logs higher in young kittens with primary infection than adult cats with primary infections. The mean levels of FECV shedding among adults were the same for primary and chronic infections. Although shelters were not the primary source of these viruses for many relinquished cats, factors intrinsic to the shelter environment were critical in amplifying shedding and spread to susceptible individuals. Extrinsic factors were especially important for the spread of FHV and FECV. FHV shedding rates increased from 4% to 50% in 1 week's time. The speed and magnitude of the increase in FHV shedding suggested that there was reactivation of latent infections as well as acquisition of new infections. FECV shedding increased 10 to 1,000,000 fold in 1 week among cats that were already infected at entry, and more than one-half of initially negative cats were shedding FECV a week later. Feline calicivirus infection was the least likely to spread in the shelter. The infection rate only increased from 11 to 15% in 1 week.     

Changes in some acute phase protein and immunoglobulin concentrations in cats affected by feline infectious peritonitis or exposed to feline coronavirus infection

The possible role of some acute phase proteins (APPs) and immunoglobulins in both the pathogenesis and diagnosis of feline infectious peritonitis (FIP) has been investigated. Serum protein electrophoresis and the concentration of haptoglobin (Hp), serum amyloid A (SAA), alpha(1)-acid glycoprotein (AGP), IgG and IgM were evaluated in cats exposed to feline coronavirus (FCoV) and in cats with FIP. The highest concentration of APPs was detected in affected cats, confirming the role of these proteins in supporting a clinical diagnosis of FIP. Repeated samplings from both FIP affected and FCoV-exposed cats showed that when FIP appeared in the group, all the cats had increased APP levels. This increase persisted only in cats that developed FIP (in spite of a decrease in alpha(2)-globulins) but it was only transient in FCoV-exposed cats, in which a long lasting increase in alpha(2)-globulins was observed. These results suggest that changes in the electrophoretic motility of APPs or APPs other than Hp, SAA and AGP might be involved in the pathogenesis of FIP or in protecting cats from the disease.