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Immunofluorescent and electron microscopic studies of feline small intestinal organ cultures infected with feline infectious peritonitis virus.

The pathogenesis, morphology, and morphogenesis of feline infectious peritonitis virus were studied in feline small intestinal organ cultures. Characteristic coronaviruses were demonstrated by electron microscopy in the extracellular organ culture fluid taken at 1 through 12, 15, 22, 31, and 43 days after the cultures were inoculated. Viral particles had a mean diameter of 94 nm. Numerous petal-shaped projections approximately 15 nm long were seen on the envelopes. Specific immunofluorescence was detected in the absorptive epithelial cells of villi of small intestinal organ cultures at 1 through 12 days after they were inoculated. By electron microscopy, coronavirus particles were observed entirely in the cytoplasm of the infected columnar epithelial cells of villi with no apparent nuclear involvement. Thin sections of the villous epithelium of small intestinal organ cultures showed a number of ultrastructural alterations, and morphogenesis of feline infectious peritonitis virus in the absorptive epithelial cells of villi was consistent with that for other known human animal coronaviruses.

Animals↗

Feline infectious peritonitis viruses arise by mutation from endemic feline enteric coronaviruses.

Feline infectious peritonitis virus (FIPV) strains from six cats and three different geographic areas were compared genetically with feline enteric coronavirus (FECV) isolates obtained from cats inhabiting the same environments. Sequence comparisons were made from 1.2- to 8.9-kb segments on the 3' end of the genome. FECV/FIPV pairs from the same catteries or shelters were 97.3-99.5% related but were genetically distinct from FIPV and FECV strains obtained from cats living in geographically distinct environments. The high genetic similarity between FECVs and FIPVs from the same environment strongly suggested a common ancestry. Based on the presence of deletion mutations in the FIPVs and not in the FECVs, it was concluded that FIPVs evolved as mutants of FECVs. The mutations are deletions in the FIPVs and not insertions in the FECVs since similar sequences are present in other strains that have segregated earlier from a common ancestor. Therefore, the order of descent is form FECV to FIPV. Mutations unique to FIPVs were found in open reading frames (ORFs) 3c in 4 of 6 isolates and/or 7b in 3 of 6 isolates. When the study was extended to include 7 additional FIPV isolates, 11/13 of the FIPVs sequenced were found to have mutated 3c ORFs.

Amino Acid Sequence↗

Development of a nested PCR assay for detection of feline infectious peritonitis virus in clinical specimens.

A diagnostic test for feline infectious peritonitis virus (FIPV) infection based on a nested PCR (nPCR) assay was developed and tested with FIPV, feline enteric coronavirus (FECV), canine coronavirus (CCV), and transmissible gastroenteritis virus (TGEV) and clinical fluid samples from cats with effusive feline infectious peritonitis (FIP). The target sequence for the assay is in the S1 region of the peplomer protein E2 gene. A vaccine strain of FIPV and two wild-type FIPV strains tested positive, but FECV, TGEV, and CCV tested negative. Preliminary tests with 12 cats with clinical evidence of effusive FIP and 11 cats with an illness associated with effusions, but attributed to other causes, were performed. Eleven of the 12 cats with effusive FIP tested positive, while 1 was negative. Ten of the 11 cats ill from other causes tested negative, while 1 was positive. On the basis of clinical laboratory and histopathologic criteria, the preliminary sensitivity and specificity of the assay were 91.6 and 94%, respectively.

Animals↗

Risk of feline infectious peritonitis in cats naturally infected with feline coronavirus.

A longitudinal survey of 820 cats in 73 households was conducted over a period of 6 years to establish the fate of pet cats that were seropositive after natural exposure to feline coronavirus (FCoV). In particular, their risk of developing feline infectious peritonitis (FIP) was determined. The seropositive cats were assigned to 1 of 3 groups: cats from households in which FIP had recently been diagnosed; cats from households in which FIP had not been diagnosed, but from which kittens had been relocated and subsequently died of FIP; and cats from households in which FIP had not been diagnosed. Cats in the first group were not at greater risk of developing FIP than were cats in the other 2 groups. Consequently, any household in which seropositive cats live must be considered a potential source of FCoV that can cause FIP. There was no evidence that the enhanced disease, which has been described after experimentally induced infection of seropositive cats, exists in nature. Thus, analysis of the survival of the seropositive cats over periods of up to 36 months indicated that their risk of developing FIP decreased with time, suggesting the development of immunity rather than increased susceptibility to disease. In addition, of 56 cats deemed to have been naturally reinfected because their anti-FCoV antibody titers decreased and subsequently increased, only 3 developed FIP.

Animal Feed↗

Characterization of monoclonal antibodies against feline infectious peritonitis virus type II and antigenic relationship between feline, porcine, and canine coronaviruses.

Seven monoclonal antibodies (MAbs) with neutralizing activity against feline infectious peritonitis virus (FIPV) strain 79-1149 (type II) were prepared. When the polypeptide specificity recognized by these monoclonal antibodies (MAbs) was investigated by Western immunoblotting, all of the MAbs reacted with peplomer glycoprotein (S) of the virus. By competitive binding assay these MAbs were found to recognize at least 3 different epitopes. The reactivity of these MAbs with 6 viruses classified as FIPV type I (UCD-1, UCD-2, UCD-3, UCD-4, NW-1, and Black), feline enteric coronavirus (FECV) type II strain 79-1683, canine coronavirus (CCV) strain 1-71, and transmissible gastroenteritis virus (TGEV) strains TO-163 and SH was examined by neutralization tests. All MAbs neutralized FECV strain 79-1683, CCV strain 1-71, and TGEV strains TO-163 and SH, while they did not neutralize the 6 FIPV type I viruses. Moreover, the MAb against TGEV strain TO-163, which has strong neutralizing activity against 7 TGEV viruses, neutralized CCV strain 1-71, FECV strain 79-1683, and FIPv strain 79-1146, but did not neutralize the 6 FIPV type I viruses. These results demonstrated that there are at least 3 epitopes involved in the neutralization of FIPV type II strain 79-1146, and that these epitopes are not present in FIPV type I viruses but are present in FECV strain 79-1683 which does not induce feline infectious peritonitis, TGEV strains TO-163 and SH, and CCV strain 1-71. These results suggest the presence of 2 serotypes of FIPV which can be clearly distinguished by the neutralization test using MAbs.

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cDNA cloning and sequence analysis of the gene encoding the peplomer protein of feline infectious peritonitis virus.

The peplomer gene of feline infectious peritonitis virus (FIPV) strain 79-1146 was isolated from a genomic cDNA library by differential hybridization with RNA 2 and 3 as probes. From the nucleotide sequence a primary translation product of 1452 residues (Mr 160,472) was predicted, containing an N-terminal signal sequence, a C-terminal transmembrane segment and 35 potential N-linked glycosylation sites. By S1 nuclease analysis the 5' end of the presumptive RNA 2 body was located at about 30 nucleotides upstream from the initiating AUG codon. At approximately the same position a nine nucleotide sequence ACUAAACUU was found, which was also present 37 nucleotides downstream from the open reading frame. Comparison of the sequences of the FIPV, murine hepatitis virus and infectious bronchitis virus peplomer proteins showed about 27% overall homology, with most conservation in the C-terminal half.

Amino Acid Sequence↗

Cellular composition, coronavirus antigen expression and production of specific antibodies in lesions in feline infectious peritonitis.

Twenty-three cats with spontaneous feline infectious peritonitis (FIP) were examined by light microscopy including immunohistology and histochemistry in order to determine the cellular composition and the expression of viral antigen in lesions in FIP. Furthermore, the presence of plasma-cells producing coronavirus-specific antibodies was evaluated in situ. Macrophages and neutrophils were demonstrated by an antibody against calprotectin (leukocyte protein L1, myeloid/histiocyte antigen), neutrophils were recognized due to their chloroacetate esterase activity, and B- and T-lymphocytes were identified by antibodies against the CD3 antigen and the CD45R antigen, respectively. Expression of viral antigen was immunohistologically demonstrated by a monoclonal antibody (mAb) against coronavirus while coronavirus-specific antibodies in situ were identified by the application of feline coronavirus prior to the coronavirus antibody. Lesions were classified as diffuse alterations at serosal surfaces, granulomas with areas of necrosis, granulomas without extended necrosis, focal and perivascular lymphoplasmocytic infiltrates, and granulomatous-necrotizing vasculitis. Diffuse alterations on serosal surfaces were represented either by activated mesothelial cells with single coronavirus antigen-bearing macrophages or by layers of precipitated exudate containing single to numerous granulomas with areas of necrosis. In liver and spleen, the exudate was often underlaid by a small band of subcapsular B-cells with an occasional plasma-cell producing coronavirus-specific antibodies. In other locations, a variably broad band of B-cells and plasma-cells, often infiltrating between underlying muscle fibers, separated the exudate from the unaltered tissue. Some of these plasma-cells were positive for coronavirus-specific antibodies. In granulomas with areas of necrosis, the central necrosis was surrounded by macrophages usually expressing considerable amounts of viral antigen. Few B-cells and plasma-cells were found in the periphery. In granulomas without extended necrosis, the number of macrophages were lower. Only few macrophages expressing low amounts of viral antigen were present. B-cells and plasma-cells formed a broad rim. Few plasma-cells stained positive for coronavirus-specific antibodies. In both types of granulomas, few neutrophils were found between macrophages. Few T-cells were seen scattered throughout the lesions. Focal and perivascular lymphoplasmocytic infiltrates were mainlyseen in omentum and leptomeninx. B-cells were the predominant cells; some plasma-cells were positive for coronavirus-specific antibodies. Viral antigen was not readily detected in these alterations. Granulomatous-necrotizing vasculitis was occasionally found in kidneys and leptomeninx. It was dominated by macrophages which often stained strongly positive for coronavirus antigen. Different types of alteration were often seen in the same animal and even the same tissue. There was no obvious correlation between the cat's age, gross pathological changes, and the histological types of alteration. Single plasma-cells positive for coronavirus-specific antibodies were found around blood vessels distant from inflammatory alterations, within the lung parenchyma, as infiltrating cells in the mucosa of the small intestine, and in spleen and mesenteric lymph node. Results show that alterations in FIP are heterogeneous concerning cellular composition and expression of viral antigen. The dominance of B-cells in part of the lesions together with the presence of plasma-cells positive for coronavirus-specific antibodies indicate that these cells may play a role in the maintenance of inflammatory processes in FIP.

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Induction and enhancement of feline infectious peritonitis by canine coronavirus.

Preexisting antibody to feline infectious peritonitis virus (FIPV) causes acceleration and enhancement of disease on subsequent infection of cats with FIPV. Other workers have shown that canine coronavirus (CCV) can infect cats subclinically, but have found no evidence of enhancement of, or protection against, subsequent FIPV infection. With various isolates of CCV, we determined that 1 strain of CCV can induce transient mild diarrhea in cats and, furthermore, that previous infection with CCV causes acceleration and enhancement of subsequent infection with FIPV. In addition, sequential inoculation of cats with another strain of CCV caused lesions indistinguishable from those of FIP, without exposure at any time to FIPV.

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Biological and pathological consequences of feline infectious peritonitis virus infection in the cheetah.

An epizootic of feline infectious peritonitis in a captive cheetah population during 1982-1983 served to focus attention on the susceptibility of the cheetah (Acinoyx jubatus) to infectious disease. Subsequent observations based upon seroepidemiological surveys and electron microscopy of fecal material verified that cheetahs were indeed capable of being infected by coronaviruses, which were antigenically related to coronaviruses affecting domestic cats, i.e. feline infectious peritonitis virus/feline enteric coronavirus. Coincident with the apparent increased susceptibility of the cheetah to infectious diseases, were observations that the cheetah was genetically unusual insofar as large amounts of enzyme-encoding loci were monomorphic, and that unrelated cheetahs were capable of accepting allogenic skin grafts. These data provided the basis for a hypothesis that the cheetah, through intensive inbreeding, had become more susceptible to viral infections as a result of genetic homogeneity.

Acinonyx↗

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.

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Immunohistological demonstration of feline infectious peritonitis virus antigen in paraffin-embedded tissues using feline ascites or murine monoclonal antibodies.

Feline infectious peritonitis (FIP) virus antigen was demonstrated after methanol, ethanol or formalin fixation in paraffin-embedded tissues by means of monoclonal and polyclonal antibodies. The monoclonal antibody was induced by immunization with transmissible gastroenteritis virus. Polyclonal antibodies were obtained by purification on protein A-Sepharose of ascites fluid from a cat with FIP. Almost all cats diagnosed as suffering from FIP by postmortem and histological examination exhibited FIP virus (FIPV) antigen in macrophages in granulomas whereas FIPV antigen was only once demonstrable in another location.

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Feline leucocyte antigen class II polymorphism and susceptibility to feline infectious peritonitis.

There are four outcomes to feline coronavirus (FCoV) infection: the development of feline infectious peritonitis (FIP, which is immune-mediated), subclinical infection, development of healthy lifelong carriers and a small minority of cats who resist infection (Addie and Jarrett, Veterinary Record 148 (2001) 649). Examination of the FCoV genome has shown that the same strain of virus can produce different clinical manifestations, suggesting that host genetic factors may also play a role in the outcome of infection. FIP is most prevalent amongst pedigree cats, although how much of this is due to them living in large groups (leading to higher virus challenge and stress which predisposes to FIP) and how much is due to genetic susceptibility is not known. If host genetics could be shown to play a role in disease, it may allow the detection of cats with a susceptibility to FIP and the development of increased population resistance through selective breeding. The feline leucocyte antigen (FLA) complex contains many genes that are central to the control of the immune response. In this preliminary study, we used clonal sequence analysis or reference strand conformational analysis (RSCA) to analyse the class II FLA-DRB of 25 cats for which the outcome of FCoV exposure was known. Individual cats were shown to have between two and six FLA-DRB alleles. There was no statistically significant association between the number of alleles and the outcome of FCoV infection. No particular allele appeared to be associated with either the development of FIP, resistance to FCoV, or the carrier status. However, the analysis was complicated by apparent breed variation in FLA-DRB and the small number of individuals in this study.

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The sites of early viral replication in feline infectious peritonitis.

The sites of early replication of feline infectious peritonitis virus were studied following oral inoculation of specific-pathogen-free (SPF) cats with virus grown in cell cultures. Viral antigen was first detected by immunofluorescence in the tonsils and small intestine within 24 h of inoculation, and was later found in caecum, colon, mesenteric lymph nodes and liver. However, histological changes in the gut did not appear until relatively late in the course of infection. Virus was recovered from the oropharynx and the faeces from as early as the second or third day after inoculation, and shedding continued until euthanasia.

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Inhibition of feline infectious peritonitis virus replication by recombinant human leukocyte (alpha) interferon and feline fibroblastic (beta) interferon.

Replication of feline infectious peritonitis virus (FIPV) in feline cell cultures was inhibited after incubation of cells with either human recombinant leukocyte (alpha) interferon (IFN) or feline fibroblastic (beta) IFN for 18 to 24 hours before viral challenge exposure. Compared with virus control cultures, FIPV yields were reduced by ranges of 0.1 to 2.7 log10 or 2 to 5.2 log10 TCID50 in cultures treated with human alpha- or feline beta-IFN, respectively; yield reductions were IFN dose dependent. Sensitivity to the antiviral activities of IFN varied with cell type; feline embryo cells had greater FIPV yield reductions than did similarly treated feline kidney or feline lung cells. Comparison of the virus growth curves in IFN-treated and virus control cultures indicated marked reduction in intracellular and extracellular FIPV in IFN-treated cultures. Compared with virus control cultures, intracellular and extracellular infectivity in IFN-treated cultures was delayed in onset by 12 and 30 hours, respectively, and FIPV titers subsequently were reduced by 3 to 3.5 and 5 log10 TCID50, respectively. Frequently, immunofluorescent and electron microscopy of IFN-treated cells or cell culture fluids did not reveal virus; however, even in cultures without viral cytopathic changes, small amounts of virus occasionally persisted in cells.

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Characterization of a feline infectious peritonitis virus isolate.

A virus isolated in cell culture from the spleen of a cat with feline infectious peritonitis was identified by physicochemical, morphological and antigenic criteria as a coronavirus. The feline infectious peritonitis virus was compared in vitro with canine coronavirus, a reported enteric pathogen of dogs. The feline isolate was characterized, by chloroform sensitivity and resistance to 5-iododeoxyuridine, respectively, as containing essential lipid and an RNA genome. Other traits of the isolate included resistance to acidic conditions, heat lability, and resistance to trypsin. Electron microscopy showed viral particles with a structure consistent with that of the prototype of the coronavirus group, infectious bronchitis virus. Indirect immunofluorescence with canine coronavirus monospecific antiserum showed the viral isolate to be antigenically related to canine coronavirus. Specific-pathogen-free cats inoculated by various routes with cell-culture-propagated virus had both clinical symptoms and lesions consistent with feline infectious peritonitis.

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Laboratory profiles in cats with different pathological and immunohistochemical findings due to feline infectious peritonitis (FIP).

Blood was collected from 55 cats with feline infectious peritonitis (FIP) and from 50 control cats in order to define whether differences in pathological findings and in distribution of feline coronaviruses (FCoV) can be associated with changes in haemograms, serum protein electrophoresis, and antibody titres. Compared to controls, the whole group of FIP-affected cats had blood changes consistent with FIP. Based on the pathological findings or on the immunohistochemical distribution of viral antigen, FIP-affected cats were divided in the following groups: subacute against acute lesions; low against strong intensity of positivity; intracellular against extracellular positivities; positive against negative lymph nodes. Lymphopenia was more evident in cats with acute forms, strong intensity of positivity, extracellular antigen and negative lymph nodes. Cats with positive lymph nodes had the most evident changes in the protein estimations. These results suggest that differences in pathological findings might depend on different reactive patterns to the FCoVs.

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Acquisition of macrophage tropism during the pathogenesis of feline infectious peritonitis is determined by mutations in the feline coronavirus spike protein.

In feline coronavirus (FCoV) pathogenesis, the ability to infect macrophages is an essential virulence factor. Whereas the low-virulence feline enteric coronavirus (FECV) isolates primarily replicate in the epithelial cells of the enteric tract, highly virulent feline infectious peritonitis virus (FIPV) isolates have acquired the ability to replicate efficiently in macrophages, which allows rapid dissemination of the virulent virus throughout the body. FIPV 79-1146 and FECV 79-1683 are two genetically closely related representatives of the two pathotypes. Whereas FECV 79-1683 causes at the most a mild enteritis in young kittens, FIPV 79-1146 almost invariably induces a lethal peritonitis. The virulence phenotypes correlate with the abilities of these viruses to infect and replicate in macrophages, a feature of FIPV 79-1146 but not of FECV 79-1683. To identify the genetic determinants of the FIPV 79-1146 macrophage tropism, we exchanged regions of its genome with the corresponding parts of FECV 79-1683, after which the ability of the FIPV/FECV hybrid viruses to infect macrophages was tested. Thus, we established that the FIPV spike protein is the determinant for efficient macrophage infection. Interestingly, this property mapped to the C-terminal domain of the protein, implying that the difference in infection efficiency between the two viruses is not determined at the level of receptor usage, which we confirmed by showing that infection by both viruses was equally blocked by antibodies directed against the feline aminopeptidase N receptor. The implications of these findings are discussed.

Amino Acid Sequence↗