key: cord-0836338-492xi3ee
authors: Evermann, J. F.; Heeney, J. L.; Roelke, M. E.; McKeirnan, A. J.; O'Brien, S. J.
title: Biological and pathological consequences of feline infectious peritonitis virus infection in the cheetah
date: 1988
journal: Arch Virol
DOI: 10.1007/bf01310822
sha: 120d23660fed4a3bd15050de3ccf4205ffa4f85c
doc_id: 836338
cord_uid: 492xi3ee

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.

Research aimed toward understanding the pathogenesis of viral infections of endangered species constitutes a major component of the species survival management plan for the cheetah [38, 56] . One of the primary reasons for this emphasis was the recent occurrence of a devastating epizootic of feline coronavirus infection (feline infectious peritonitis [FIP] ) in captive cheetahs in 1982-1983 [26, 29, 72] . Since that time, there have been other reports on the apparent susceptibility of the cheetah to infectious diseases with emphasis on FIP [-4, 10, 38, 62, 63, 93] . Since there are currently no vaccines available for control of FIP in either domestic or exotic cat populations it is essential that biologists, diagnosticians, and veterinarians be aware of the impact that this infection has upon the cheetah, so that appropriate management steps can be taken to minimize the chance for infection and thereby lessen the risk of fatal disease. The purpose of this review is to present desciptions of the various forms of coronaviral infections in the cheetah relying upon studies of both natural infections, as well as experimental infections in other species with coronaviruses, such as mouse hepatitis virus (MHV), canine coronavirus (CCV), transmissible gastroenteritis virus (TGEV) of swine, and bovine coronavirus (BCV) of neonatal calves [28, 36, 39, 49, 57, 60, 61, 75, 79, 92] .

The biology of the feline coronaviruses has a short history since isolates were not available for laboratory studies until 1976 [see 66 for review]. Prior to that time, the disease was referred to as feline systemic proliferative and exudative vasculitis [30, 40] , and later feline infectious peritonitis (FIP) [91] . Recognition of the first coronavirus associated with FIP in cats was determined by electron microscopy in 1970 [86] . Inasmuch as cell culture-adapted isolates of the FIP virus (FIPV) were not yet available, the disease condition was experimentally transmitted by the inoculation of liver homogenates from diseased cats into the peritoneal cavity of susceptible cats. During the period of experimental transmission of FIP by tissue homogenates, serologic assays were developed which utilized indirect immunofluorescence on cryostat sections of liver obtained from cats with clinical FIP [66] . Subsequent serologic studies relied upon cell culturegrown homologous virus, FIPV, or heterologous cross-reacting coronavirus strains, such as CCV and TGEV [25, 33, 42~44, 59, 66] .

Seroprevalence studies conducted during the 1970s indicated that certain populations of cats had a high percentage of antibody, suggesting that either FIPV was not 100% fatal as was generally believed, or that cats were also being [4, 66, 68, 69] . Since those initial reports on the divergent nature of feline coronaviruses and the designation of FIPV and FECV strains, there have been additional reports on the detection and/or isolation of feline coronaviruses [4, 66] . The spectrum of disease resulting from these various isolates is presented in Table 1 . Although there are marked phenotypic differences in terms of in vivo virulence, efforts to distinguish FECV strains from FIPV strains in vitro have not been successful [8, 31, 32, 85] . It is conceivable that molecular technology will reveal differences at the genomic level, which reflect variation in strain virulence or pathology [18, 19, 46, 7] . Diagnostic molecular procedures would be useful in the detection of cats that were shedding FIPV into the environment. However, if FIPV is the progeny of a random mutation from FECV, or a recombinant of two enteric coronaviruses, then the application would vary according to the frequency of these mutational events [51, 54, 66, 70, 78, 80, 90] .

Although the majority of research on the feline coronaviruses has been conducted in domestic cats, access to serologic tests has allowed investigators to test serum from other Felidae, as well as from closely related members of the cat family, such as the cheetah [43, 66] . The serologic results, as well as reports of sporadic cases of FIP in cheetahs, indicated that they were susceptible to infection and in some cases succumbed to disease [10, 26, 29, 43, 62] . The susceptibility of the cheetah to FIP was of particular interest during an outbreak of the disease in a wildlife park in 1982-1983 [26, 29] . In this incident, an infected cheetah was imported to the facility, which was a leading center for breeding cheetahs in North America. 

Infectious agents were isolated in Crandell feline kidney (CrFK) cells from tissues submitted from 3 cheetahs that died during the FIP epizootic of 1982-1983 [26, 29] . A common feature of the isolates was their reduced cytopathic effect (CPE) in CrFK cells and cytoplasmic immunofluorescence when stained with antisera to a strain of moderate virulence, FIPV UCD-1 ( Fig. 1 a, The cheetah virus has been maintained in cell culture as a persistent noncytolytic infection [27, 29] . However, infected cells do express periods of cytopathogenicity referred to as "crisis periods", but following each crisis, cells emerge and the infection is maintained. Assays for cell-free, virus have been hampered due to the low release of extracellular virus. Ultrastructural studies conducted on the cells persistently infected by the cheetah coronavirus have revealed the presence of virus particles in cytoplasmic vacuoles, but minimal virus at the cell surface [27] . This is in contrast to the high number of virus particles observed, both within virus-infected cells, as well as at the surface of cells with the cytopathogenic coronaviruses, FECV 79-1683 and FIPV 79-1146 [27] .

The phenotypic parameters of the cheetah coronavirus in vitro indicate that the virus is a biological variant of a virulent coronavirus or is in a partially nonpermissive host cell [27] . This is further suggested by its cell-associated nature and the lack of cell fusion capability, which would indicate incomplete expression of the essential E2 protein (fusion protein) [21, 37, 39, 82] . This observation may be due to a partially defective virus [55] or to a virus which fails to mature properly due to the absence of a host-cell protease [39, 89] . The cheetah agent represents the least virulent in cell culture as compared with other feline coronaviruses (Table 1 ) except for FECV UCD, which has not yet been cultured in vitro, but must be maintained by passage through cats [66, 683. Enzymatic enhancement of coronavirus replication and expression of CPE in vitro have been reported for a number of different coronaviruses including BCV and TGEV [2, 13, 14, 58, 84] . The addition of low amounts of trypsin (5 Ixg/ml) to serum-free culture media enhances the CPE of the feline corona- viruses FIPV UCD-1 and FIPV UCD-4, and also appears to augment the expression of the cheetah coronavirus [27] . Studies on the effects of trypsin on various coronavirus strains, including FECV and FIPV, have suggested that trypsin-sensitive and trypsin-resistant phenotypes occur in nature [13, 58] . Those strains that are highly resistant have been hypothesized to survive longer in the gastrointestinal tract and therefore be infectious longer, both for the infected host, as well as for other susceptible animals in the population which come in contact with the shedding host [15, 16] .

Initially, definitive information on the natural spread of the feline coronaviruses was hampered due to the lack of cell-free virus; however, subsequent studies with cell-free The incubation periods for feline coronavirus infections are variable depending upon the dose and strain of virus and the age of and route of entry into the host. Pedersen has conducted the most definitive studies to date on the pathogenicity of the viruses [66, 70] . His results indicated an incubation period of 4-12 days for FECV-induced enteric infection, and 8 days to 3 months for the FIPV strains. In some cases the outcome of FIPV infection is dependent upon concurrent immunosuppression such as may occur with feline leukemia virus infection [70] .

The clinical features of the feline coronaviruses in domestic cats and cheetahs appear to be very similar ]-4, 7, 29, 66, 72, 74] . Symptoms in domestic cats following FECV infection may range from subclinical to a mild diarrhea in uncomplicated cases [66] . Although not well documented in cats, multiple enteric infections with other viruses or bacteria should be considered in making a differential diagnosis. This has been reported to be the case in dogs with concurrent CCV and canine parvovirus infections [28] .

The clinical manifestations of FIP include anorexia, icterus, and elevated serum proteins in fluids within the thoracic/abdominal cavities (wet form). Although experimental studies of FIP in cheetahs has not been conducted, information obtained from naturally occurring cases has indicated that the majority show early signs of liver dysfunction followed by periods of anorexia, dehydration and death [26, 29] . It is important to recognize that although cheetahs appear to be very susceptible to FIP-related disease, there are other factors which may contribute to a generalized liver dysfunction, such as dietary deficiencies [34, 76] .

Pathologic studies of cheetahs diagnosed as having FIP have many features in common with the lesions reported in domestic cats [1, 25, 26, 29, 66] . A predominant feature in the FIP lesions in cheetahs is multi-focal necrosis throughout many organs including the liver, kidneys, pancreas, spleen, lymph nodes, and thymus. The necrotic areas are characterized by karyorrhexis, karyolysis, cytolysis, and infiltration by lymphocytes and macrophages. Fibronecrotic plaques are present on many organs in both the pleural and peritoneal cavities. Necrotic foci containing lymphocytic infiltrates, macrophages, and neutrophils are also present in mesenteric fat and within the muscular layers of the small and large bowel. Lymphoid aggregates in the spleen and lymph nodes were depleted. The fundamental histopathological lesions are a generalized vasculitis and perivasculitis [1, 44, 66, 72, 88, 91] .

Although the course of the disease and distribution of lesions bear a close resemblance to FIP in domestic cats, it is important that a complete necropsy and detailed histological examination be conducted in order to confirm the occurrence of FIP in the cheetah [93] .

Several other lesions have been noted in cheetahs in addition to changes attributed to FIPV [26, 29] . The gastric mucosa of four cheetahs contained superficial erosions of mucosal epithelium. In most cases, the changes were relatively mild with hyperemia of superficial mucosal vessels, mild focal hemorrhages and mild fibrosis of the interstitium. Gastric ulcers have been observed in one cheetah. Epithelium within the gastric pits was generally intact, although fibroblastic response bridged gastric pits in some areas. A moderate interstitial infiltrate of lymphocytes, plasma cells and macrophages was associated with areas of erosion.

The immune response of the cat to the feline coronaviruses presents a critical factor in the pathogenesis of FIPV infection. Many of the lesions observed in cases of FIP can be directly attributed to the immune response; i.e., an immunemediated vasculitis [1, 4, 41, 47, 66] . These findings are supported by the reports of enhancement of FIP by prior infusion of passively acquired antibody and the failure of conventional vaccine preparations to protect cats against subsequent challenge [see 66, for review] .

Currently, at least three distinct cellular lineages of the cat's immune system appear to be critical in determining the outcome of FIPV infection [4, 4t, 47, 48, 66, 83] . These include the macrophage, the B-cell and the T-cell populations. The macrophage appears to be one of the primary sites for FIPV replication in vivo and was demonstrated to be a source of virus in vitro prior to the isolation of viruses in conventional cell cultures [66] . Although definitive studies have not been reported yet with FIPV, it may be predicted that cells of the monocyte-macrophage lineage are very important in conferring resistance to infection, and in subsequent immune reactions of the processed FIPV antigens with the B-and T-cell populations. Coronaviral infection of the macrophage has been reported to be one of the major criteria for distinguishing resistance and susceptibility in mice with the murine coronavirus, MHV [3, 5, 6, 9, 11, 20, 22, 52, 53] . FIP-inducing strains apparently infect and replicate in feline macrophages while non-FIP strains, e.g., the FECV strains, are primarily restricted to mucosal infections without replication in macrophages [see 66, for review].

In the majority of cases of FIP, serum antibody has been measured by the indirect immunofluorescence assay (IFA), a group-specific test that does not distinguish between antibody to FIPV and FECV [4, 26, 33, 38, 69] . Serum from cats with high IFA antibody titers has been demonstrated to enhance the pathogenesis of FIPV when passively administered to cats six hours prior to virus inoculation [66] . The role of antibody in the pathogenesis of FIP has been controversial, but is not without precedent since there are several viral infections which have been documented to have an immune-mediated disease sequelae. These include dengue fever of humans [17, 65, 87] , and yellow fever virus [35] . The mechanism(s) of antibody-mediated enhancement may take the form of non-neutralizing antibodies, enhancing antibodies, or blocking antibodies (bind to/or block T-cell receptors) [see 73 for review]. A likely explanation is that certain strains of feline coronavirus, i.e., FIPV variants or mutants [51, 78, 80, 90] , infect and replicate in macrphage cells, perhaps altering normal B-and T-cell interactions. The B-cell response is polyclonal and antibodies that are formed appear to detect all the major viral proteins as determined by Western blot techniques [38] . Certain types of these antibodies may serve as enhancing antibodies in terms of increased viral uptake and replication within macrophages, which are subsequently spread throughout the body.

The effectiveness of the T-cell response appears to play a critical role in controlling FIPV infection, but may also play a part in the subsequent immunemediated pathogenesis. Impairment of the T-cell response, such as may occur during concurrent viral infection with either feline panleukopenia virus or feline leukemia virus, appears to predispose cats to FIP [66, 70, Evermann, unpublished observation]. Without T-cell surveillance of virus-infected macrophages, cell-associated viremia occurs and dispersal of virus occurs throughout the body [88] . The actual mechanism(s) of FIPV-induced tissue damage is being investigated, but may be a combination of inflammatory response with neutrophils imparting tissue damage alone or in combination with cytotoxic T cells attempting to rid tissues of virus-infected macrophages [23] . The basic lesions in FIP are a result of the cat's immune response producing large quantities of antibodies and the formation of immune complexes which are subsequently deposited within the vessels of the serosa [41, 44, 66, 87, 88] . Persistence of viral antigens in the serosal blood vessels provokes a hypersensitivity reaction with the migration and infiltration of mononuclear cells into vessel adventitia and media, resulting in severe vascular damage [87, 88] . The outcome from the vessel damage usually includes a serous effusion and accumulation of fluid in the major body cavities. This type of immune response is characteristic of a type III hypersensitivity reaction [44, 66, 87, 88] .

The genetic regulation of the immune response has been of interest in studying viral infections of animals, since it has been shown that resistance to certain viruses appears to be under genetic control [3, 6, 9, 24] . More recently, the cheetah has been shown to be genetically unusual insofar as large amounts of enzyme-encoding loci are monomorphic in natural populations [63, 64, 94] . This observation, coupled with the extraordinary finding that unrelated cheetahs would immunologically accept allogenic skin grafts, was interpreted in the context of a hypothesis that the cheetah had undergone intensive inbreeding in its recent natural history [63] . Such a situation would have significant effect upon the immune defenses against microbial pathogens. By analogy to other species, natural populations have multiple loci which are polymorphic for functions which influence the outcome of viral-induced disease outbreaks [-62] . Notable amongst such genes are those forming the major histocompatibility complex, whose gene products function directly in monitoring immune response to viral infections [3, 5, 6, 94] . The genetic homogeneity of the cheetah may be an important contributing factor in the increased susceptibility of the species to viral infections, such as FIPV and feline herpesvirus [26, 71, 72] .

Since the fatal outcome of the FIP epizootic of 1982-1983, it has been apparent that cheetahs are highly vulnerable to infection and disease from the feline coronaviruses [26, 29, 62, 63] . In an effort to determine the prevalence of feline coronavirus infection in cheetahs, a serologic survey and an electron microscopic analysis were conducted on captive and wild-caught cheetahs [38] . The serological results from captive cheetahs in 14 zoologic parks in North America (Table2) indicated that 25 of 89 (28%) captive cheetahs were seropositive to feline coronavirus by IFA (group-specific serology test). These results were higher than those of captive cheetahs located in southern Africa and Europe, but similar to captive cheetahs in eastern Africa as well as to wild-caught cheetahs ( Table 3 ). The serologic results indicated that the cheetahs were being infected by a virus which was antigenically related to the feline coronavirus group, and that there was a comparable risk of acquiring infection whether 

Efforts to control FIP in captive cheetahs are structured around periodic serologic testing and the segregation of cheetahs that are seropositive to feline coronaviruses by IFA. The limitations of these recommendations are the possible lack of specificitly of IFA for identifying exposure to pathological variants, and the lack of complete understanding of the coronaviruses affecting cheetahs [4, 27, 38, 66] . However, until further information is reported regarding coronaviral diseases of exotic cats, especially the cheetah, then the management should follow basic guidelines for control of an infectious disease (Table 4 ). 

The cheetah has assumed a prominent position in the zoologic and wildlife communities due to its endangered status and its apparent increased susceptibility to microbial infections [56, 62] . These factors have emphasized the need for further research into the genetics and the diseases that may affect the cheetah in captivity, as well as in the wild [63] . Although coronaviral infections of the domestic cat have been recognized for over 2 decades, the emergence of pathogenic variants in the cheetah populations has only recently been acknowledged as having a potentially severe impact on its survival [56, 62, 66] .

Primary amongst the coronaviruses affecting domestic cats is FIPV, which is regarded as being 100% fatal once clinical signs are manifested [4, 66] . The occurrence of coronaviral infections of the cheetah have now been documented based on serology, electron microscopy of fecal contents, and the occurrence of fatal forms of infectious peritonitis compatible with the clinicopathologic signs observed in domestic cats with FIP [7, 10, 26, 29, 38, 43, 56, 72] .

These observations may indicate that the cheetah has acquired a unique group of coronaviruses which have antigenic similarity to the domestic cat coronaviruses or that the cheetah is susceptible to cross-species transmission of domestic cat coronaviruses [27, 38, 45, 81] . Serological studies support the contention that both situations may be important in the epizootiology of FIPV in cheetahs [26, 38, 43] . Although cheetahs in the wild have a comparable seroprevalence to the feline coronavirus group, their antibody titers are invariably lower than those titers detected in captive cheetahs during a disease outbreak [38, 43] . The seroprevalence of feline coronavirus infection in cheetahs in captivity, both in North America and Africa, indicates that the virus is transmitted with equal frequency. While evidence in support of cross-species transmission of feline coronaviruses to the cheetah is lacking, it is known that the cheetah cells are susceptible to some of the domestic cat coronaviruses in vitro [27] .

It is apparent that further analysis of cheetahs in captivity needs to be conducted in order to determine more about their microbial flora and their immune response [12] . This information is essential in order to determine the risk of infection and disease from viruses such as FIPV and feline herpesvirus [71, 72] . Until more is known regarding the pathogenesis of coronaviral infections of the cheetah, it would be prudent to establish surveillance programs which utilize a combination of serologic monitoring and quarantine procedures in order to insure the survival of the cheetah in captivity [38]. 

Feline infectious peritonitis. An immune-mediated coronaviral vasculitis

Transmissible gastroenteritis (TGE) of swine: survivor selection of TGE virus mutants in stomach juice of adult pigs

Genetics of resistance of animals to viruses. I. Introduction and studies in mice

Feline coronaviral infections

Host age and genotypic effects on enterotropic mouse hepatitis virus infection

Response of genetically susceptible and resistant mice to intranasal inoculation with mouse hepatitis virus JHM

Feline infectious peritonitis in South Africa

Plaque assay, polypeptide composition and immunochemistry of feline infectious peritonitis virus and feline enteric coronavirus isolates

Genetic resistance to mouse hepatitis virus correlates with absence of virus-binding activity on target tissues

Feline infectious peritonitis. An update of a captive cheetah population

Intraepithelial leukocytes contain a unique subpopulation of NK-like cytotoxic cells active in the defense of gut epithelium to enteric murine coronavirus

Health of adult free-living cheetahs

Enzymatic and acidic sensitivity profiles of selected virulent and attenuated transmissible gastroenteritis viruses of swine

A double-protease-resistant variant of transmissible gastroenteritis virus and its ability to induce lactogenic immunity

Shedding of eneteric coronavirus in adult cattle

Chronic shedding of bovine enteric coronavirus antigen-antibody complexes by clinically normal cows

Evidence for two mechanisms of dengue virus infection of adherent human monocytes: trypsin-sensitive virus receptors and trypsin-resistant immune complex receptors

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

Intracellutar RNAs of the feline infectious peritonitis coronavirus strain 79-1146

Susceptibility/resistance to mouse hepatitis virus strain 3 and macrophage procoagulant activity are genetically linked and controlled by two non-H-2-1inked genes

Neutralizing secretory IgA and IgG do not inhibit attachment of transmissible gastroenteritis virus

Interactive influence of infectious disease and genetic diversity in natural populations

Genetic basis for species vulnerability in the cheetah

The cheetah is depauperate in genetic variation

Delayed-type hypersensitivity: probable role in the pathogenesis of dengue hemorrhagic fever/dengue shock syndrome

Coronavirus disease (coronavirus enteritis, feline infectious peritonitis). In: Holzworth J (ed) Diseases of the cat

Infection studies in kittens using feline infectious peritonitis virus propagated in cell culture

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

Pathogenicity studies of feline coronavirus isolates 79-1146 and 79-1683

Experimental studies with three new strains of feline infectious peritonitis virus: FIPV-UCD 2, FIPV-UCD 3 and FIPV-UCD 4

Eosinophilic ulcers in association with herpetic dermatitis in sibling cheetahs

Feline infectious peritonitis in a captive cheetah

Antibody-dependent enhancement of viral infectivity

Infectious disease of nondomestic cats

Experimentally induced coronavirus infections in calves: viral replication in the respiratory and intestinal tracts

Dietary estrogen--a probable cause of infertility and liver disease in captive cheetahs

Diagnosis of porcine and bovine enteric coronavirus infections using cloned cDNA probes

The mutation rate and variability of eukaryotic viruses: an analytical review

Intestinal, pulmonary, and serum antibody responses of feeder pigs exposed to transmissible gastroenteritis virus by the oral and the oralintranasal routes of inoculation

Rapid evolution of RNA viruses

Experimental studies of a coronavirus and coronavirus-like agent in a barrier-maintained feine breeding colony

Proteolytic cleavage of the E 2 glycoprotein of murine coronavirus: activation of cell-fusing activity of virions by trypsin and separation of two different 90K cleavage fragments

Role ofT cells in feline infectious peritonitis virus infection of suckling mice

Trypsin-enhanced replication of neonatal calf diarrhea coronavirus in bovine embryonic lung cells

Antigenic and biological diversity of feline coronaviruses: feline infectious peritonitis and feline enteritis virus

Morphogenesis of a virus in cats with experimental feline infectiuos peritonitis

Antibody-mediated enhancement of disease in feline infectious peritonitis: comparisons with dengue hemorrhagic fever

Pathogenesis of feline infectious peritonitis: nature and development of viremia

Proteases involved in the processing of viral polyproteins

Viral interference-dominance of mutant viruses over wild-type viruses in mixed infections

Feline infectious peritonitis: review of gross and histopathologic lesions

Lesions in the small intestine of newborn pigs inoculated with porcine, feline and canine coronaviruses

Feline coronavirus. In: Appel MJ (ed) Virus infections of carnivores

Genetic variation of major histocompatibility complex class I genes in various species: the lack of polymorphism in the African cheetah

The authors wish to express their appreciation to Drs. Niels Pedersen