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

Two feline coronavirus isolates were characterized by their disease-causing potential in cats. The 79-1683 feline coronavirus isolate caused an inapparent-to-mild enteritis when given oronasally to specific-pathogen-free kittens and was not a cause of feline infectious peritonitis (FIP). Target tissues for the virus were the mature apical epithelium of the small intestine, mesenteric lymph nodes, tonsils, thymus, and (to a lesser extent) the lungs. Inoculated kittens shed high numbers of virus in their feces for 14 to 17 days, but remained infectious to susceptible kittens for longer periods of time, as evidenced by contact-exposure studies. Because the 79-1683 isolate induced only enteritis, it was designated feline enteric coronavirus (FECV) 79-1683. The 79-1146 feline coronavirus isolate induced effusive abdominal FIP in specific-pathogen-free kittens after oronasal and intraperitoneal inoculation. Clinical signs of disease appeared within 12 to 14 days in almost all inoculated kittens. Because this isolate caused FIP, it was designated FIP virus (FIPV) 79-1146. Cross-protective immunity was not induced by the various coronavirus infections. Kittens preimmunized with the UCD strain of FECV (FECV-UCD) or with FECV-79-1683 were not immune to infection with FIPV-79-1146. Likewise, kittens previously inoculated with FECV-79-1683 were not immune to infection with FIPV-UCD1. In fact, preexisting heterologous FECV-79-1683 immunity often accelerated and enhanced the severity of disease caused by inoculation with FIPV-UCD1.(ABSTRACT TRUNCATED AT 250 WORDS)     

A study of naturally occurring feline coronavirus infections in kittens.

Feline coronavirus is a common infection in cats, as indicated by the high prevalence of antibodies against the virus, especially in multicat households. Approximately 5 to 12 per cent of seropositive cats develop classical feline infectious peritonitis. A survey of kittens born into households of seropositive cats demonstrated the existence of healthy coronavirus carriers. Seronegative animals did not appear to excrete virus. No specific antibody titre could be linked to carrier status and some carrier cats subsequently became seronegative. The management of the kittens strongly influenced whether they became infected, and some degree of protection appeared to be conferred by maternally derived antibody. At present, feline infectious peritonitis virus and feline enteric coronavirus can only be differentiated by their different clinical histories in infected catteries. In this survey, cases of feline infectious peritonitis occurred in kittens from households where the initial presentation had been enteritis and vice versa. Therefore no difference in epidemiology could be found.     

Comparison of serologic assays for measurement of antibody response to coronavirus in cats

Serologic virus neutralization tests, indirect immunofluorescence tests, and ELISA, using tissue culture-adapted feline infectious peritonitis virus (FIPV) or feline enteric coronavirus (FECV) were compared for their ability to distinguish specific virus exposure in cats. Sera of specific-pathogen-free cats inoculated with virulent or modified FIPV or FECV were used to compare the sensitivity and specificity of the homologous assays to a heterologous assay that measures antibody reactivity with transmissible gastroenteritis virus of swine. The geometric means of the serologic titers in FIPV and FECV assays were higher for FIPV- or FECV-infected specific-pathogen-free cats than the geometric means of the transmissible gastroenteritis virus assays for most groups. None of the assays was specific enough to discern the virus to which a cat had been exposed. However, the FIPV virus neutralization test appeared to be more sensitive for detection of an early response to FIPV infection than did the FIPV immunofluorescence test or FIPV-ELISA.     

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

The infectivity and pathogenicity to newborn pigs of antigenically related coronaviruses from pigs (transmissible gastroenteritis virus; TGEV), cats (feline infectious peritonitis virus; FIPV), and dogs (canine gastroenteritis virus; CGEV) were studied by light, scanning electron, and immunofluorescence microscopy. Hysterectomy-derived, 12-hour-old pigs were orally given tissue culture or frozen preparations of 6 coronavirus strains (3 porcine, 2 feline, and 1 canine). The pigs were killed at regular intervals between 24 and 144 hours after exposure. Virulent TGEV and virulent FIPV produced necrosis of villous epithelium, resulting in villous atrophy in the jejunum and the ileum. Similar, but less extensive and severe lesions, were produced by the 4 other viruses. Coronaviral antigens were identified by immunofluorescence in villous epithelial cells of pigs that had been inoculated with virulent TGEV, attenuated TGEV, virulent FIPV, and tissue culture-adapted FIPV. In contrast, coronaviral antigens were not induced by the small plaque variant TGEV and virulent CGEV in the villous epithelium, but rather in cells of the lamina propria and crypt epithelium.     

Attempted immunization of cats against feline infectious peritonitis, using avirulent live virus or sublethal amounts of virulent virus

Kittens vaccinated with an avirulent biotype of the Black strain of feline infectious peritonitis virus (FIPV; given oronasally) developed both indirect fluorescent and virus-neutralizing antibodies, but were not protected against oronasal challenge exposure with virulent virus. In fact, kittens vaccinated with avirulent virus were more readily infected than were nonvaccinated cats. A proportion of kittens could be immunized to FIPV by giving sublethal amounts of virulent virus. This technique, however, was too inconsistent and hazardous to have clinical relevance. The results of these studies indicated that humoral immunity was not protective in FIPV infection. There was no correlation between fluorescent and virus-neutralizing antibodies and either disease or immunity. Immune serum from FIPV-resistant cats failed to passively protect susceptible animals against virulent virus given intraperitoneally or oronasally, and as expected, actually sensitized them to infection. It was concluded that cell-mediated immunity was probably responsible for protection.     

Differential role for low pH and cathepsin-mediated cleavage of the viral spike protein during entry of serotype II feline coronaviruses

Feline infectious peritonitis (FIP) is a terminal disease of cats caused by systemic infection with a feline coronavirus (FCoV). FCoV biotypes that cause FIP are designated feline infectious peritonitis virus (FIPV), and are distinguished by their ability to infect macrophages and monocytes. Antigenically similar to their virulent counterparts are FCoV biotypes designated feline enteric coronavirus (FECV), which usually cause only mild enteritis and are unable to efficiently infect macrophages and monocytes. The FCoV spike protein mediates viral entry into the host cell and has previously been shown to determine the distinct tropism exhibited by certain isolates of FIPV and FECV, however, the molecular mechanism underlying viral pathogenesis has yet to be determined. Here we show that the FECV strain WSU 79-1683 (FECV-1683) is highly dependent on host cell cathepsin B and cathepsin L activity for entry into the host cell, as well as on the low pH of endocytic compartments. In addition, both cathepsin B and cathepsin L are able to induce a specific cleavage event in the FECV-1683 spike protein. In contrast, host cell entry by the FIPV strains WSU 79-1146 (FIPV-1146) and FIPV-DF2 proceeds independently of cathepsin L activity and low pH, but is still highly dependent on cathepsin B activity. In the case of FIPV-1146 and FIPV-DF2, infection of primary feline monocytes was also dependent on host cell cathepsin B activity, indicating that host cell cathepsins may play a role in the distinct tropisms displayed by different feline coronavirus biotypes.     

Differentiation of canine coronavirus and porcine transmissible gastroenteritis virus by neutralization with canine,porcine and feline sera

Monospecific antisera were prepared in rabbits against canine coronavirus (CCV) and transmissible gastroenteritis virus of pigs (TGEV), and in 24 pigs and 3 cats against TGEV alone. Neutralizing antibody titres were higher for the immunizing than the heterologous virus, although cross-neutralization usually was detected. This confirmed that CCV and TGEV are distinct, but antigenically related coronaviruses. In sera from 41 dogs, CCV-neutralizing titres were on average 2.7 fold higher than TGEV-neutralizing titres, suggesting that CCV was the causal agent. Sera from 29 cats in colonies with feline infectious peritonitis (FIP) and known to contain TGEV-neutralizing antibody, were found to have titres 12.3 fold higher against CCV. The FIP virus (FIPV) is probably more closely related to CCV than TGEV as judged by antigens involved in virus neutralization.Antisera to two isolates of bovine coronavirus, three isolates of haemagglutinating encephalomyelitis virus, seven strains of avian infectious bronchitis virus and the 229E strain of human coronavirus all failed to neutralize CCV and TGEV. Thus CCV, TGEV and probably FIPV fall into a group of antigenically related agents, separable from other members of the family Coronaviridae, by both virus neutralization and immunofluorescence tests.     

Feline congenital myotonia

FELINE infections peritonitis (FIP) is a systemic, fatal, immune-mediated vasculitis caused by a feline coronavirus (FCoV). Historically, FIP virus (FIPV) and feline enteritis by a feline enteric coronavirus (FECV). Recent studies have shown that there is essentially only one FCoV in the field, although laboratory strains may vary in virulence.     

Comparative properties of feline coronaviruses in vitro.

Two feline coronaviruses were characterized to determine their biological properties in vitro and their antigenic relatedness to a previously recognized feline infectious peritonitis virus and canine coronavirus. The viruses, designated WSU 79-1146 and WSU 79-1683, were shown to have comparable growth curves with the prototype feline infectious peritonitis virus. Treatment of the feline infectious peritonitis virus strains with 0.25% trypsin indicated that they were relatively resistant to proteolytic inactivation when compared with the feline enteric coronavirus strain. This observation may serve as a useful in vitro marker to distinguish closely related members of the feline coronavirus group. Plaque assay results indicated that the feline infectious peritonitis virus strains produced large homogeneous plaques in comparison to the feline enteric coronavirus strain and canine coronavirus, which showed a heterogenous plaque size distribution. No naturally temperature sensitive mutants were detected in either of the feline coronavirus populations. Both of the viruses were antigenically related to feline infectious peritonitis virus and to a lesser extent to canine coronavirus by virus neutralization.     

Identification of viral antigens that induce antibody responses on exposure to coronaviruses

Various techniques were used to look for protective, non-cross-reactive antibodies in the sera of cats exposed to virulent feline infectious peritonitis virus (FIPV). Antibodies reactive with feline enteric coronavirus (FECV) from FIPV-exposed cats were adsorbed by several passages over an FECV-Sepharose column. In an ELISA against FECV and FIPV, the activity against both viruses was removed at the same rate; thus, no FIPV-specific antibodies could be identified. By gel electrophoresis-derived ELISA, the responses of cats surviving FIPV exposure were compared with those of cats succumbing to FIPV exposure to determine whether survival could be correlated with an antibody response against a particular virus protein. Results indicated that both groups responded in the same way to the matrix envelope protein and nucleocapsid proteins. Even though the response to peplomer in each group was weak, the survivor group responded better to this protein. Furthermore, the response of this group to the peplomer protein had the highest correlation with virus neutralization titer.     

Disseminated intravascular coagulation in experimentally induced feline infectious peritonitis

Disseminated intravascular coagulation was induced in kittens by intraperitoneal inoculation of feline infectious peritonitis virus (FIPV). Kittens seronegative to FIPV survived significantly (P less than 0.05) longer than those seropositive to FIPV. Pyrexia, anemia, icterus, hyperbilirubinemia, and elevated concentrations of liver-specific enzymes were detected in the inoculated cats. Lesions induced included disseminated fibrinonecrotic and pyogranulomatous inflammation, hepatic necrosis, and widespread phlebitis and thrombosis. Localization of FIP viral antigen and immunoglobulin G was demonstrated in foci of heptic necrosis by immunofluorescence miroscopy. Lymphopenia, thrombocytopenia, hyperfibrinogenemia, and increased quantities of fibrin-fibrinogen degradation products were present in cats after the onset of clinical illness. Depression of factor VII, VIII, IX, X, XI, and XII plasma activities and prolongation of prothrombin and partial thromboplastin times also developed in infected cats. The accelerated onset of clinical disease and mortality in seropositive kittens vs seronegative kittens and the association of virus and antibody in multiple foci of hepatic necrosis suggest an immune-mediated component is involved in the pathogenesis of this disease.     

Attempted immunisation of cats against feline infectious peritonitis using canine coronavirus

Specific pathogen free kittens were vaccinated with an unattenuated field isolate of canine coronavirus (CCV) either by aerosol or subcutaneously, and received boosting vaccinations four weeks later. Aerosolisation elicited a homologous virus-neutralising (VN) antibody response that increased steadily over a four-week period and levelled off one to two weeks after revaccination. The initial aerosolised dose produced an asymptomatic infection with excretion of CCV from the oropharynx up to eight days after vaccination; virus shedding was not detected, however, after the second inoculation. Cats vaccinated subcutaneously developed low VN antibody titres after the first CCV dose and experienced a strong anamnestic response after the second dose. Neutralising antibody titres then levelled off one to two weeks after revaccination at mean values somewhat lower than in cats vaccinated by aerosol. CCV was not isolated from the oropharynx after either subcutaneous dose. Four weeks after CCV boosting inoculations, vaccinated cats and sham-vaccinated control cats were divided into three subgroups and challenged by aerosol with the virulent UCD1 strain of feline infectious peritonitis virus (FIPV UCD1) at three different dosage levels. Five of six cats (including sham-vaccinated controls) given the lowest challenge dose showed no signs of disease, while all other cats developed lesions typical of feline infectious peritonitis (FIP). The five surviving cats developed FIP after subsequent challenge with a fivefold higher dose of FIPV. Thus heterotypic vaccination of cats with CCV did not provide effective protection against FIPV challenge.     

An update on aspects of viral gastrointestinal diseases of dogs and cats

Viruses commonly cause gastrointestinal illnesses in dogs and cats that range in severity from mild diarrhoea to malignant neoplasia. Perpetual evolution of viruses is reflected in changing disease patterns, so that familiar viruses are sometimes discovered to cause new or unexpected diseases. For example, canine parvovirus (CPV) has regained the ability to infect felids and cause a panleucopenia-like illness. Feline panleucopenia virus (FPV) has been shown to cause fading in young kittens and has recently been implicated as a possible cause of feline idiopathic cardiomyopathy. Molecular scrutiny of viral diseases sometimes permits deeper understanding of pathogenesis and epizootiology. Feline gastrointestinal lymphomas have not, in the past, been strongly associated with retroviral infections, yet some of these tumours harbour retroviral proviruses. Feline leukaemia virus (FeLV) may play a role in lymphomagenesis, even in cats diagnosed as uninfected using conventional criteria. There is strong evidence that feline immunodeficiency virus (FIV) can also be oncogenic. The variant feline coronaviruses that cause invariably-fatal feline infectious peritonitis (FIP) arise by sporadic mutation of an ubiquitous and only mildly pathogenic feline enteric coronavirus (FECV); a finding that has substantial management implications for cat breeders and veterinarians. Conversely, canine enteric coronavirus (CECV) shows considerable genetic and antigenic diversity but causes only mild, self-limiting diarrhoea in puppies. Routine vaccination against this virus is not recommended. Although parvoviruses, coronaviruses and retroviruses are the most important known viral causes of canine and feline gastrointestinal disease, other viruses play a role. Feline and canine rotaviruses have combined with human rotaviruses to produce new, reassortant, zoonotic viruses. Some companion animal rotaviruses can infect humans directly. Undoubtedly, further viral causes of canine and feline gastrointestinal disease await discovery.     

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.     

Delayed-type hypersensitivity skin responses associated with feline infectious peritonitis in two cats

Two cats previously challenge-exposed and seropositive to feline infectious peritonitis virus (FIPV) were evaluated for delayed-type hypersensitivity (DTH) skin responses to intradermal FIPV. Before testing, cat 1 (FIP-resistant) had survived a severe experimental FIPV challenge-exposure and had remained asymptomatic, whereas cat 2 (FIP-susceptible) developed acute fulminant FIP after a considerably smaller virus challenge-exposure. Cat 1 developed a focal thickened plaque at the FIPV-injected skin site at 48 hours after injection. Histological examinations of serial punch biopsies from virus-inoculated skin revealed perivascular and diffuse dermal infiltrations of macrophages, lymphocytes and polymorphonuclear leucocytes which were maximal at 48 to 72 hours after injection. In contrast, cat 2 did not react grossly and showed only very mild dermal infiltrates at 72 hours after injection. The present findings of strong DTH responses to FIPV in a resistant cat and minimal responses in a cat with acute fulminant FIP suggest that certain in vivo cellular immune reactions may be associated with disease resistance.     

Virus shedding and immune responses in cats inoculated with cell culture-adapted feline infectious peritonitis virus

Eight specific pathogen-free cats were inoculated orally or parenterally with a cell culture-adapted strain of feline infectious peritonitis virus (FIPV). Faeces and oropharyngeal swabs were monitored daily for infectious virus by inoculation of feline embryo lung cells. Virus was recovered from both sites for approximately 2 weeks after inoculation, before clinical signs of disease developed. Peripheral blood lymphocytes collected from these cats were tested in an in-vitro blastogenic assay using concanavalin A (con A) and FIPV antigen. All cats showed a profound suppression of the response to con A which only recovered to pre-inoculation levels in 2 cats, one of which survived. These 2 cats also responded to FIPV antigen on the 21st day after infection, the greater response being in the survivor. The other cats, surviving 16-18 days, developed no response to FIPV antigen. Antibody titres, measured by immunofluorescence and by virus neutralization, rose rapidly to very high levels in all cats, regardless of the route of inoculation.     

Differentiation between transmissible gastroenteritis virus and porcine respiratory coronavirus using a cDNA probe.

A plasmid, pG3BS, containing a cDNA clone from the 5' coding region of the peplomer glycoprotein gene appears to be specific for enteric transmissible gastroenteritis virus (TGEV) strains and for live-attenuated TGEV vaccines. This cDNA probe is used to differentiate porcine respiratory coronavirus (PRCV) isolates from TGEV field and vaccine strains by a slot blot hybridization assay. Probe pG3BS also hybridizes to canine coronavirus (CCV) RNA but does not hybridize to antigenically related feline infectious peritonitis virus (FIPV) RNA. The RNAs of 13 enteric TGEV isolates from the United States, Japan, and England, 4 US-licensed live-attenuated TGEV vaccines, and antigenically closely related CCV were detected by pG3BS. The RNAs of FIPV and 3 US isolates of PRCV did not react with pG3BS but were detected by a TGEV-derived plasmid, pRP3. Pigs infected with either PRCV or TGEV test serologically positive for TGEV antibody by the serum neutralization test. Characterization of the virus circulating in a swine herd by the pG3BS probe will differentiate between an enteric TGEV and a respiratory PRCV infection.     

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.     

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

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