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.     

Antigenic analysis of feline coronaviruses with monoclonal antibodies (MAbs): preparation of MAbs which discriminate between FIPV strain 79-1146 and FECV strain 79-1683.

We prepared 31 monoclonal antibodies (MAbs) against either FIPV strain 79-1146 or FECV strain 79-1683, and tested them for reactivity with various coronaviruses by indirect fluorescent antibody assay (IFA). Sixteen MAbs which reacted with all of the 11 strains of feline coronaviruses, also reacted with canine coronavirus (CCV) and transmissible gastroenteritis virus (TGEV). In many of them, the polypeptide specificity was the recognition of transmembrane (E1) protein of the virus. We succeeded in obtaining MAbs which did not react with eight strains of FIPV Type I viruses (showing cell-associated growth) but reacted with FIPV Type II (79-1146, KU-1) and/or FECV Type II (79-1683) (showing non-cell associated growth). These MAbs also reacted with CCV or TGEV. These MAbs recognized peplomer (E2) glycoprotein, and many antigenic differences were found in this E2 protein. These results suggest that FIPV Type II and FECV Type II viruses are antigenically closer to TGEV or CCV than to FIPV Type I viruses. Furthermore, the MAb prepared in this study has enabled discrimination between FIPV strain 79-1146 and FECV strain 79-1683, which was thought to be impossible by the previous serological method.     

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.     

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.     

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.     

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.     

Immunologic phenomena in the effusive form of feline infectious peritonitis

The effusive form of feline infectious peritonitis (FIP) was reproduced by injecting 12- to 16-week-old kittens intraperitoneally with a cell-free inoculum derived from the tissues of infected cats. The kittens used for the study were either positive for FIP virus-reacting antibodies before inoculation or they were seronegative. Seropositive kittens were obtained from a cattery where the natural infection was enzootic, and seronegative kittens were obtained from a specific-pathogen-free cattery. Only about half the kittens that were seronegative before inoculation developed disease or serum antibodies to the tissue-derived virus. Seronegative kittens that developed disease showed no signs of illness until 8 to 10 days after inoculation, and they lived for 7 to 14 days after clinical signs appeared. The onset of clinical disease coincided with the appearance of serum antibodies. In contrast, all of the seropositive kittens became ill within 36 to 48 hours after inoculation, and died within 5 to 7 days. If seronegative kittens were treated with immune serum or immunoglobulin (Ig)G, they developed disease with the same frequency, acuteness, and severity as seropositive kittens. Foci of hepatitis and serositis in seropositive kittens contained viral antigen, IgG bound to antigen, and complement. Serum complement activity also decreased several days before death in seropositive kittens inoculated with tissue-derived FIP virus. The temporal relationship of clinical disease and the appearance of serum antibodies, the more acute and severe nature of the disease produced in seropositive kittens, and the presence of antibody and complement in the lesions indicated that effusive FIP is immunologically mediated.     

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

The propagation of feline infectious peritonitis virus (NW1-FIPV strain) in cell culture is described. Tissue culture-propagated virus was used to inoculate specific-pathogen-free kittens intraperitoneally, intratracheally, or orally. Intraperitoneal inoculation caused seroconversion and effusive peritonitis in 100% of the kittens. Intratracheal inoculation produced disease in 60% of the kittens, and oral inoculation in only 20%. Seroconversions without production of disease occurred in 10% of the kittens inoculated by either the intratracheal or the oral route. The remainder of the kittens inoculated by the intratracheal (30%) and oral (70%) routes did not develop serum antibodies or disease.     

The prevalence of types I and II feline coronavirus infections in cats.

The types of feline coronaviruses that are prevalent throughout Japan were determined by competitive enzyme-linked immunosorbent assay (ELISA) using a monoclonal antibody (MAb) to feline infectious peritonitis virus (FIPV) Type II and neutralizing test using Type II FIPV as challenge virus. A total of 1,079 cat serum samples were tested by indirect fluorescent antibody (IFA) assay for FIPV Type II antigen, all 42 sample from natural cases of FIP, 138 of 647 (21.3%) from cases with some chronic diseases and 57 of 390 (14.6%) from apparently non-diseased cases were positive. Of the 42 cases with FIP, 29 (69%) and 13 (31%) were found to have infection with FIPV Types I and II, respectively. Of the cases with chronic diseases, 111 (80.4%) were shown to have infection with FIPV or FECV Type I, while 14 (10.1%) with FIPV or FECV Type II. All of the 57 apparently non-diseased cases seemed to have been infected with FIPV or FECV Type I. These results indicated that feline coronavirus Type I is more high prevalent in Japan.     

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.     

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.     

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.     

Pathogenesis of feline enteric coronavirus infection

Fifty-one specific pathogen-free (SPF) cats 10 weeks to 13 years of age were infected with a cat-to-cat fecal-oral passed strain of feline enteric coronavirus (FECV). Clinical signs ranged from unapparent to a mild and self-limiting diarrhea. Twenty-nine of these cats were FECV na?ve before infection and followed sequentially for fecal virus shedding and antibody responses over a period of 8-48 months. Fecal shedding, as determined by real-time polymerase chain reaction (RT-PCR) from rectal swabs, appeared within a week and was significantly higher in kittens than older cats. FECV shedding remained at high levels for 2-10 months before eventually evolving into one of three excretion patterns. Eleven cats shed the virus persistently at varying levels over an observation period of 9-24 months. Eleven cats appeared to have periods of virus shedding interlaced with periods of non-shedding (intermittent or recurrent shedders), and seven cats ceased shedding after 5-19 months (average 12 months). There was no change in the patterns of virus shedding among cats that were excreting FECV at the time of a secondary challenge exposure. Four cats, which had ceased shedding, re-manifested a primary type infection when secondarily infected. Cats with higher feline coronavirus (FCoV) antibody titers were significantly more likely to shed virus, while cats with lower titers were significantly less likely to be shedding. Twenty-two kittens born to experimentally infected project queens began shedding virus spontaneously, but never before 9-10 weeks of age. Natural kittenhood infections appeared to be low grade and abortive. However, a characteristic primary type infection occurred following experimental infection with FECV at 12-15 weeks of age. Pregnancy, parturition and lactation had no influence on fecal shedding by queens. Methylprednisolone acetate treatment did not induce non-shedders to shed and shedders to increase shedding.     

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 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.     

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.     

In vitro inhibition of feline coronavirus replication by small interfering RNAs

Infection with virulent biotypes of feline coronavirus (FCoV) can result in the development of feline infectious peritonitis (FIP), a typically fatal immune mediated disease for which there is currently no effective antiviral treatment. In this study we demonstrate the ability of small interfering RNA (siRNA) mediated RNA interference (RNAi) to inhibit the replication of virulent FCoV strain FIPV WSU 79-1146 in an immortalised feline cell line. A panel of eight synthetic siRNAs targeting four different regions of the FCoV genome were tested for antiviral effects. Efficacy was determined by qRT-PCR of intracellular viral genomic and messenger RNA, TCID50 infectivity assay of extracellular virus, and direct IFA for viral protein expression. All siRNAs demonstrated an inhibitory effect on viral replication in vitro. The two most effective siRNAs, targeting the untranslated 5' leader sequence (L2) and the nucleocapsid gene (N1), resulted in a >95% reduction in extracellular viral titre. Further characterisation of these two siRNAs demonstrated their efficacy when used at low concentrations and in cells challenged with high viral loads. Taken together these findings provide important information for the potential therapeutic application of RNAi in treating FIP.     

Diagnostic Features of Clinical Neurologic Feline Infectious Peritonitis

Feline infectious peritonitis (FIP) is a fatal Arthus-type immune response of cats to infection with FIP virus, a mutant of the ubiquitous feline enteric coronavirus (FECV). The disease may occur systemically or in any single organ system, and primary neurologic disease is a common subset of such manifestations. We examined 16 domestic cats with clinical neurologic FIP and 8 control cats with nonneurologic FIP, with the intention of identifying the ante-and postmortem diagnostic tests that most contribute to accurate diagnosis. Of the 16 cats with neurologic FIP, 15 were less than 2 years of age and all 16 originated from large multiplecat households. The most useful antemortem indicators of disease were positive anti-coronavirus IgG titer in cerebrospinal fluid, high serum total protein concentration, and findings on magnetic resonance imaging suggesting periventricular contrast enhancement, ventricular dilatation, and hydrocephalus. Postmortem diagnosis was facilitated by FIP monoclonal antibody staining of affected tissue and coronavirus-specific polymerase chain reaction. Most cats with neurologic and ocular forms of FIP had patchy, focal lesions, suggesting that recently developed technologies described in this report may be useful for evaluation of cats with suspected FIP.