Natural FCoV infection: cats with FIP exhibit significantly higher viral loads than healthy infected cats

Natural feline coronavirus (FCoV) infection has been shown to not only induce intestinal infection with viral shedding, but also systemic infection which either remains without clinical signs or leads to feline infectious peritonitis (FIP). As systemic infection is not the key event in the development of FIP, the question arises as to whether a potential difference in viral load might be of importance. Therefore, the purpose of this study was to quantitatively assess feline coronavirus (FCoV) RNA loads in haemolymphatic tissues of healthy, long-term FCoV-infected cats and cats with FIP. In cats that died from FIP, viral loads were significantly higher, indicating a higher rate of viral replication or a reduced capacity for viral clearance in cats developing and/or suffering from FIP.     

Tissue distribution of a feline AGP related protein (fAGPrP) in cats with feline infectious peritonitis (FIP)

Feline alpha(1)-acid glycoprotein (fAGP) increases during feline infectious peritonitis (FIP). We have recently identified a 29 kDa protein that we named feline AGP-related protein (fAGPrP) due to its cross-reactivity with an anti-human AGP monoclonal antibody. In this work we describe the tissue distribution of fAGPrP during FIP, and its relationship with feline coronavirus (FCoV) and myeloid cells. Tissues from five control cats and from 15 cats with FIP were examined by immunohistochemistry using monoclonal antibodies against human AGP, FCoV and myeloid antigens. Diffuse fAGPrP positivity within the lesions, likely due to vascular plasma leakage, endothelial and epithelial lining were detectable. Compared to controls, fAGPrP-expressing cells often increased in number and were diffusely distributed in lymph nodes, as usually occurs for IgM-producing plasma cells during early immune responses. These findings did not depend on the presence of FCoVs or of myeloid cells, suggesting that fAGPrP is not directly involved in the pathogenesis of FIP.     

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.     

Shifts in circulating lymphocyte subsets in cats with feline infectious peritonitis (FIP): pathogenic role and diagnostic relevance

Cats with feline infectious peritonitis (FIP) are usually lymphopenic and have lymphoid depletion evident in spleen and lymph nodes. In particular, the number of CD4+ lymphocytes in tissues decreases during the evolution of FIP lesions. This decrease is most likely due to increased lymphocyte apoptotic rate. In contrast, cats infected with the Feline Coronavirus (FCoV) develop a follicular hyperplasia in the peripheral lymph nodes. The current study was devised to evaluate the possible pathogenic role of shifts in circulating lymphocyte subsets in FIP. Peripheral blood from cats with FIP was evaluated and compared with peripheral blood from clinically healthy cats living in both FCoV-free and FCoV-endemic catteries. Blood from cats with diseases other than FIP was also examined in order to define the diagnostic relevance of the changes. Lymphocyte subsets were analysed by flow cytometry, using a whole blood indirect immunofluorescence technique and mAbs specific for feline CD5, CD4, CD8, CD21. The results of the current study suggest that cats recently infected with FCoV that do not develop the disease have a transient increase in T cells; cats from groups with high prevalence of FIP have a moderate but persistent decrease in T cell subsets; cats with FIP have a very severe decrease in all the subsets of lymphocytes. Moreover, during FIP many lymphocytes do not express any membrane antigen, most likely due to early apoptosis. Cats with diseases other than FIP also had decreased number of lymphocytes: as a consequence, the diagnostic relevance of these findings is very low. Nevertheless, the lack of flow cytometric changes had a high negative predictive value (NPV), thus allowing to exclude FIP from the list of possible diagnoses in cats with normal cytograms.     

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

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

Cellular composition and interferon-gamma expression of the local inflammatory response in feline infectious peritonitis (FIP)

Feline infectious peritonitis (FIP) is one of the most important viral diseases of cats. International studies estimate that approximately 80% of all purebred cats are infected with the causative agent, feline coronavirus (FCoV). Out of these, 5-12% develop clinical symptoms of FIP. The pathogenesis of the disease is complex with many unresolved issues relating to the role of the immune system. The aim of the present study was to determine the proportions of various inflammatory cell types in FIP lesions by using a panel of cat specific, thoroughly validated, monoclonal antibodies. In addition, the expression of interferon-gamma within the inflammatory lesions was examined by RT-PCR. Our results confirm the mixed nature of the inflammatory reaction in FIP, involving B cells and plasma cells as well as CD4+ and CD8+ T cells. However, one cell type stands out as being the key element in both the "wet" and "dry" forms of FIP: the macrophage. Upregulation of IFN-gamma expression within the inflammatory lesions suggests a local activation of macrophages, which might result in increased viral replication.     

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.     

Flow cytometric detection of alpha-1-acid glycoprotein on feline circulating leucocytes

To assess whether alpha‐1‐acid glycoprotein (AGP) can be detected on the membrane of feline circulating leucocytes. Design The presence of AGP on circulating leucocytes was investigated in both clinically healthy cats and cats with different diseases. A group of feline coronavirus (FCoV)‐positive cats, comprising cats with feline infectious peritonitis (FIP) and cats not affected by FIP but seropositive for FCoV, were included in this study because the serum concentration of AGP increases during FCoV infection. Procedure Flow cytometry (using an anti‐feline AGP antibody), serum protein electrophoresis, routine haematology and measurement of the serum AGP concentration were performed using blood samples from 32 healthy cats (19 FCoV‐seropositive), 13 cats with FIP and 12 with other diseases (6 FCoV‐seropositive). The proportion of cats with AGP‐positive leucocytes in the different groups (e.g. controls vs sick; FIP vs other diseases, etc.) or in cats with different intensities of inflammatory response was compared using a Chi‐square test. Results AGP‐positive leucocytes were found in 23% of cats. Compared with controls, the proportion of patients with positive granulocytes and monocytes was higher among sick cats (especially cats with diseases other than FIP) and cats with high serum AGP concentration, but not in cats with leucocytosis or that were FCoV‐seropositive. Conclusion AGP‐positive leucocytes can be found in feline blood, especially during inflammation. Conversely, no association between AGP‐positive leucocytes and FIP was found. Further studies are needed to elucidate the mechanism responsible for this finding and its diagnostic role in cats with inflammation.     

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.     

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.     

Serum alpha1-acid glycoprotein (AGP) concentration in non-symptomatic cats with feline coronavirus (FCoV) infection

Previous studies have demonstrated that the concentration of alpha1-acid glycoprotein (AGP) transiently increases in asymptomatic cats infected with feline coronavirus (FCoV). In order to establish whether these fluctuations depend on the FCoV status, the serum concentration of AGP and anti-FCoV antibody titres and/or faecal shedding of FCoVs in clinically healthy cats from catteries with different levels of prevalence of FCoV infection were monitored over time. Serum AGP concentrations fluctuated over time in clinically healthy cats from the cattery with the highest prevalence of feline infectious peritonitis (FIP) and significantly increased just before an outbreak of FIP. Further studies are required to clarify whether the observed increase of AGP concentration is a consequence of the increased viral burden or a protective response against mutated viral strains. Nevertheless, the results of the present study suggest that AGP might be useful in monitoring FCoV-host interactions in FCoV-endemic catteries.     

Treatment of cats with feline infectious peritonitis

Feline infectious peritonitis (FIP) infection resulting in clinical signs is invariably fatal despite clinical intervention. As FIP is an immune-mediated disease, treatment is mainly aimed at controlling the immune response triggered by the infection with the feline coronavirus (FCoV). Immune suppressive drugs such as prednisone or cyclophosphamide may slow disease progression but do not produce a cure. In nearly every published case report of attempted therapy for clinical FIP, glucocorticoids have been used; there are, however, no controlled studies that evaluate the effect of glucocorticoids as a therapy for FIP. Some veterinarians prescribe immune modulators to treat cats with FIP with no documented controlled evidence of efficacy. It has been suggested that these agents may benefit infected animals by restoring compromised immune function, thereby allowing the patient to control viral burden and recover from clinical signs. However, a non-specific stimulation of the immune system may be contraindicated as clinical signs develop and progress as a result of an immune-mediated response to the mutated FCoV.     

Recommendations from workshops of the second international feline coronavirus/feline infectious peritonitis symposium

In August 2002, scientists and veterinarians from all over the world met in Scotland to discuss feline coronavirus (FCoV) and feline infectious peritonitis (FIP). The conference ended with delegates dividing into three workshops to draw up recommendations for FCoV control, diagnosis and treatment and future research. The workshops were chaired by the three authors and the recommendations are presented in this paper.     

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.     

Prevalence of antibodies against feline coronavirus and Chlamydophila felis in Swedish cats

Serum samples from 214 Swedish cats with no signs of infectious disease were analysed for the presence of antibodies against Chlamydophila felis (Cp felis), while 209 of these were also analysed for feline coronavirus (FCoV) antibodies. The prevalence of antibodies against Cp felis was 11%, with no significant difference between purebred and mixed breed cats. The overall prevalence of antibodies against FCoV was 31%, significantly higher among pure breed cats (65%) than among mixed breed cats (17%). A high proportion of cats with antibodies against FCoV had relatively high antibody titres, and was therefore likely to be shedding FCoV in faeces. For Cp felis, the majority of seropositive animals had relatively low antibody titres, and the risk of these animals infecting others is not known.     

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

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

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

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