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

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

Feline coronavirus serotypes 1 and 2: seroprevalence and association with disease in Switzerland

To determine the prevalence of antibodies to feline coronavirus (FCoV) serotypes 1 and 2 in Switzerland and their association with different disease manifestations, a serological study based on immunofluorescence tests was conducted with Swiss field cats using transmissible gastroenteritis virus (TGEV), FCoV type 1 and FCoV type 2 as antigens. A total of 639 serum samples collected in the context of different studies from naturally infected cats were tested. The current study revealed that, with an apparent prevalence of 83%, FCoV serotype 1 is the most prevalent serotype in Switzerland. FCoV type 1 viruses induced higher antibody titers than FCoV type 2, and were more frequently associated with clinical signs and/or feline infectious peritonitis. The antibody development in seven cats experimentally infected with FCoV type 1 revealed that, with progressing duration of infection, antibodies to FCoV type 1 significantly increased over those to FCoV type 2. There was a significant relationship between antibody titers against TGEV, FCoV 1, and FCoV 2 and TGEV antigen detected the highest proportion of seropositive cats. We conclude that a vaccine against FCoV should be based on FCoV type 1-related antigens and that for serodiagnosis of FCoV infection TGEV should be used to attain the highest diagnostic efficiency. When serology is used in addition to clinical signs, hematology, and clinical chemistry results as an aid to diagnose clinical FIP, TGEV shows a diagnostic efficiency equal to that of a FCoV antigen.     

Feline Coronavirus Serotypes 1 and 2: Seroprevalence and Association with Disease in Switzerland

To determine the prevalence of antibodies to feline coronavirus (FCoV) serotypes 1 and 2 in Switzerland and their association with different disease manifestations, a serological study based on immunofluorescence tests was conducted with Swiss field cats using transmissible gastroenteritis virus (TGEV), FCoV type 1 and FCoV type 2 as antigens. A total of 639 serum samples collected in the context of different studies from naturally infected cats were tested. The current study revealed that, with an apparent prevalence of 83%, FCoV serotype 1 is the most prevalent serotype in Switzerland. FCoV type 1 viruses induced higher antibody titers than FCoV type 2, and were more frequently associated with clinical signs and/or feline infectious peritonitis. The antibody development in seven cats experimentally infected with FCoV type 1 revealed that, with progressing duration of infection, antibodies to FCoV type 1 significantly increased over those to FCoV type 2. There was a significant relationship between antibody titers against TGEV, FCoV 1, and FCoV 2 and TGEV antigen detected the highest proportion of seropositive cats. We conclude that a vaccine against FCoV should be based on FCoV type 1-related antigens and that for serodiagnosis of FCoV infection TGEV should be used to attain the highest diagnostic efficiency. When serology is used in addition to clinical signs, hematology, and clinical chemistry results as an aid to diagnose clinical FIP, TGEV shows a diagnostic efficiency equal to that of a FCoV antigen.     

Neutrophil survival factors (TNF-alpha,GM-CSF,and G-CSF) produced by macrophages in cats infected with feline infectious peritonitis virus contribute to the pathogenesis of granulomatous lesions

Feline infectious peritonitis (FIP) is a feline coronavirus (FCoV)-induced fatal disease of domestic and wild cats. The infiltration of neutrophils into granulomatous lesions is unusual for a viral disease, but it is a typical finding of FIP. This study aimed to investigate the reason for the lesions containing neutrophils in cats with FIP. Neutrophils of cats with FIP were cultured, and changes in the cell survival rate were assessed. In addition, the presence or absence of neutrophil survival factors was investigated in specimens collected from cats with FIP. Furthermore, it was investigated whether macrophages, one of the target cells of FIPV infection, produce neutrophil survival factors (TNF-alpha, GM-CSF, and G-CSF). We showed that virus-infected macrophages overproduce neutrophil survival factors, and these factors act on neutrophils and up-regulate their survival. These observations suggest that sustained production of neutrophil survival factors by macrophages during FCoV infection is sufficient for neutrophil survival and contributes to development of granulomatous lesions.     

Full genome analysis of a novel type II feline coronavirus NTU156

Infections by type II feline coronaviruses (FCoVs) have been shown to be significantly correlated with fatal feline infectious peritonitis (FIP). Despite nearly six decades having passed since its first emergence, different studies have shown that type II FCoV represents only a small portion of the total FCoV seropositivity in cats; hence, there is very limited knowledge of the evolution of type II FCoV. To elucidate the correlation between viral emergence and FIP, a local isolate (NTU156) that was derived from a FIP cat was analyzed along with other worldwide strains. Containing an in-frame deletion of 442 nucleotides in open reading frame 3c, the complete genome size of NTU156 (28,897 nucleotides) appears to be the smallest among the known type II feline coronaviruses. Bootscan analysis revealed that NTU156 evolved from two crossover events between type I FCoV and canine coronavirus, with recombination sites located in the RNA-dependent RNA polymerase and M genes. With an exchange of nearly one-third of the genome with other members of alphacoronaviruses, the new emerging virus could gain new antigenicity, posing a threat to cats that either have been infected with a type I virus before or never have been infected with FCoV.     

Expression patterns in feline blood and tissues of α<Subscript>1</Subscript>-acid glycoprotein (AGP) and of an AGP-related protein (AGPrP)

₁-Acid glycoprotein (AGP) is an acute-phase protein (APP) that modulates immune responses, probably – at least in humans – owing to the modification of its glycosylation pattern. On this perspective, feline AGP can be a useful comparative model, as it has different concentrations in cats susceptible or resistant to some disease. As a preliminary approach to the study of feline AGP (fAGP) we have purified this protein from feline serum by HPLC using human AGP (hAGP) as a model. Immunoblotting with a polyclonal antibody against fAGP and with a monoclonal antibody against hAGP was performed on serum from healthy cats, from cats exposed to feline coronavirus (FCoV) infection and from cats with purulent inflammations, such as feline infectious peritonitis (FIP), feline immunodeficiency virus (FIV) and feline leukemia virus (FeLV). Immunohistochemistry on tissues from healthy cats and from cats with different diseases (FIP, FIV, FeLV, locally extensive inflammation) was also performed with the same antibodies. Both hAGP and fAGP have been purified to homogenity as determined by SDS-PAGE. fAGP did not react with the anti-hAGP antibody which, in contrast, detected in feline serum a low MW protein that we called fAGP-related protein (fAGPrP). This protein was underexpressed in cats with FeLV and FIP. Both fAGP and fAGPrP were immunohistochemically detected in plasma and hepatocytes with a stronger intensity in cats with FIP and some inflammatory conditions. Moreover, fAGPrP was detected in the cytoplasm of tissue cells, most likely identifiable with plasma cells. These cells were rarely detectable in cats with FIV and FeLV, and numerous in cats with FIP and with locally extensive inflammation. In conclusion, purified fAGP has physicochemical characteristics similar to those of hAGP, but does not cross-react with anti-hAGP antibodies. In contrast, the anti-hAGP detected an AGP-related protein whose blood concentration and tissue distribution was not related to that of fAGP. Moreover, both fAGP and fAGPrP were differently expressed in cats with pathologic conditions compared to controls. Further study of these proteins by analysing their structural characteristics is required.Abbreviations AGP ₁-Acid glycoprotein - APP acutephase protein - FC0V feline coronavirus - FeLV feline leukemia virus - FIP feline infectious peritonitis - FIV feline immunodeficiency virus Originally presented at the ACCA meeting 2002, Sandwich, Kent, UK.     

Detection of subgenomic mRNA of feline coronavirus by real-time polymerase chain reaction based on primer-probe energy transfer (P-sg-QPCR)

Feline infectious peritonitis is one of the most severe devastating diseases of the Felidae. Upon the appearance of clinical signs, a cure for the infected animal is impossible. Therefore rapid and proper diagnosis for both the presence of the causative agent, feline coronavirus (FCoV) and the manifestation of feline infectious peritonitis is of paramount importance. In the present work, a novel real-time RT-PCR method is described which is able to detect FCoV and to determine simultaneously the quantity of the viral RNA. The new assay combines the M gene subgenomic messenger RNA (sg-mRNA) detection and the quantitation of the genome copies of FCoV. In order to detect the broadest spectrum of potential FCoV variants and to achieve the most accurate results in the detection ability the new assay is applying the primer-probe energy transfer (PriProET) principle. This technology was chosen since PriProET is very robust to tolerate the nucleotide substitutions in the target area. Therefore, this technology provides a very broad-range system, which is able to detect simultaneously many variants of the virus(es) even if the target genomic regions show large scale of variations. The detection specificity of the new assay was proven by positive amplification from a set of nine different FCoV strains and negative from the tested non-coronaviral targets. Examination of faecal samples of healthy young cats, organ samples of perished animals, which suffered from feline infectious peritonitis, and cat leukocytes from uncertain clinical cases were also subjected to the assay. The sensitivity of the P-sg-QPCR method was high, since as few as 10 genome copies of FCoV were detected. The quantitative sg-mRNA detection method revealed more than 10-50,000 times increase of the M gene sg-mRNA in organ materials of feline infectious peritonitis cases, compared to those of the enteric FCoV variants present in the faeces of normal, healthy cats. These results indicate the applicability of the new P-sg-QPCR test as a powerful novel tool for the better detection and quantitation of FCoV and for the improved diagnosis of feline infectious peritonitis, this important disease of the Felidae, causing serious losses in the cat populations at a global scale.     

Activation of p38 MAPK by feline infectious peritonitis virus regulates pro-inflammatory cytokine production in primary blood-derived feline mononuclear cells

Feline infectious peritonitis (FIP) is an invariably fatal disease of cats caused by systemic infection with a feline coronavirus (FCoV) termed feline infectious peritonitis virus (FIPV). The lethal pathology associated with FIP (granulomatous inflammation and T-cell lymphopenia) is thought to be mediated by aberrant modulation of the immune system due to infection of cells such as monocytes and macrophages. Overproduction of pro-inflammatory cytokines occurs in cats with FIP, and has been suggested to play a significant role in the disease process. However, the mechanism underlying this process remains unknown. Here we show that infection of primary blood-derived feline mononuclear cells by FIPV WSU 79-1146 and FIPV-DF2 leads to rapid activation of the p38 MAPK pathway and that this activation regulates production of the pro-inflammatory cytokine tumor necrosis factor alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta). FIPV-induced p38 MAPK activation and pro-inflammatory cytokine production was inhibited by the pyridinyl imidazole inhibitors SB 203580 and SC 409 in a dose-dependent manner. FIPV-induced p38 MAPK activation was observed in primary feline blood-derived mononuclear cells individually purified from multiple SPF cats, as was the inhibition of TNF-alpha production by pyridinyl imidazole inhibitors.     

Seroprevalence of Bartonella henselae infection and correlation with disease status in cats in Switzerland.

The prevalence of infection with Bartonella henselae was investigated in cats from different areas of Switzerland. Serum samples of 728 cats were examined for antibodies to B. henselae by immunofluorescent antibody testing, and the results were analyzed with a view to a possible correlation between a positive titer and signalment, clinical signs, infection with feline leukemia virus (FeLV), feline immunodeficiency virus (FIV), feline coronavirus (FCoV), or feline spumavirus (FeSFV), and the living environments of the cats. The seroprevalence in all cats was 8.3%. No significantly different prevalence was found in sick versus healthy cats (9.2 versus 7.2%); however, in sick cats seropositive for B. henselae, there was an increased frequency of stomatitis and a variety of diseases of the kidneys and the urinary tract. There was an increased prevalence of B. henselae in cats positive for FCoV (P = 0.0185) or FeSFV (P = 0.0235) and no statistically significant increased prevalence in cats infected with FeLV or FIV. There was no correlation between a positive titer and sex or breed. The same prevalence of B. henselae antibodies was found in cats with and without access to the outdoors and in cats from single- and multicat households. The seroprevalence was increased in cats living south of the Alps (12.1%); however, this difference was not significant (P = 0.0616).     

B-cell activation in cats with feline infectious peritonitis (FIP) by FIP-virus-induced B-cell differentiation/survival factors

It has been suggested that antibody overproduction plays a role in the pathogenesis of feline infectious peritonitis (FIP). However, only a few studies on the B-cell activation mechanism after FIP virus (FIPV) infection have been reported. The present study shows that: (1) the ratio of peripheral blood sIg⁺ CD21⁻ B-cells was higher in cats with FIP than in SPF cats, (2) the albumin-to-globulin ratio has negative correlation with the ratio of peripheral blood sIg⁺ CD21⁻ B-cell, (3) cells strongly expressing mRNA of the plasma cell master gene, B-lymphocyte-induced maturation protein 1 (Blimp-1), were increased in peripheral blood in cats with FIP, (4) mRNA expression of B-cell differentiation/survival factors, IL-6, CD40 ligand, and B-cell-activating factor belonging to the tumor necrosis factor family (BAFF), was enhanced in macrophages in cats with FIP, and (5) mRNAs of these B-cell differentiation/survival factors were overexpressed in antibody-dependent enhancement (ADE)-induced macrophages. These data suggest that virus-infected macrophages overproduce B-cell differentiation/survival factors, and these factors act on B-cells and promote B-cell differentiation into plasma cells in FIPV-infected cats.     

Two related strains of feline infectious peritonitis virus isolated from immunocompromised cats infected with a feline enteric coronavirus.

Two groups of cats were experimentally infected orally with the cat-passaged RM strain of feline enteric coronavirus (FECV-RM). One group of cats (n = 19) had been chronically infected with feline immunodeficiency virus (FIV) for over 6 years, while a second control group (n = 20) consisted of FIV-naive siblings. Fecal virus shedding of FECV occurred in both groups starting on day 3 postinfection, nearly ceased by 4 weeks in FIV-uninfected cats, but remained at high levels in FIV-infected animals. FIV-infected cats shed virus for a longer period of time and at levels 10 to 100 times greater than those for FIV-uninfected cats. The coronavirus antibody response of the FIV-infected cats was delayed and of reduced titer compared with that of the FIV-uninfected animals. Cats in both groups remained asymptomatic for the first two months following FECV-RM infection; however, 8 to 10 weeks postinfection two cats in the FIV-infected group developed feline infectious peritonitis (FIP). The FIP viruses (designated FIPV-UCD9 and -UCD10) isolated from these two cats had almost complete genetic homology to each other and to the infecting FECV-RM. However, unlike FECV-RM, they readily induced FIP when inoculated intraperitoneally into specific-pathogen-free cats. This study confirms that FIPVs are frequently and rapidly arising mutants of FECV. Immunosuppression caused by chronic FIV infection may have enhanced the creation and selection of FIPV mutants by increasing the rate of FECV replication in the bowel and inhibiting the host's ability to combat the mutant viruses once they occurred.     

Replication of feline coronaviruses in peripheral blood monocytes

Summary. Feline infectious peritonitis virus (FIPV) (Coronaviridae) causes the most lethal viral infection in cats: FIP. The related feline enteric coronavirus (FECV) causes mild enteritis. Why these feline coronaviruses manifest so differently in vivo is not known. In this study, infection kinetics (titres and antigen expression) of FIPV 79-1146, and FECV 79-1683, were determined in peripheral blood monocytes from 3 donor cats and compared to those in Crandell feline kidney (CrFK) cells. The infection kinetics in monocytes were host dependent. Monocytes from 1 cat were resistant to both FIPV- and FECV-infection. Monocytes from the other 2 cats could initially be infected by both FIPV and FECV but FIPV infection was sustained in monocytes of only one cat. FECV-infection was never sustained and viral production was up to 100 times lower than in FIPV-infected monocytes. In CrFK cells, FIPV and FECV infection kinetics did not differ. In monocytes of a larger cat population (n = 19) the 3 infection patterns were also found. Considering all 22 investigated cats, 3/22 were not susceptible for FIPV and FECV. The rest could be infected with FECV and FIPV but 10/22 cats had monocytes that only sustained FIPV infection and 9/22 sustained neither FIPV nor FECV infection. Both authors contributed equally.     

Serological recognition of feline infectious peritonitis virus spike gene regions expressed as synthetic peptides andE. coli fusion protein

Summary Cats exposed to feline infectious peritonitis virus (FIPV) or feline enteric coronavirus (FECV) cannot be differentiated by serological analysis. Three synthetic peptides and anE. coli recombinant fusion protein generated from FIPV 79-1146 spike gene sequence were produced. Coronavirus positive cat sera reacted to peptide aa 950–990 but were non-reactive to aa137–151 and aa 150–180 peptides as demonstrated by ELISA. Amino acid sequence 97–222 expressed as a galk fusion protein inE. coli was tested against coronavirus positive cat sera by western blot analysis. Only sera from cats exposed to the FIPV type-II strains DF-2 or 79–1146 that were exhibiting signs of FIP recognized the fusion protein. Sera from FECV exposed cats did not recognize the 97–222 fusion protein in western blot analysis.     

Detection of transmissible gastroenteritis virus neutralising antibody in cats

Summary High titres of neutralising activity to transmissible gastroenteritis virus (TGEV), a porcine coronavirus, were found in sera and peritoneal fluids from cats infected with feline infectious peritonitis (FIP). A small proportion of cats, from a hospital population unaffected by FIP, also had neutralising activity. Procedures to remove non-specific viral inhibitors, including treatment by heat inactivation, trypsin, sulphydryl reagent and kaolin absorption were unsuccessful. The active component was unable to neutralise another porcine coronavirus, haemagglutinating encephalomyelitis virus or the porcine enterovirus, Talfan. Gel filtration of feline sera and peritoneal fluid demonstrated high levels of the neutralising activity in the area corresponding to 7S IgG, which could be removed by absorption with specific anti-IgG serum and these properties are suggested to be consistent with those of antibody. These findings imply that there is a coronavirus in cats which is antigenically related to TGEV and its possible nature is discussed.With 4 Figures     

Feline infectious peritonitis: role of the feline coronavirus 3c gene in intestinal tropism and pathogenicity based upon isolates from resident and adopted shelter cats

Feline infectious peritonitis virus (FIPV) was presumed to arise from mutations in the 3c of a ubiquitous and largely nonpathogenic feline enteric coronavirus (FECV). However, a recent study found that one-third of FIPV isolates have an intact 3c and suggested that it is not solely involved in FIP but is essential for intestinal replication. In order to confirm these assumptions, 27 fecal and 32 FIP coronavirus isolates were obtained from resident or adopted cats from a large metropolitan shelter during 2008-2009 and their 3a-c, E, and M genes sequenced. Forty percent of coronavirus isolates from FIP tissues had an intact 3c gene, while 60% had mutations that truncated the gene product. The 3c genes of fecal isolates from healthy cats were always intact. Coronavirus from FIP diseased tissues consistently induced FIP when given either oronasally or intraperitoneally (i.p.), regardless of the functional status of their 3c genes, thus confirming them to be FIPVs. In contrast, fecal isolates from healthy cats were infectious following oronasal infection and shed at high levels in feces without causing disease, as expected for FECVs. Only one in three cats shed FECV in the feces following i.p. infection, indicating that FECVs can replicate systemically, but with difficulty. FIPVs having a mutated 3c were not shed in the feces following either oronasal or i.p. inoculation, while FIPVs with intact 3c genes were shed in the feces following oronasal but not i.p. inoculation. Therefore, an intact 3c appears to be essential for intestinal replication. Although FIPVs with an intact 3c were shed in the feces following oronasal inoculation, fecal virus from these cats was not infectious for other cats. Attempts to identify potential FIP mutations in the 3a, 3b, E, and M were negative. However, the 3c gene of FIPVs, even though appearing intact, contained many more non-synonymous amino acid changes in the 3' one-third of the 3c protein than FECVs. An attempt to trace FIPV isolates back to enteric strains existing in the shelter was only partially successful due to the large region over which shelter cats and kittens originated, housing conditions prior to acquisition, and rapid movement through the shelter. No evidence could be found to support a recent theory that FIPVs and FECVs are genetically distinct.     

Vascular endothelial growth factor (VEGF), produced by feline infectious peritonitis (FIP) virus-infected monocytes and macrophages, induces vascular permeability and effusion in cats with FIP

Feline infectious peritonitis virus (FIPV) causes a fatal disease called FIP in Felidae. The effusion in body cavity is commonly associated with FIP. However, the exact mechanism of accumulation of effusion remains unclear. We investigated vascular endothelial growth factor (VEGF) to examine the relationship between VEGF levels and the amounts of effusion in cats with FIP. Furthermore, we examined VEGF production in FIPV-infected monocytes/macrophages, and we used feline vascular endothelial cells to examine vascular permeability induced by the culture supernatant of FIPV-infected macrophages. In cats with FIP, the production of effusion was related with increasing plasma VEGF levels. In FIPV-infected monocytes/macrophages, the production of VEGF was associated with proliferation of virus. Furthermore, the culture supernatant of FIPV-infected macrophages induced hyperpermeability of feline vascular endothelial cells. It was suggested that vascular permeability factors, including VEGF, produced by FIPV-infected monocytes/macrophages might increase the vascular permeability and the amounts of effusion in cats with FIP.     

Molecular cloning of feline tumour necrosis factor receptor type I (TNFR I) and expression of TNFR I and TNFR II in lymphoid cells in cats.

Tumour necrosis factor (TNF)-alpha is a pro-inflammatory cytokine produced by many types of cells. It has been shown that two distinct TNF receptors (TNFRs), TNFR type I (TNFR I) and TNFR type II (TNFR II), have different functions in signal transduction, which is possibly associated with the development of a variety of diseases. In this study, we isolated a feline TNFR I cDNA clone and analysed the expression of TNFR I and TNFR II mRNA in feline lymphoid cells. The deduced amino acid sequence of feline TNFRI cDNA showed 75.8, 62.5 60.9 and 72.1% similarity with those of its human, mouse, rat, and pig counterparts, respectively. The feline TNFR I cDNA was shown to encode extracellular, transmembrane and intracellular domains fundamentally conserved in the homologues of other species. Expression of TNFR I and TNFR II mRNAs was shown to be up-regulated in feline peripheral blood mononuclear cells (PBMC) by stimulation with concanavalin A. Five of six feline lymphoma cell lines were shown to express both TNFR I and TNFR II mRNAs. The expression of TNFR I in PBMC was up-regulated in cats infected with feline immunodeficiency virus (FIV), whereas the expression of TNFR II in PBMC was not different between FIV-infected cats and uninfected cats. The present study indicate that expression of TNFR I and TNFR II may be associated with disease progression, especially in retrovirus infections in cats.