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

Summary Antigenically related feline coronaviruses cause two distinct disease manifestations in infected cats. The diseases are feline infectious peritonitis (FIP), in which the virus is widely disseminated, and feline enteric coronavirus (FECV), a mild disease in which the virus is usually limited to the villi. These two viruses were found to differ in their growth in cell culture. FIPV grows to higher titer, forms larger plaques and switches off host cell protein synthesis more effectively than FECV. Cross neutralization studies showed antigenic differences between the strains. There also appeared to be a difference in the nucleoprotein molecular weight of the viruses causing these two different disease syndromes.This work was supported by grant NIH/RR 01203-07.     

Differential effects of virulent and avirulent equine infectious anemia virus on macrophage cytokine expression

Equine infectious anemia virus (EIAV) causes rapid development of acute disease followed by recurring episodes of fever, thrombocytopenia, and viremia. Most infected equid eventually bring the virus under immunological control. We recently reported the development of an equine-specific ribonuclease protection assay (RPA) to quantitate mRNA levels of 10 cytokines. Using this newly developed RPA, we now show significant differences in cytokine induction in equine monocyte-derived macrophages (EMDM) exposed to virulent and avirulent EIAV. Virulent EIAV17 induced significant increases in interleukin (IL)-1alpha, IL-1beta, IL-6, IL-10, and tumor necrosis factor (TNF)-alpha by 0.5-1 h postinfection (hpi). In contrast, the avirulent virus failed to induce any of the tested cytokines above that of control levels. These data show a direct correlation between cytokine dysregulation and EIAV pathogenesis.     

Antigenic relationship of the feline infectious peritonitis virus to coronaviruses of other species

Summary Utilizing the direct and indirect fluorescent antibody procedure, the antigenic relationship of the feline infectious peritonitis virus (FIPV) to 7 other human and animal coronaviruses was studied. FIPV was found to be closely related to transmissible gastroenteritis virus (TGEV) of swine. Transmissible gastroenteritis virus and FIPV were in turn antigenically related to human coronavirus 229E (HCV-229E) and canine coronavirus (CCV). An interesting finding in the study was that the 8 coronaviruses selected for this study fell into one of two antigenically distinct groups. Viruses in each group were antigenically related to each other to varying degrees, but were antigenically unrelated to coronaviruses of the second group. The first antigenically related group was comprised of mouse hepatitis virus, type 3 (MHV-3), hemeagglutinating encephalomyelitis virus 67N (HEV-67N) of swine, calf diarrhea coronavirus (CDCV), and human coronavirus OC43 (HCV-OC43). The second antigenically related group was comprised of FIPV, TGEV, HCV-229E and CCV.With 2 FiguresThis research was supported by grants from the Winn Foundation of the Cat Fanciers of America and the S.O.C.K. Organization, Lafayette, California.     

Competitive enzyme immunoassays for the rapid detection of antibodies to feline infectious peritonitis virus polypeptides.

Monoclonal antibodies specific for the envelope (E1), peplomer (E2), and nucleocapsid (N) polypeptides of feline infectious peritonitis virus (FIPV) were used in rapid, competitive enzyme-linked immunosorbent assays (ELISA) to study the humoral immune response of cats to FIPV infection. Results from the competitive ELISAs were correlated with those from immunofluorescent antibody assays (IFAs) on 203 samples obtained from 64 individual cats. The IFA results correlated best with those obtained with the anti-E1 specific competitive ELISA (85.7%). In contrast, anti-N and anti-E2 competitive ELISA results correlated with IFA results only 65.5 and 2.4% of the time, respectively. The results of the anti-E1 specific competitive ELISA were not influenced by the total immunoglobulin concentration or the possible presence of free viral antigens in the serum. These results suggest that a competitive ELISA involving the use of enzyme-conjugated monoclonal antibody to the E1 glycoprotein of FIPV is a simple and rapid replacement for the more cumbersome IFA.     

Differences in virus receptor for type I and type II feline infectious peritonitis virus

Summary.  Feline infectious peritonitis viruses (FIPVs) are classified into type I and type II serogroups. Here, we report that feline aminopeptidase N (APN), a cell-surface metalloprotease on the intestinal, lung and kidney epithelial cells, is a receptor for type II FIPV but not for type I FIPV. A monoclonal antibody (MAb) R-G-4, which blocks infection of Felis catus whole fetus (fcwf-4) cells by type II FIPV, was obtained by immunizing mice with fcwf-4 cells which are highly susceptible to FIPV. This MAb also blocked infection of fcwf-4 cells by type II feline enteric coronavirus (FECV), canine coronavirus (CCV), and transmissible gastroenteritis virus (TGEV). On the other hand, it did not block infection by type I FIPVs. MAb R-G-4 recognized a polypeptide of relative molecular mass 120–130 kDa in feline intestinal brush-border membrane (BBM) proteins. The polypeptide possessed aminopeptidase activity, and the first 15 N-terminal amino acid sequence was identical to that of the feline APN. Feline intestinal BBM proteins and the polypeptide reacted with MAb R-G-4 (feline APN) inhibited the infectivity of type II FIPV, type II FECV, CCV and TGEV to fcwf-4 cells, but did not inhibit the infectivity of type I FIPVs. Accepted January 19, 1998 Received November 17, 1997     

Intriguing interplay between feline infectious peritonitis virus and its receptors during entry in primary feline monocytes

Two potential receptors have been described for the feline infectious peritonitis virus (FIPV): feline aminopeptidase N (fAPN) and feline dendritic cell-specific intercellular adhesion molecule grabbing non-integrin (fDC-SIGN). In cell lines, fAPN serves as a receptor for serotype II, but not for serotype I FIPV. The role of fAPN in infection of in vivo target cells, monocytes, is not yet confirmed. Both serotype I and II FIPVs use fDC-SIGN for infection of monocyte-derived cells but how is not known. In this study, the role of fAPN and fDC-SIGN was studied at different stages in FIPV infection of monocytes. First, the effects of blocking the potential receptor(s) were studied for the processes of attachment and infection. Secondly, the level of co-localization of FIPV and the receptors was determined. It was found that FIPV I binding and infection were not affected by blocking fAPN while blocking fDC-SIGN reduced FIPV I binding to 36% and practically completely inhibited infection. Accordingly, 66% of bound FIPV I particles co-localized with fDC-SIGN. Blocking fAPN reduced FIPV II binding by 53% and infection by 80%. Further, 60% of bound FIPV II co-localized with fAPN. fDC-SIGN was not involved in FIPV II binding but infection was reduced with 64% when fDC-SIGN was blocked. In conclusion, FIPV I infection of monocytes depends on fDC-SIGN. Most FIPV I particles already interact with fDC-SIGN at the plasma membrane. For FIPV II, both fAPN and fDC-SIGN are involved in infection with only fAPN playing a receptor role at the plasma membrane.     

Biological and pathological consequences of feline infectious peritonitis virus infection in the cheetah

Summary An epizootic of feline infectious peritonitis in a captive cheetah population during 1982–1983 served to focus attention on the susceptibility of the cheetah (Acinoyx jubatus) to infectious disease. Subsequent observations based upon seroepidemiological surveys and electron microscopy of fecal material verified that cheetahs were indeed capable of being infected by coronaviruses, which were antigenically related to coronaviruses affecting domestic cats, i.e. feline infectious peritonitis virus/feline enteric coronavirus. Coincident with the apparent increased susceptibility of the cheetah to infectious diseases, were observations that the cheetah was genetically unusual insofar as large amounts of enzyme-encoding loci were monomorphic, and that unrelated cheetahs were capable of accepting allogenic skin grafts. These data provided the basis for a hypothesis that the cheetah, through intensive inbreeding, had become more susceptible to viral infections as a result of genetic homogeneity.     

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.     

Epitope-specific antibody response against glycoprotein E of pseudorabies virus.

In this study we investigated the epitope-specific antibody response against glycoprotein E (gE) of pseudorabies virus. Epitope-specific antibody responses were investigated by enzyme-linked immunoperoxidase monolayer assays. In a vaccinated crossbred pig population, most pigs responded to antigenic domain E and to a lesser degree to antigenic domains C and D. Only few pigs responded to antigenic domains F, A, and B. Using vaccinated pigs, we investigated the influence of two different pseudorabies virus strains and the genetic background of the host on the epitope-specific antibody response. More pigs infected with the virulent NIA-3 strain had a detectable antibody response against antigenic domains C, F and A than did pigs infected with the mildly virulent Sterksel strain (P < or = 0.05; Fisher's exact test). No differences in the epitope-specific antibody responses of two genetically different pig breeding lines were observed (P > or = 0.1; Fisher's exact test). In both breeding lines the incidence of the epitope-specific antibody response was comparable to that in the crossbred pig population. In addition, we studied the epitope-specific antibody responses were strikingly different and indicated that genetic background influenced the epitope-specific antibody response. Of the serum samples of mice with C57BL and a BALB background, 40 and 17% respectively, were positive in the one of the epitope-specific immunoassays. In contrast to pigs, mice responded predominantly to antigenic domain D and to a lesser degree to antigenic domains E and B. Only few mice had a detectable antibody response against antigenic domains C and A, and none had a detectable antibody response against antigenic domain F.     

Morphological and biochemical comparison of virulent and avirulent isolates of Haemophilus pleuropneumoniae serotype 5.

Capsular structure and biochemical composition varied between two isolates (virulent and avirulent) of Haemophilus pleuropneumoniae serotype 5. The presence of capsule was determined by transmission electron microscopy with glutaraldehyde-osmium, ruthenium red, alcian blue, and phosphotungstic acid staining procedures. The virulent isolate of H. pleuropneumoniae had a distinct, adherent capsule. The avirulent isolate had a fragile, easily removed capsule. Capsular material (CM) and a lipopolysaccharide (LPS) were isolated from each bacterial isolate and were compared biochemically and biologically. CM from both isolates contained carbohydrates, no detectable protein, and no detectable to trace amounts of lipid A. Each LPS contained heptose, hexose, galactose, glucosamine, 2-keto-3-deoxyoctonate, and lipid A. Biological responses to CM and LPS from both isolates were demonstrated in the proclotting enzyme of Limulus polyphemus amebocyte lysate activation and in serological cross-reactions by immunofluorescence and immunodiffusion precipitation. The virulent isolate contained approximately 10 mg of LPS per g more on an original dry weight basis than the avirulent isolate. LPS from the virulent isolate contained approximately 13 times more galactose than LPS from the avirulent isolate. The differences of capsular structure and biochemical composition may contribute to the role of CM in porcine H. pleuropneumoniae infections.     

Immunization of cats against feline infectious peritonitis with anti-idiotypic antibodies.

Anti-idiotypic antibodies (Ab2s) generated against neutralizing antibodies (Ab1s) specific for feline infectious peritonitis virus (FIPV) were shown to be specific for paratope-associated idiotopes of the Ab1s and not against isotypic determinants. In a study to determine the efficacy of an anti-idiotypic vaccine against feline infectious peritonitis (FIP), cats that were immunized with a pool of monoclonal Ab2s developed Ab3s that recognized the variable regions of the Ab2s as well as the natural antigen. In cats challenged with a lethal dose of virus the control group followed a predictable course of infection ultimately succumbing to FIP. Two immunized cats survived virus challenge and a third cat lived twice as long as the controls. The fourth immunized cat showed no evidence of protection. The ability to induce levels of protection against FIP lends support to the concept of using anti-idiotypic antibodies as a prophylactic vaccine.     

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.     

Differentiation between virulent and avirulent Yersinia enterocolitica isolates by using Congo red agar

Cultivation of clinical isolates of Yersinia enterocolitica of diverse geographical origin on a medium containing 5 micrograms of Congo red per ml disclosed two colony types. These were designated CR+ and CR- according to their ability to bind Congo red. CR+ strains bore plasmids of between 40 and 50 megadaltons and were positive in several tests of Y. enterocolitica virulence, including autoagglutination, reduced growth on magnesium oxalate agar, resistance to the bactericidal effect of serum, and lethality for iron-overloaded mice. CR- strains were plasmidless and were negative in all these assays. The Congo red reaction provides a simple and efficient means of screening Y. enterocolitica for virulence and is the best available method for identifying individual plasmid-bearing colonies.     

Phylogeographic distribution of very virulent infectious bursal disease virus isolates in the Iberian Peninsula

Viral population dynamics of very virulent infectious bursal disease virus (vvIBDV) field strains isolated in the Iberian Peninsula since the first outbreak in the 1990s have been analysed. Low levels of genetic variability and a global purification selection pattern were reported in 480 base pairs of the hypervariable region of the VP2 gene, indicating a lack of a selection-driven immune escape in the evolutive pathway of the virus. The viral population structure of vvIBDV strains in the Iberian Peninsula showed a strong relationship between geography and phylogeny, with two main groups observed. A global comparison among vvIBDV strains also showed an association with sequences from the same country. The low variability, the strong purifying selection and the geographical pattern observed point to a picture where the virus evolves slowly, occupying the same geographical niche for a long time. The scenario depicted fits well with the biological features of the virus: being able to remain viable for long periods of time due to a strong environmental resistance, and as an immunosuppressive agent, capable per se of annihilating temporally the immune system of the host.