URA INRA de Génétique Moléculaire et Cellulaire, Génétique Virale, Ecole Nationale Vétérinaire d'Alfort, 94704 Maisons Alfort, France1
Virbac, BP 27, 06516 Carros, France2
Author for correspondence: M. Eloit.Fax +33 1 43 96 71 31. e-mail eloit{at}vet-alfort.fr
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Abstract |
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Preliminary observations showed that naturally seropositive cats, following isolation to avoid successive reinfections, follow two types of behaviour. Some of them maintain an antibody response for several months, suggesting a chronic infection. In others, a decrease in antibody titres is observed until a negative response is recorded, which may be a sign of elimination of the virus (V. Gonon, unpublished data). To analyse if such differences between virushost interactions could be associated with immune recognition targeted to specific virus proteins, we planned to investigate the antibody response against the major structural proteins of the virus.
To this aim, 13 catteries from different areas of France that comprised 150 seropositive cats, with previous (eight catteries) or no (five catteries) recorded cases of FIP, were included in the protocol. All the cats were isolated from each other by their owners at the beginning of the protocol, so as to minimize superinfection events. The antibody responses were regularly screened (every 2 months) for 1 to 2 years by an immunofluorescence assay. Because, in certain cases, the owners did not maintain strict conditions of isolation, we discarded some samples and conserved only those of 90 strictly isolated cats, which were processed for further analysis. At the end of the observation period, we ranked the cats into two groups (I and II), depending on their antibody titres. Group I included the 42 cats that became antibody-negative. RTPCR identification of virus sequences as described in Herrewegh et al. (1995a ) was conducted using samples of blood and faeces from each cat at the end of the observation period, and gave negative results (data not shown). Group II included the 48 cats that did not show any evidence of decrease in antibody titres. RTPCR analysis was conducted on samples from 36 cats at the end of the observation period and gave positive results in all cases. A sample of serum corresponding to the isolation date, or close to it, was used for Western blot analysis. The cats were ranked in groups I and II independently of previous records of clinically expressed FIP. To these two groups, we added a third group (III) of 43 naturally sick cats that were diagnosed as having the humid form FIP on the basis of symptoms, age and necropsy (when available). In this group, we tested by Western blot the sera sent by the practitioner for routine analysis.
Sera of cats from these three groups were analysed by Western blot as follows. The 79-1146 type II FCoV virus was amplified in Fcwf cells. The supernatant was clarified by low-speed centrifugation, then concentrated by ultracentrifugation. Virus proteins were resolved by SDSPAGE in a 7·5% acrylamide gel, then transferred to a nitrocellulose membrane (Optitran BA-S83, Schleicher & Schuell). Sera diluted to 1 in 7 were incubated for 4 h at room temperature. Bound antibodies were revealed through the phosphatase alkaline reaction using alkaline-phosphatase conjugated anti-cat (H+L) antibodies (Jackson Immunoresearch). The spot intensity was evaluated with NIH Image software after membrane scanning. A control positive serum, from a naturally infected cat, which gave clearly defined spots for each structural virus protein was used for the calibration of each membrane (definition of the basal line and location of each protein). (Glyco)proteins S, M and N were identified on the basis of their relative molecular mass (about 200, 25 and 50 kDa, respectively). Some sera also recognized a protein with a relative molecular mass of 6069 kDa, possibly of cellular origin (labelled protein X). The absolute intensities of anti-S, -M and -N glycoprotein responses that reflected the absolute level of antibodies against the virus varied from cat to cat independently of their group of origin. So, the results were expressed as the ratio of the intensity of each virus protein spot to the sum of the intensity of all the spots.
Fig. 1 shows the results obtained for the cats of groups I, II and III. For the X protein, which was not recognized by all sera, the depicted value is the average between the responders and the non-responders. In all groups, the anti-N response corresponded to about 30% of the total antibody response, and could not be used to define a typology of cats. In contrast, antibody responses against M and S permitted us to define two categories, as summarized in Fig. 2
, by the S/M ratio. The first category, characterized by a high S/M ratio (mean 1·6±0·8, P=0·05) includes the cats from group I, which cleared virus infection. The second category, defined by a very low S/M ratio, includes the cats from groups II (mean 0·05±0·01, P=0·05) and III (mean 0·03±0·01, P=0·05), which established a chronic FCoV infection that eventually led to FIP disease. No obvious difference could be found between cats from groups II and III.
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At this time, it is not clear whether the anti-S antibody response is a mechanism or only a marker of virus clearance. To address this question, we plan to identify the domains of the S glycoprotein preferentially recognized by the antibodies from cats of group I and to test them in vaccination experiments. In fact, as this antibody response was identified by Western blot in denaturing conditions, it is likely that the antigenicity of corresponding domain(s) is not highly dependent upon the glycoprotein conformation. The antibody response against conformational epitopes was not investigated in this study.
The results presented here show that, in natural conditions, the anti-S response does not seem to be a risk factor in the development of FIP disease, which should challenge the widely accepted idea of the detrimental effect of the S glycoprotein in FIP recombinant vaccines.
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References |
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Received 4 March 1999;
accepted 13 May 1999.