Grouping of Feline Calicivirus Field Isolates Using Monoclonal Antibodies

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1 SHORT REPORT Grouping of Feline Calicivirus Field Isolates Using Monoclonal Antibodies Tomoko TAJIMA, Erika NAKATA, Yukinobu TOHYAI1), Kazuyo YURI2), Hiromi KATAE2), and Takeshi MIKAMI3) Department of Veterinary Microbiology, College of Agriculture, Osaka Prefecture University, 1-1, Gakuen-cho, Sakai Osaka , Japan,1) Department of Veterinary Microbiology, Faculty of Agriculture, Kagoshima University, , Korimoto, Kagoshima 890, Japan,2) Animal Science Division, Dainippon Pharmaceutical Co.,Ltd., 103, Fushio-cho, Ikeda, Osaka 563, Japan and3) Department of Veterinary Microbiology, Faculty of Agriculture, The University of Tokyo, 1-1, Yayoi, Bunkyo-ku, Tokyo 113, Japan (Received 6 June 1997/Accepted 10 October 1997) Summary Immuno-serological characterizations of feline calicivirus (FCV) field isolates and vaccine strains were examined using neutralizing monoclonal antibodies (MAbs) raised against the F4 strain of FCV and polyclonal antisera. Neutralizing test (NT) with present MAbs showed that 29 isolates and 2 vaccine strains of FCV were divided into 4 groups, but 8 isolates could not be classified into any groups. The results of NT using polyclonal antisera were varied and indicated that the neutralizing activities of these sera were not related to the expression of neutralizing epitopes detected by MAbs used in the present study. These results demonstrated a heterogeneity among FCV isolates circulating concurrently in Japan and the possibility of grouping of calicivirus field isolates using MAbs. Key words: Feline calicivirus, Monoclonal antibody Feline calicivirus (FCV) infects cats via oral and mainly causes upper respiratory diseases3). A number of different strains of FCV have been isolated in the world and the serological relationships among these isolates have been analyzed since 1970)s using polyclonal antibodies ( MAbs )1,2,4,6,7) and monoclonal antibodies5,9,10) Although these strains are closely related each other and constitute one serotype, differences can be detected by serum cross-neutralization tests and enzyme-linked immunosorbent assay6,7,5,9,10) Previously the serological differences between the viruses isolated from the different clinical groups were examined2,4,5). However, there are no reports a- bout the grouping of FCV field isolates by serological characteristics. If the grouping of FCVs is possible, Corresponding address: Tomoko TAJIMA Department of Veterinary Microbiology, College of Agriculture, Osaka Prefecture University, 1-1, Gakuen-cho, Sakai Osaka 593, Japan, TEL.: ext.2491 FAX.: tajima@vet.osakafu-u.ac.jp it may be useful for seroepidemiological analysis of FCV field isolates, such as the trend of epidemic virus type or the change of antigenicity of the viruses in the field, and the development of effective vaccine. Therefore, we planned the immuno-serological characterizations of FCVs isolated in 1991 using neutralizing MAbs9-11) and tried to divide them into groups by the expression of neutralizing epitopes. Crandell feline kidney (CRFK) cells were used for the propagation of viruses. Thirty-six field isolates of FCV were derived from the oral or nasal swabs of cats suspected of having viral respiratory diseases8). Protocols of these isolates are summarized in Table 1. The F4 strain of FCV isolated in 1968 is corresponded to the prototype strain of FCV in Japan. The F 9 strain is a vaccine strain, widely used in Japan. The 255 strain is the vaccine strain supplied by Rhone Merieux (France). All of the field isolates were purified by three rounds of plaque cloning and propagated in CRFK cells. Twelve MAbs prepared against the FCV F4 strain

2 58 Tabel 1 Origin of feline calicivirus field isolates were used in the present study. All MAbs were characterized previously and the epitope that each antibody recognized was determined9-11). For neutralization test (NT) using MAbs, we prepared twofold serial dilutions steps of the MAbs (ascitic fluids) starting from 1:200 to 1:25,600. The dilutions were mixed with an equal volume of virus fluid containing 2 ~103 TCID50/ml, and incubated at CRFK monolayer cells on microplate wells. After 4 days of cultivation, the results were read and the NT titer was expressed as the reciprocal of the highest dilution of serum which neutralized the virus in more than 50% of the wells. Repeated tests were carried out and the geometric means were calculated. In order to compare the results, r value is calculated by using the formula of 37t for 1 hr. The mixture was then inoculated into

3 Grouping of feline calicivirus with monoclonal antibodies 59 Tabel 2 Results of feline calicivirus neutralization test using monoclonal antibodies a) Classification of epitopes [9-11]. b) r value is calculated by using the following formula. r= antibody titer against test strain /a ntibody titer against F4 strain 0.05>r : -,0.05 <2 : +, 2 r : ++ r=antibody titer against test strain / antibody titer against F4 strain. According to the 20-unit concept1,6), ć value higher than 0.05 was considered as positive. Polyclonal antisera was prepared in guinea pigs. Six viruses were selected as the antigens according to their reactivity with MAbs. One field isolate from each group A to D,vaccine strains F9 and 255 were

4 60 Table 3 Results of feline calivivirus neutralizing test using polyclonal antisera a) Grouping of the virus according to the results of neutralization b) Geometric mean of the reciprocal of 50% neutralization end point dilution. - :Negative at the serum concentration of 1:40. selected and propagated in CRFK cells. As the antigen, the supernatant of virus-inoculated cells was collected and concentrated using 33.3% of ammonium sulfate. After dialysing against PBS, the antigen was injected intraperitoneally into each guinea pig. Fur ther, virus antigen was administered intramuscularly for 4 times at 2-3 week interval. One week after the last injection, antisera was obtained by heart puncture. For NT using polyclonal antisera, sera was diluted

5 Grouping of feline calicivirus with monoclonal antibodies 61 1 in 10, heat-inactivated and absorbed with equal volume of normal CRFK cells and then diluted from 100 to 12,800 in two-fold serial dilutions. The method of NT is the same as NT using MAbs. In the case that the result was negative at the serum dilution of 1:100, two-fold serial dilutions starting from 1:40 was prepared and tested in the same manner again. The dilution 1:40 was selected as some antisera showed cytotoxicity against CRFK cells in the dilution lower than 1: 40. The test was done twice and geometric means were calculated. The results of NT using MAbs are shown in Table 2. From their reactivity in the NT, the field isolates of FCV were divided into 4 groups. Group A contained 12 field isolates which were not neutralized by any MAb. The vaccine strain, F9 was placed in this group. Eleven field isolates and a vaccine strain, 255, in group B were only neutralized by 1D7, which recognizes epitope 1. Group C contained 3 isolates which were neutralized by all MAbs except 1D7 and 8C7. The 3 isolates in group D were only neutralized by 10F9 and 16C10, which recognize the epitope 3B. The remaining 7 isolates which were neutralized by various MAbs could not be classified into any groups. All antibodies neutralized F4 strain, the antigen for the MAbs. MAb 8C7 reacted with only F4 strain. The results of NT using polyclonal antisera are shown in Table 3. Antisera against 91-5, F9, 91-17, 255 and neutralized about 40% of the viruses used. Antiserum against 91-7 neutralized 7 isolates (18%), less than the other sera. Thirteen isolates (30%) were not neutralized by any antisera including antisera against vaccine strains. The field isolates of FCV were divided into 4 groups according to the reactivities with neutralizing MAbs. Differences in the expression of neutralizing epitopes were observed among the groups. Previously Tohya et al. examined the reactivities of neutralizing MAbs with 10 strains of FCV from Japan, 5 from America, 1 from New Zealand and 2 from Switzerland and found different reactivities with these strains 9,10). Present results are different from their results. Although they reported that MAb 1D7 could detect all FCV strains used9,10), 23 isolates and F9 (62%) were not neutralized by 1D7 in the present study. Furthermore, 12 field isolates and F9 did not react with any MAbs. The reason for this is unknown, but one possibility is the differences of the virus isolation time. The Japanese strains they used in the previous study were isolated from 1968 to Antigenic change might occurr in field isolates during 20 years period. The results of NT using polyclonal antisera were contrary to our expectations. We expected that the viruses in group B, C and D were neutralized by the antisera against the viruses in the same group. However, there was no relationship between the grouping and reactivities with polyclonal antisera. For example, 3 isolates in group C were neutralized by many MAbs as shown in Table 2. However only one isolate (91-19) was neutralized only by homologous antiserum. These results indicate that the neutralizing activities of polyclonal antisera are not related to the expression of neutralizing epitopes detected by MAbs against F4 strain. The polyclonal antisera used in the present study neutralized about 40% of the viruses except anti-91-7 serum at the dilution of 1:40. The positive rate was a little higher than the previous report using 5 units of antibody4). However, many strains that were not neutralized by any antiserum are still existing and these results indicate the variety in the field. of FCV antigenicity For the development of an effective vaccine, the serological survey of the viruses prevalent in the field is important, especially for the viruses which show a wide variety of antigenicity. This is the first trial to characterize many FCV field isolates in Japan and divide into groups by the expression of neutralizing epitopes. Present results demonstrate a heterogeneity among FCV isolates circulating concurrently in Japan and the results are similar to the previous reports4,6). However it was possible to divide FCV field isolates into 4 groups according to the expression of neutralizing epitopes. Though the exact biological meaning of the grouping is not known in the present study, the data obtained here will be useful for further seroepidemiological and genetic analysis of FCVs. Also the periodical surveillance like this is important and will be of help in elucidating the mechanisms of antigenic changes of FCVs and to develop more effective vaccine.

6 62 Acknowledgement We would like to thank Dr. R. H. Piyankarage, Osaka Prefecture University, for reading the manuscript. References 1) Burki, F., Starustka, B. and Ruttner, O.: Attempts to serologically classify feline caliciviruses on a national and an international basis. Infect. Immun., 14, , ) Dawson, S. et al.: Typing of feline calicivirus i- solates from different clinical groups by virus neutralisation tests. Vet. Rec., 133, 13-17, ) Fenner, F.J. et al.: Caliciviridae. In: Veterinary Virology, 2nd ed., , Academic Press, San Diego, ) Knowles, J.O et al.: Neutralisation patterns a- mong recent British and North American feline calicivirus isolates from different clinical origins. Vet. Rec., 121, , ) McArdle, F. et al.: Feline calicivirus strain differentiation using monoclonal antibody analysis in an enzyme-linked immuno-flow-assay. Vet. Microbiol., 51, , ) Povey, R.C.: Serological relationships among feline caliciviruses. Infect. Immun.,10, , ) Povey, C. and Ingersoll, J.: Cross-protection a- mong feline caliciviruses. Infect. Immun., 11, , ) Tajima, T. et al.: Virus isolation from feline respiratory disease from November 1990 to May J. Anim. Clin. Res. Found., 1, 19-23, ) Tohya, Y. et al.: Preparation and characterization of neutralizing monoclonal antibodies to feline calicivirus. Jpn. J. Vet. Sci. 52, , ) Tohya, Y.et al.: Neutralizing epitopes of feline calicivirus. Arch. Virol., 111, , ) Tohya, Y. et al.: Mapping of antigenic sites involved in neutralization on the capsid protein of feline calicivirus. J. Gen. Virol.,18, ,1997.

7 Grouping of feline calicivirus with monoclonal antibodies