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1、Arch Virol (1989) 109:185-196 Archives Vi rology by Springer-Verlag 1989 Characterization of a temperature sensitive feline infectious peritonitis coronavirus K. K. Christianson, J. D. Ingersoll, R. M. Landon, N. E. Pfeiffer, and J. D. Gerber Biological Research and Development, Norden Laboratories,
2、 Lincoln, Nebraska, U.S.A. Accepted September 20, 1989 Summary. The characteristics of a temperature sensitive feline infectious peri- tonitis virus (TS-FIPV) were examined. TS-FIPV, unlike its parent strain, DF2 wild type FIPV (WT-FIPV), propagated at 31 C (permissive temperature) but not at 39 C (
3、nonpermissive temperature). This temperature preference of TS- FIPV was also demonstrated in cats by the ability of the virus to replicate only at the lower temperature in the upper respiratory tract and not at systemic sites where higher temperatures (38-39 C) prevail. Viral structural proteins and
4、 RNA were synthesized at 39 C but some undefined maturational defect prevented the formation of infectious TS-FIPV at its nonpermissive temperature. TS- FIPV was more thennolabile than WT-FIPV which indicated alterations in the structural proteins of TS-FIPV, and a difference in the envelope protein
5、 of the two viruses was revealed by Western blot analysis. Plaque assay characterization showed that TS-FIPV produced small plaques in comparison to the large plaques of WT-FIPV. These unique characteristics possessed by TS-FIPV may account for its nonvirulent nature and ability to stimulate protect
6、ive immune responses in cats. Introduction Feline infectious peritonitis (FIP) is a complex and fatal disease of cats caused by infection with feline infectious peritonitis virus (FIPV). Previous attempts to consistently protect cats by immunization with other antigenically related coronaviruses suc
7、h as porcine transmissible gastroenteritis virus (TGEV) 26, canine coronavirus (CeV) 2, and human coronavirus 1 have been unsuc- cessful. Inconsistent protection was found when cats were given a sublethal dose of virulent FIPV and cats vaccinated with an avirulent FIPV were more easily infected than
8、 were nonvaccinated cats 14. Recently, it has been dem- onstrated that an intranasally (IN) administered temperature sensitive FIP (TS-FIPV) vaccine is efficacious and safe upon FIPV challenge 5. 186 K.K. Christianson et al. The pathogenesis of FIP is complicated and not fully understood. Evidence i
9、ndicates that FIP is an immune-mediated disease 10. The virus has been shown to replicate initially in the upper respiratory tract and small intestine 20. Macrophages, the primary FIPV target cell, may then cross the mucosal barrier and spread virus throughout the cat 8, 24, 25. Furthermore, a cor-
10、relation between FIPV virulence in vivo and ability to infect macrophages in vitro has been observed 18. It has been suggested that a strong cell-mediated immune response to FIPV may be more important than humoral immunity in protecting cats from this disease 15, 19. However, the role of local immun
11、e responses in the upper respiratory tract and intestinal tract has not been carefully evaluated and may represent an important immune defense mechanism against FIP. TS-FIPV was developed by serial passages at a reduced temperature, fol- lowed by ultraviolet irradiation. The selected virus will prop
12、agate at its per- missive temperature (31 C) but not at its nonpermissive temperature (39 C). Temperature sensitivity was accompanied by the appearance of various char- acteristics that distinguish TS-FIPV from its virulent parent strain. The purpose of this report is to present these distinguishing
13、 characteristics which include plaque size, temperature stability, ability to synthesize RNA, expression of structural proteins and temperature dependent replication in vitro and in vivo. Materials and methods Cell culture Norden Laboratories feline kidney (NLFK) cells were used from passage 80 to 9
14、2. Cells were propagated in Basal Medium Eagle (BME) supplemented with 5% fetal bovine serum (FBS) and 10raM Hepes buffer. Virus isolation The DF2 wild type FIP virus (WT-FIPV) was originally isolated from a cat liver explant. After several passages of tissue homogenates in specific pathogen free (S
15、PF) cats, the virus was adapted to NLFK cells by cocultivation with infected primary spleen cells. The DF2 WT-FIPV strain was grown on NLFK cells at 39 C for passages I through 60 and then passed at 31 C up to passage 99. The virus collected at passage 99 was ultraviolet irradiated (5 cm distance wi
16、th a Westinghouse 782-30 lamp, 118 V, 60 cycles, 0.5 amps) for 5 min. The virus was plaque purified and designated TS-FIPV. Passage 13 of the plaque purified TS- FIPV was used in the characterization studies. Plaque assay, growth curve, and virus titration The plaque assay was done with confluent cell monolayers inoculated with 0.2 ml of a tenfold virus dilution. After adsorption for 1 h at 31 C or 39 C, the cells were overlaid with 4ml of 0.75% carboxymethylcellulose in 199
