| Feline infectious peritonitis virus(FIPV)can cause fatal immune-mediated diseases in felines.FIPV infection in domestic or wild felids usually leads to the development of pyogranulomatous lesions in the organs or the accumulation of fluid in the chest or abdomen,with a mortality rate approaching 100%after disease onset.There is a lack of clinically effective vaccine protection.Therefore,the design of an effective FIPV vaccine is crucial for controlling the disease and protecting wild felids.Among the many types of vaccines,live attenuated vaccines not only stimulate a good humoral immune response in the host,but also induce cellular immunity similar to natural infection in vivo.Studies have shown that the enzyme activities of coronavirus nonstructural proteins nsp14 and nsp16 can affect the virulence.In this study,the nsp14(N415)and nsp16(D129)methyltransferase(MTase)active sites of the strongly pathogenic virulent strain FIPV QS-79 were mutagenically weakened by reverse genetics techniques.Two mutants of FIPV QS-79 dnsp14 and FIPV QS-79 dnsp16(hereinafter referred to as dnsp14 and dnsp16)were successfully saved in CRFK cells.And their biological characteristics in vitro,pathogenicity,immunogenicity and protective effect in vivo were evaluated,the results are as follows:1.Biological characteristics of FIPV mutant strains in vitroThe plaque-forming ability of the mutant strains dnsp14 and dnsp16 and the growth kinetics on CRFK and Fcwf-4 cells were not significantly different from the parental strains,which proved that the two mutant strains did not have replication defects in vitro.After 15consecutive passages,the mutation sites of dnsp14 and dnsp16 did not recover,indicating the genetic stability of the two mutant strains was good.After infecting Fcwf-4 cells with dnsp14,dnsp16 and QS-79,it was found both mutant strains induce elevated expression of interferonβ(IFN-β)and interferon-stimulated gene 15(ISG 15).In addition,after detecting inflammation related cytokines,the expression level of Tumor Necrosis Factorα(TNF-α)was not significantly different in dnsp14 and QS-79 infected cells,while the expression level of TNF-αwas significantly enhanced in dnsp16 infected cells.The above data suggest that dnsp14 and dnsp16 can activate the host’s antiviral response.2.Pathogenicity study of FIPV mutant strain in vivoPathogenicity tests showed that adult cats oral infected with the QS-79 parental strain resulted in 100%mortality,and the mortality rate of both dnsp14 and dnsp16 oral infection was 25%.The pathogenicity of both mutant strains in adult cats was weakened.The clinical manifestation after infection in dnsp14 group were milder than those in QS-79 group.In75%of cats,the clinical manifestation gradually relief until they disappear in the later stages of infection;in 25%of cats,the clinical manifestation gradually worsen until all of these cats die.According to clinical symptoms and routine biochemical blood tests,75%of surviving cats in the dnsp16-infected group were always healthy;25%of the cats in chronic infection showed mild clinical symptoms and eventually died.The results of tissue viral load detection verified that dnsp16 mutant strains had replication defects in vivo.The virus load in the brain of the dnsp14-infected group was lower than that of the QS-79 group,and the virus load in other organs were not significantly different from those in the QS-79 group;the virus load in each organ of the dnsp16-infected group were significantly lower than those in the dnsp14 group and QS-79 group,indicating that dnsp16 mutant strains are more attenuated.3.Comparison of immunogenicity and protective efficacy of FIPV mutant strainsBoth dnsp14 mutant virus and dnsp16 mutant virus were used to immunize adult cats with high dose(10~5 TCID50)and low dose(10~4 TCID50).dnsp14 produced high levels(>2~8)of neutralizing antibody titers at both immunization doses.The dnsp16 group could only produce low levels(<2~6)of neutralizing antibodies at both immunization doses.Both high and low dose immunizations with dnsp14 provided 50%protection for the cats in challenge.f SAA is an inflammatory predictor of infection that is elevated after challenge.This was accompanied by mild clinical symptoms,suggesting that the adaptive immune response can be rapidly activated after exposure to dnsp14,producing an antiviral effect.The clinical symptoms of surviving cats gradually recovered in the infection,and the clinical symptoms of dead cats did not recover but the survival period was prolonged.Immunization with high and low doses of dnsp16 does not provide protection in adult cats.All adult cats died after the challenge.There was no significant difference in the lifecycle between the immunized group and the nonimmunized group for dead cats.Compared with the control group,the immune group had more obvious organ lesions,and the viral load in the tissue was higher.The above data show that inoculation with dnsp16mutant strain is more conducive to FIP.4.Immune protection of cured animals in secondary challengeIn order to explore whether inoculation of dnsp14 mutant strains can provide long-term protection to adult cats,a second challenge experiment was carried out on cats in the high-dose immunization group who recovered from the first challenge.All cats still maintained a high level of neutralizing antibody titers before the rechallenge.After the challenge,all cats showed reversible clinical manifestations and survived.This suggests that the adaptive immune responses induced by dnsp14 protect cats that have survived a primary FIPV challenge against subsequent challenges with FIPV.In summary,this study constructed two FIPV mutant strains dnsp14 and dnsp16 that proliferated well in vitro and were genetically stable.The pathogenicity of these two mutant strains is reduced in adult cats,and dnsp14 has better humoral immune response and protective effect than dnsp16.The above results will provide a reference for future FIPV attenuated vaccine research. |
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