Establishment And Application Of Reverse Genetics System For Feline Calicivirus 2280

Feline Calicivirus(FCV) belongs to the family Caliciviridae,the genus Vesivirus and is a common pathogen of domestic cats and wild cats.FCV infection is usually associated with respiratory disease,oral ulcer,conjunctivitis,chronic enteritis.Highly virulent,systemic strains of FCV,cause high mortality in infected cats.At present,using of vaccines for the prevention of FCV is still a major measure,but the existing vaccines fail to provide complete protection.Animals recoved from the disease can release FCV for long periods of time makes it more difficult to prevention.FCV 2280 is a moderate-virulent strain that can cause pantropic infection.To further understand the pathogenesis of FCV 2280,we need to use the reverse genetics system to study the relationship between viral genome structure and function.We established reverse genetics system of FCV 2280 strain.First,according to the characteristics of restriction sites in FCV genome,we divided the genome into three fragements and cloned them into p OK-12,respectively.At the same time,T7 RNA polymerase promoter and ribozyme sequence of hepatitis delta virus(Hdv Rz)were introduced into 5? end and 3? end of the FCV genomic c DNA respectively,and a genetic marker was created by the synonymous mutation to disable the Xba I cleavage site in the viral genome at554 position.Then the vector expressing viral genomic p OK-2280 was obtained.RNAs with cap structure were prepared by in vitro transcription after linearized,and we transfected it into CRFK cells and then obtained P1 recombinant virus.In addition,the recombinant viral genomic RNA was extracted and prepared as c DNA template.As following,we identified the viral c DNA fragment by Xba I digesting.As a consequence,the rescued virus was recombinant virus.Moreover,the replication kinetics of the r FCV was similar to the parental virus.The established FCV reverse genetic system provided a basis for further study on biological characteristics and pathogenic mechanism of FCV.To develop an effective vector vaccine,we selected different positions to identify which could insert foreign gene.First,the 5' end of p5.6,the 3' end of p76,the 5' end of VP1 gene and the position of VP1 gene at 264 to 265 were selected respectively base on reverse genetics of FCV2280 to construct a recombinant vector expressing the enhanced green fluorescent protein(e GFP).Then the recombinant vector was transfected into CRFK cells after in vitro transcription to rescue the recombinant virus.The rescued virus infected CRFK cells respectively,and were identified through the expressing of e GFP.As a result,the fluorescence was observed after insertion into the5 'end of the VP1 gene and the 264 to 265 site of VP1 gene.At last,we identified the stability of the e GFP gene and found no fluorescence when the recombinant virus with the 5' end of the VP1 gene inserted in P2 generation,and no fluorescence until P5 generation in other recombinant virus.Therefore,the stability needs to be further improved.In conclusion,we established the reverse genetics for FCV 2280,and screened out the insertion sites of foreign genes.It will set the foundation for the study of the pathogenesis of FCV,and improve the the development of live vector vaccine.