Establishment Of Reverse Genetics System For Chimeric FCV F9 And Screening Of Replication Differential Of Genes

Feline Calicivirus(FCV)belongs to Vesivirus in the Caliciviridae family,which is mainly harmful to felines and distributed widely in the world.The Feline Calicivirus mainly causes upper respiratory diseases and is most often detected in feline stomatitis.The immunity protection of feline provided by the Feline Calicivirus vaccine is poor,what?s more,felines sometimes get Feline Calicivirus disease clinically after immunization and possibly have a long-term or even lifelong detox.So far,there is no domestic vaccine for the Feline Calicivirus that has been approved for sale in China.Thus,the study of Feline Calicivirus is very urgent,but the related research is still limited and our understanding of all aspects of feline goblet virus is still insufficient.The attenuated strain FCV F9 is a classic Feline Calicivirus vaccine strain which is widely used in the study of Feline Calicivirus vaccine as a parent strain of most current FCV live vaccines.We spliced FCV F9 genome-wide sequence by restrict enzymes and ligases in order to construct FCV F9 infectious clones.By optimizing the codons of the sequence,we solved the problem of frameshift mutation and obtained the recombinant FCV F9 clone plasmid p OK-F9 H with the exact amino acid.The linearized p OK-F9 H was transcribed in vitro,capped RNA was transfected into F81 cells,and the virus r FCV F9 was rescued.After analyzing the plaque forming ability,CPE forming ability and multi-step growth curve of the rescue virus and the parent strain,r FCV F9 and the parental strain were basically identical in terms of replication ability and vitro growth ability,which indicated the success of constructing infectious clone of FCV F9 strain and rescuing the virus.The reverse genetic system of the virulent strain FCV 2280 has been constructed in the early stage of our experiment,and the two reverse genetic systems of virulent and attenuated strains were used to construct a chimeric virus for further experiments.We studied the influence of ORFs of Feline Calicivirus on the difference in FCV in vitro replication by constructing ORFs chimeric viruses.We divided the three ORFs into ORF1 and ORF2-3,and used FCV 2280 and FCV F9 as the viral backbones used in replacement.And then we constructed four chimeric plasmids,rescued the chimeric virus as well.Through the biological analysis of the ORF chimeric virus and the parental virus,we found that three ORFs all had an effect on the growth of the virus.In order to screen out the genes affecting FCV replication in ORF1,six non-structural proteins in ORF1 of FCV F9 were replaced with FCV 2280 as the viral backbone,and four chimeric plasmids were constructed,the chimeric virus was rescued as well.After screening for P39 and p30-VPg proteins,the p39 and p30-VPg proteins in ORF1 of FCV 2280 were chimerically replaced with FCV F9 as the viral backbone,and the chimeric virus was rescued and the results were verified.Through the biological analysis of the chimeric virus of ORF1 gene and the parental virus,the results showed that the replacement of p30-VPg in virulent and attenuated strains of Feline Calicivirus had the greatest influence on the growth ability of the virus.In this study,we successfully constructed FCV F9 infectious clones and rescued ten chimeric viruses using a reverse genetic systems of virulent and attenuated strains.Biological analysis showed that p30-VPg are the most non-structural proteins that had the greatest impact on FCV replication.