Complete Genome Sequence Analysis And Establishment Of Reverse Genetic Operating System Of Feline Calicivirus

Feline calicivirus?FCV?is a highly prevalent pathogen that can cause an infectious felid upper respiratory tract disease.FCV circulates worldwide and can affect all felids in any age group.All felids are susceptible to FCV.In this study three FCV strains CH-JL1,CH-JL2 and CH-JL3 were sequenced by PCR technique,the length of amplified complete sequence was 7 687nt,7 710nt,7 710nt,respectively,and the three FCV full-length genomic sequences for CH-JL 1,2 and 3 were submitted to the GenBank database,respectively.Sequence analysis showed that in the 3?UTR,CH-JL2 and CH-JL3 were approximately more than twenty nucleotides longer than31 reference isolates,except strains HRB-SS,WZ-1,XH,12Q087-1 and 12Q087-5.Each CH-JL strain was compared with other 32 reference strains over the complete genomic sequence,ORFs nucleotide sequence and ORFs amino acid sequence respectively by DNAstar software,the result based on the complete genomic sequence alignment showed that each CH-JL strain had similarities in the range of76.2%⁸2.2%,76.8%⁹6.4 and 76.8%⁹6.4%.Phylogenetic analysis showed that CH-JL1 strain formed a branch with FB-NJ-13,GD,12Q087-1 and 12Q087-5 strains.CH-JL2 strain was the most closely related to CH-JL3 strain and formed another branch together with the other isolates.CH-JL1 strain shared a long nucleotide span with CH-JL2 and CH-JL3 strains.The availability of complete genomic sequences will serve as a reference for future epidemiological studies of FCV.The establishment of reverse genetic system can provide a platform for the research of the viral genomic function,the molecular pathogenesis,the new vaccine,the new RNA virus vector and so on.In this study,FCV CH-JL2 strain isolated from Jilin Province was selected as the research object,FCV reverse genetic manipulation platform was constructed using RNA polymerase ? in vivo transcription system.pcDNA3.1^?+?plasmid was adopted,the hammerhead ribozyme?HamRz?was add into5'end of the genome of FCV CH-JL2 strain,Hepatitis delta virus ribozyme?HdvRz?was add into 3'end of virus genome,and a genetic marker was introduced into virus genome,the infectious cDNA clone of FCV CH-JL2 strain was constructed successfully by segmented-PCR cloning,and then was transfected into the feline kidney cell F81.The result showed that there was no cytopathic effect.Then the sequence of HamRz was removed from plasmid,and the first base of the FCV genome is placed at the initiation site of transcription,later transfected F81 cells,The result showed that there was the same cytopathic effect as that of parental virus.At last,genetic marker of rescue virus was testd by RT-PCR and enzyme digestion.Specific fluorescence of rescue virus was testd by indirect immunofluorescence assay.These results showed that the reverse genetic operating system of FCV CH-JL2 strain was established successfully.The titer of rescue virus was 6.3×10⁶ IU/ml,rescue virus had almost the same increment curve as parental virus.