Isolation And Identification Of Feline Calicivirus And Analysis Of Gene Sequences In Hangzhou Area

Feline Calicivirus(FCV)is a common pathogeny for respiratory diseases in cats.It is distributed all over the world and often breaks out among cats.In this study,in order to understand the prevalence and molecular ecology of FCV,48 samples of suspected to be infected with FCV from Hangzhou area were identified by PCR,applied for FCV isolation and propagation,followed by phylogenetic tree analyzing based on VP1 gene sequences.The RT-PCR results showed that 32 samples were determined to be FCV positive.All of the F81 cells,inoculated with the 32 samples,showed obvious CPE.The sequence analysis of VP1 gene showed that the nucleotide homologies between the 32 isolates are between 69.2%and 99%,and the amino acid homologies are between 82.1%and 99.9%.The 32 isolates display higher homology with domestic isolates strains than that with abroad isolates.Phylogenetic tree analysis showed that 12 of the 32 isolates are closely related to the domestic isolates,15 are closely related to the UK isolates,and the other four strains with the multinational isolates form a separate branch.Analysis of the linear B cell epitopes in D-E region of VP1 gene showed that two epitopes in D region and conE region are relatively conservative,while the epitopes in 5'HRV region were highly variable.Although the 32 strains were isolated from the similar,time period of the same region,VP1 gene,the major antigenic of FCV,still dispiayed significant variance,most of which ranged from 20%to 30%.Then,the adaptability of these isolates to F81 cells was studied,the titer of the selected isolates was determined,and the multistep growth curve was drawn.The results of the adaptability of 32 isolates to F81 cells showed that DF018,DF019 and DF026 had poor adaptability to F81 cells.The multistep growth curve showed that the titers of the three isolates reached a peak at 24 h,but there were replication differences among the three isolates.Finally,the selected isolates were analyzed after full-length genome sequencing.The full-length genome sequence of DF018,DF019 and DF026 were amplified,and the full lengths obtained were 7 687 bp,7 687 bp and 7 689 bp,respectively.Further genetic and evolutionary analysis showed that DF018 was closely related to DF019,and the three isolates were closely related to the reference strains 12Q087-1 and 12Q087-5 in South Korea.The above results indicate that FCV is widely prevalent in Hangzhou area with obvious genetic polymorphic,which brings great challenges to molecular epidemiological surveillance and vaccine prevention and control of FCV infection.