Molecular Characterization Isolation And Identification Of Feline Calicivirus In Sichuan And Chongqing Regions

Feline calicivirus(FCV)is an important pathogen causing oral and upper respiratory diseases in felines and causes serious economic losses to the cat industry.Therefore,understanding the prevalence and molecular characteristics of FCV can help prevent or reduce the transmission of FCV.At present,the prevalence and molecular characteristics of FCV in Sichuan and Chongqing are still unclear,and the purpose of this study was to perform molecular detection and genomic study of FCV in feline nasopharyngeal swab samples from Sichuan and Chongqing from 2017 to2020,and the results are as follows.1.Detection of Feline Calicivirus and VP1 Typing in Sichuan and Chongqing AreasA total of 162 feline nasopharyngeal swab samples were collected from 14 cat houses and 2 animal hospitals in Sichuan and Chongqing,including 110 samples in Chengdu and 52 samples in Chongqing.Nested RT-PCR was used to detect FCV,and the genetic subtypes of FCV-positive samples were identified by VP1(B-F)partial sequence.The results showed that there were 38 FCV positive samples,the positive rate was 23.46%(38/162,95% CI: 17.2%~30.7%),in which the positive rate of cat house was 18.52%(30/162,95% CI: 12.9%~25.5%),the positive rate of animal hospital was 4.94%(8/162,95% CI: 2.2%~9.5%),the positive rate of cat house was significantly higher than that of animal hospital(p < 0.01).The results showed that FCV was prevalent in Sichuan and Chongqing areas,and the aggregation sites were more susceptible to FCV infection than animal hospitals.Thirty-eight positive samples were amplified for the partial region sequence of VP1(B-F),and due to the susceptibility of VP1 to variation,10 partial region sequences of VP1(B-F)were finally obtained,with an amplification length of 1200 bp,nucleotide similarity of 72.5%~92.8%,and deduced amino acid sequence similarity of 80.9%~96.8%.Genetic evolution analysis showed that 8 of the 10 strains clustered into the GI subtype and 2 into the GII subtype.The results showed that the main circulating GI subtypes were in Sichuan and Chongqing areas and there were variations among the strains.2.Isolation and Identification of Feline Calicivirus in Sichuan and Chongqing and Analysis of Its Whole GenomeFour FCV-positive samples were used for virus isolation using feline kidney cells(CRFK),and three FCV strains were finally isolated and cultured.These three strains were named SMU-B22-2020,SMU-F4-2020 and SMU-B5-2020,respectively,and the virus after plaque purification was 109.5 TCID50/m L,1011 TCID50/m L and 108.5TCID50/m L,respectively.With 0.5% phosphotungstic acid,the supernatant of plaque-purified virus from isolate SMU-B22-2020 was negatively stained,and round and unenveloped virions with a diameter of about 30~35 nm could be observed by transmission electron microscopy.The above results indicated that three FCV strains were successfully obtained.The three isolated FCV strains were amplified for the genome,and finally three approximate complete genome sequences were obtained,with lengths of 7653 bp,7686 bp,and 7705 bp,respectively,and the nucleotide identities of the three strains ranged from 76.8%~80.9%.Genetic evolution analysis showed that SMU-B5-2020 was most closely related to the American strain UTCVM-NH1,SMU-F4-2020 was most closely related to the Guangxi isolate GX2019 in China,SMU-B22-2020 was most closely related to the Jilin isolate CH-JL1 in China,and none of the three strains clustered into one vial with vaccine strains 2024 and F9.Recombination analysis revealed that SMU-B5-2020 had recombination phenomenon.In addition,recombination analysis revealed that there were genomic breakpoints in the structural and non-structural regions of the genome,and the recombination breakpoints were located in ORF1(3104 nt)and ORF2(5328 nt),respectively,and the parental sequences were 21223 and HRB-SS in the GI subtype.The above results showed that these three strains isolated in Sichuan and Chongqing had rich genetic diversity, which enriched the data of FCV in Sichuan and Chongqing and provided a molecular theoretical basis for understanding the genetic changes of FCV.