To comprehend the biological characteristics and genetic regulations of feline calicivirus(FCV),17 oropharyngeal samples suspected of being infected with FCV were collected from Xi'an,Shaanxi Province by PCR.The VP1 gene of FCV was amplified by PCR using the cDNA of FCV positive samples as the template and sequenced.SnapGene software was employed to analyze the amino acid sequences of VP1 of 23 FCV reference strains in this study and GenBank both within and outside China.The results showed that 6 out of 17 swab samples were FCV positive,with a positive rate of 35.3%.The amino acid sequence analysis results revealed that one VP1 sequence tested had 3 identical variations with the GII type FCV VP1 amino acid sequence,namely N377K,A539V,G557S,while the other 5 VP1 sequences were consistent with the VP1 of GI type FCV.The FCV corresponding to the VP1 gene sequence was the GII type.The FCV positive sample was inoculated into CRFK cells for virus isolation and passage,and the CPE of the cells was observed during the passage.The viruses transmitted to the 5th generation were identified by PCR and indirect immunofluorescence assay(IFA)respectively.The results showed that stable and typical CPE was produced in each generation of CRFK cells after 5 successive generations,and the specific green fluorescence of FCV was observed,indicating that a GII type FCV was isolated and named XA20-2023 strain.The whole genome sequence of the 5th generation isolated virus was amplified by PCR,and the total length of XA20-2023 strains was 7687bp after sequencing.MegAlign software was used to analyze the homology of whole genome,non-structural protein,VP1 and VP2 coding gene sequences between this virus strain and 10 FCV strains in GenBank.The phylogenetic trees of XA20-2023 strains and 85 FCV reference strains in GenBank were constructed using NJ method in MEGA11 software.The homo-logy analysis showed that the homology between XA20-2023 and 10 FCV strains in GenBank ranged from 76.4%to 84.2%,and the homology between XA20-2023 and GII type FCV SH strain was the highest at 84.2%.The homology of F9,F4 and FCV 255 with GI vaccine strains was 76.4%,77%and 77.1%,respectively.NS2 had the largest homology difference(74.6%-94.2%),while NS6 had the smallest difference(77.3%-82.9%).The homology of VP1 and VP2 coding gene sequences was 73.7%-83.8%and 76.0%-87.5%,respectively.The results of the evolutionary tree showed that XA20-2023 strains were in the same branch as the GII isolates,and the genetic distance from the GI vaccine strains F9 and F4 was relatively long.The above results further indicated that XA20-2023 strain was GII type FCV,which had low homology with the representative strains of FCV,especially the GI type vaccine strains,and the degree of variation was high.This study has enriched the gene pool of GII FCV endemic in Xi'an,and is of great significance for revealing the epidemic status of GII virus strains in China,as well as for the genetic variation of FCV and the prevention and control of diseases caused by FCV.
feline calicivirusisolation and identificationwhole genome sequencesequence analysis
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