Isolation, Identification And Passage On F81 Cells Of Feline Calicivirus

Feline calicivirus(FCV), belonging to Caliciviridae family, is an important pathogen of cats, causing a variety of clinical signs such as fever, oral ulceration,rhinitis and conjunctivitis. Severe FCV infection is fatal. FCV has a widespread distribution in the feline population worldwide. The strains were isolated from cats,tigers or cheetahs in China. FCV strains is characterized by a large variation in genetics and antigenicity, which reduced the effects of present FCV vaccines.Currently, FCV vaccines contain mainly live-attenuated and inactivated vaccine. With the wide spread of FCV, many new variants appear continuously. Therefore, our study is significant for the prevention and control of FCV.The rearch on healthy or infected cats in Jilin province was carried out.Nasopharyngeal swabs were collected, treated, and then inoculated into F81 cells.With the analysis of PCR, morphological observation and immunology assay, the isolated strains were proved to be FCV. Five FCV strains were prepared and continuingly passed in F81 cells for 20 passages. After determined by TCID50, the F81 cells were infected with FCV at a multiplicity of infection(MOI) of 0.1. We recorded the cytopathic effect time and growth curve of FCV. In addition, the ORF2 genes of these passages(P1, p5, p10, p15 and P20) were amplified by RT-PCR and analysed by sequence alignment. DNASTAR and MEGA software were used to align amino acid variation and phylogenetic trees. Also, Net NGlyc1.0, Yin OYang1.2 and Prot Scale online software were used to predict protein glycosylation site and hydrophobicity.The results showed that F81 cells had the typical cytopathic effect after the inoculation with nasopharyngeal swabs. The isolated virus were named as CH-JL1,CH-JL2, CH-JL3 and CH-JL4 after the systemic identification. The five FCV strains were adapted on F81 cells for 20 passages with the steadily increasing titers of over109.0 TCID50/mL. All of strains showed a difference on the growth curve. The prolifetation peak of CH-JL3 and CH-SH were at 16 h, and that of CH-JL2 and CH-JL4 at 20 h. CH-JL1 had the latest peak at 24 h. The results of ORF2 sequencing showed a difference in nucleotide and amino acid. The variations of amino acid werelocated in P57 S, G494 R of CH-JL1, S440 L and N519 D of CH-JL3, V614 I of CH-JL4 and K448R of CH-SH. Phylogenetic analysis based on the nucleotide of ORF2 genes indicated that CH-JL2 and CH-JL3 belonged to the same branch and the homology of nucleotide sequences was over 95.9%. Other strains were separated in one branch,such as CH-JL1 and FB-NJ-13, CH-JL4 and F65, and CH-SH and GD. The online softwares predicted that these five FCV strains had some O-glycosylation sites and similar hydrophobicity.These FCV strains were isolated from nasopharyngeal swabs of cats successfully and passaged in F81 cells continuously. We obtained FCV strains with high titers and stability on F81 cells, laying a foundation of FCV vaccine development.