Isolation And Identification Of Feline Calicivirus In Shanxi Province

Feline calicivirus?FCV?is a highly contagious pathogen that causes oral and upper respiratory disease in felines.In this study,we isolated virus strains from the nasopharyngeal swabs collected from sick cats suspected of FCV-infected in Shanxi Province,and identified the strains by RT-PCR and indirect immunofluorescence assay.We obtained the four strains of FCV and the FCV-positive strains were further subjected to plaque purification,and the purified virions were observed under electron microscope.And the viral titer of the isolates was determined by TCID₅₀.Finally,the experimental infection of kittens was carried out by four isolates.In the study,the sterile liquid of the nasopharyngeal swabs was first inoculated into F81 monolayer cells.After blind passage for three generations,the typical cytopathic effect of FCV appeared and four FCV strains were successfully isolated from the 12 nasopharyngeal swabs.A pair of primers for amplifying the FCV specific gene fragment was designed in the conserved region of the ORF1 gene by analyzing the FCV sequences published in Gen Bank for the identification of FCV strains.The isolates with typical FCV CPE were then identified by RT-PCR using FCV-specific primers and the 674 bp FCV specific fragments were amplified from all four isolates,indicating that the isolates were FCV,and the isolates were named as CH-SX1,CH-SX2,CH-SX3,and CH-SX4.The FCV-positive strains by RT-PCR were further subjected to plaque purification,and the selected plaques were suffered to five rounds of plaque purification assays to obtain four purified isolates.The supernatants of the F81 cell culture of the four plaque-purified isolates were negatively stained with0.5% phosphotungstic acid and a spherical with no capsule,capsid-like virion with an icosahedral structure with a diameter of about 35 nm was observed by electron microscopy,which was consistent with the structural characteristics of caliciviruses.The TCID₅₀ of the four isolates were 10^-7.1?CH-SX1?,10^-7.2?CH-SX2?,10^-6.5?CH-SX3?and 10^-8.3?CH-SX4?,respectively.In order to further identify whether the isolated strain is FCV,the F81 cells infected with the four isolates were analyzed by indirect immunofluorescence assay,respectively.The result showed that allfour isolates reacted positively with antibody against FCV,and specific green fluorescence were observed under the fluorescence microscope,while no fluorescence was observed in the negative control cell,indicating that all four isolates were FCV.Four FCV strains VP1 genes were amplified and sequenced by RT-PCR.Comparison of nucleotide sequences showed that all four FCV strains had higher homology with some classical FCV isolates and higher difference with vaccine strains.The kittens were infected with the isolated virus strain,Cats in each treated group began to die after the third to the fourth day of inoculation,and the death of cats reached its peak on the fifth day.The clinical signs of challenged cats tended to disappear gradually after two weeks of inoculation.The nasopharyngeal swabs collected from the second day after inoculation were positive for FCV by RT-PCR and no FCV was detected in the nasopharyngeal swabs collected after the sixth day of inoculation,except for the group challenged with CH-SX4,which the virus could be detected until the fourteenth day after infection.Serum antibodies were detected in the cats of all tested groups at eight days after inoculation.No abnormalities were observed in the control group.In this study,four FCV strains were isolated from nasopharyngeal swabs collected from cats suspected of FCV-infected in Shanxi Province,and four isolates were identified as FCV by a series of identification tests.The challenge test showed that all four strains have pathogenicity to kittens.Anti-FCV-specific antibodies were detected in all surviving cats on the eighth day after infection,indicating that the isolates can induce an immune response.This study provides a reference for the screening of FCV strains and the development of new vaccines,which can lay the foundation for the prevention of the disease.