| Feline calicivirus is a main and prevelent pathogen which can cause feline upper respiratory tractdisease, oral vesiculobullous disease and chronic gastroenteritis.The characteristic clnical symptom isoral ulceration, rhinitis, conjunctivitis, chronic gastroenteritis and febrile lameness syndrome.Occasionally, liver, spleen and pancreas injury may also be seen. One of the sever strain may even leadto the death of animals. FCV has only one serotype, and its complete genome is coded by three openreading frames, showing a diversity of its gene. FCV distributes all over the world, and has beenseparated in rare wild feline animals in some countries and regions. Recently, FCV has brought threat tothe health and survival of our country’s cats and wild animals. Therefore, the isolation and identificationand biological characteristics study of FCV will play an important scientific role in its prevention andbasic biology research.In this study, Nasopharyngeal swabs which were suspected of being infected FCV were inoculatedon CRFK. After continuous cell batches, CRFK cells appeared cytopathic effect at the third generation.By RT-PCR, the objective stripe was got from the isolate; through eva green real time RT-PCR, wedeteced the concentration of the isolate culture was 2.03×106 copies/uL; under the electron microscope,virus particles were found which were round, nonenveloped and between 35 and 39 nm, conforming tothe characteristics of FCV; indirect immunofluorescence test showed that the isolate had response withFCV positive serum. The titer of the isolate was 108 TCID50/mL. By phage assay, the isolate was purified.According to FCV sequences in Gen Bank, three pair of primers were designed to amplify the completegenome. Using DNAStar software, the genome sequence was received and was submitted to GenBank,with the registration number KM111557.In this study, biological characteristics reserch such as culture characteristics, physical andchemical factors, genetic evolution and mice pathogenicity experiment was also done. Physical andchemical properties such as temperature, acid-base, organic solvent, dry and ultraviolet irradiationshowed that 50℃ for 30 min or 70℃ for 5 min, pH value at 3.0 or more than 10.0 could inactivate theisolate. It was not sensitive to solvents. Ultraviolet irradiation was stronger than dry in inactivating thevirus during the same time. The cell pathogenicity test showed that it could make CRFK cells, Verocells and MDCK cells appear CPE, but it had no pathogenicity to DFI cells, PK-15 cell and BHK-21 cells. Complete genome sequence analysis showed that FB-NJ-13 and the other 33 reference strains hada homology of 76.2%~79.5%. Analysis of isolate ORF1, ORF2 and ORF3 amino acid sequence andphylogenetic tree analysis showed that ORF3 amino acid sequence was relativelyconservative(82.2%~86.9%). ORF1, ORF2 and ORF3 had closer genetic relationship with with GD.Thus, FB-NJ-13 may have closest relationship with GD isolate. After infection of FB-NJ-13, the micewere all alive, and only oral group appeared weight loss. Real-time fluorescent quantitative RT-PCRmethod didn’t detecte FCV in these organs.In summary, we got one FCV isolate and submitted the genome sequence to GenBank. We also didsome research on physical and chemical properties, cell pathogenic test, sequence and genetic evalutionanalysist and mice pathogenicity experimen of the isolate. Our research will provide reference for thefurther study about epidemiological study and prevention of FCV. |
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