猫杯状病毒的分离纯化及遗传进化特征分析

Purification and Genetic Evolution Characteristics of Feline Calicivirus

猫杯状病毒(FCV)作为导致猫上呼吸道感染的重要病原,具有高度传染性,严重危害猫科动物健康。为了解山西省FCV分离株变异水平及遗传发育特征,使用猫肾细胞(CRFK)分离、纯化山西省FCV分离株,用透射电镜观察病毒形态,测定病毒半数感染量(TCID_(50))和生长曲线,基于结构蛋白VP1分析猫杯状病毒基因、氨基酸变异水平与遗传发育特征。结果显示,试验纯化得3株山西省FCV分离株(SX-2021-1、SX-2021-2和SX-2021-3),病毒大小约为40 nm,TCID_(50)为10^(-8.43)/mL,FCV感染CRFK细胞的6~72 h中,在24 h时病毒滴度达到最高(SX-2021-3)。SX-2021-1与SX-2021-2株的VP1基因同源性为99.8%,SX-2021-3株VP1基因呈现较高的变异性,与其他两株的同源性约为77%。FCV山西分离株与已公布FCV毒株同源性为70.7%~83.9%,其中SX-2021-1、SX-2021-2与我国黑龙江分离株(HRB-SS)同源性最高(83.9%),SX-2021-3与我国上海分离株(SH1)同源性最高(80.5%)。FCV山西分离株VP1蛋白在E区5′高变区和3′高变区均存在多个氨基酸位点突变,其中SX-2021-3株与其他两株的VP1蛋白氨基酸差异性明显。遗传进化分析结果显示,FCV山西分离株与疫苗株和VSD毒株亲缘关系均较远,SX-2021-1、SX-2021-2与我国黑龙江地区分离株(HRB-SS、WZ-1和XH)亲缘关系较近;SX-2021-3与上海毒株(SH1)处于同一分支。综上,本研究阐明了山西省FCV分离株变异水平及遗传发育特征,为FCV病原及疫苗研究提供了重要依据。

Feline calicivirus(FCV)is an important and highly contagious pathogen that causes upper respiratory tract infections in cats and poses a serious risk to the health of felines.In order to elucidate the variation level and genetic developmental characteristics of FCV Shanxi strains,they were isolated and purified with Crandell Reese Feline Kidney cells(CRFK).The morphology of the virus was observed by transmission electron microscope,and the median infective dose(TCID_(50))and growth curve of the virus were determined.The gene,amino acid variation level and genetic development characteristics of FCV Shanxi strain were analyzed through the structural protein VP1.The results showed that three FCV Shanxi strains were isolated successfully(SX-2021-1、SX-2021-2 and SX-2021-3),with a virus size of about 40 nm and a virus titer of 10^(-8.43)TCID_(50)·mL^(-1).After infection with FCV for different times(6-72 h),the highest virus titer was reached at 24 h in CRFK cells(SX-2021-3 strain).The homology of VP1 gene between SX-2021-1 and SX-2021-2 strains is 99.8%,while the VP1 gene of SX-2021-3 strain shows high variability,with homology to the other two strains at about 77%.Compared with the published FCV strains,the homology of the FCV Shanxi isolated strains is 70.7%-83.9%.SX-2021-1 and SX-2021-2 have the highest homology(83.9%)with the Heilongjiang isolate strain(HRB-SS),and SX-2021-3 has the highest homology(80.5%)with the Shanghai isolate stain(SH1).The VP1 protein of the three FCV Shanxi isolated strains had multiple amino acid site mutations in E region,ainly concentrated in 5´hypervariable region and 3´hypervariable region.Compared with the other two strains,the amino acid sequence of the VP1 protein in SX-2021-3 strain is obviously different.The results of genetic evolution analysis showed that the FCV Shanxi isolated strains were distant from vaccine strains and VSD strains,while SX-2021-1 and SX-2021-2 were closely related to the Heilongjiang isolate strains(HRB-SS,WZ-1 and XH).SX-2021-3 and the Shanghai isolate strain(SH1)are in the same branch.In conclusion,our study elucidated the variation level and genetic development of FCV Shanxi strains,which will provide an important basis for studying FCV pathogenesis and vaccine development.