杯状病毒疫苗免疫猫中杯状病毒分离株的遗传变异分析

Genetic variation analysis of calicivirus isolates from immuned cats with calicivirus vaccine

为了解猫杯状病毒(FCV)疫苗免疫猫中FCV的感染情况及分子特征,本研究随机采集哈尔滨和大连经灭活疫苗免疫的猫口腔拭子样品29份,从中分离鉴定获得8株FCV,对分离到的病毒进行全基因组测序及同源性与遗传进化分析。全基因组序列同源性分析结果显示:8株分离病毒与国内FCV参考株的核苷酸同源性为76.1%~89.3%,与国外FCV参考株的同源性为77.1%~83.5%;8株FCV间的同源性为76.9%~97.9%,DL31株与DL37株的同源性高达97.9%;与疫苗株F4、F9、F65、255相比,8株分离病毒与其同源性在76.2%~79.8%。VP1和全基因组序列遗传进化分析显示:34株FCV可以分为2个大分支,即基因群Ⅰ和Ⅱ,8株分离病毒在两个基因群均有分布,其中分离株HRB46与韩国株12Q087-1处于同一分支,但8株FCV与疫苗株F4、F9、F65、255等均不在一个分支。将分离病毒VP1重要氨基酸位点与传统疫苗株F9比较分析,结果显示:其D区和conE区的氨基酸相对保守,但其5’高变区(5’HVR)与3’HVR的变异较大,而已有研究发现疫苗株F9在E区5’HVR的Asn-GlyThr序列(aa439~aa441)具有较强的免疫原性,8株分离株在该位点发生了1aa~3aa变异;部分B细胞抗原表位中的氨基酸也发生了突变,8株FCV在aa448~aa450这3个位点发生了连续性变异。以上结果表明FCV具有高度的遗传变异性,由此可能造成FCV疫苗免疫失败。本研究首次对FCV疫苗免疫猫开展FCV的分离鉴定,并分析了分离株与疫苗株的差异,为FCV感染的防控和疫苗的研发奠定了基础。

To investigate the prevalence of feline calicivirus(FCV) from immunized cats, 29 oral swab samples were collected from the cats immunized with inactivated FCV vaccines in Harbin and Dalian. 8 strains of FCV wereisolated and identified. The complete genome of them was sequenced and genetic evolution analysis was performed. The results showed that the isolated virus shares 76.1%-89.3% identity at the nucleotide level with the domestic reference strain and 77.1%-83.5% identity at the nucleotide level with foreign reference strains;the 8 FCVstrains share 76.9%-97.9% homology identity, and the homology identity between DL31 strain and DL37 strain is as high as 97.9%. Compared with vaccine strains F4, F9, F65, and 225, the 8 isolated viruses share76.2%-79.8% homology identity. The genetic evolution analysis of VP1 and the whole genome sequence showed that 34 strains of FCV used in this study can be divided into two branches, namely gene groups Ⅰ and Ⅱ, 8 isolated viruses are distributed in both gene groups. More recently, the isolated strain HRB46 and the Korean strain 12 Q087-1 are in the same branch, but the eight FCV strains and vaccine strains F4, F9, F65, 255, etc. are not in the same branch. Analysis of the key amino acid positions of VP1 gene of the isolated virus and the traditional vaccine strain F9 showed that the amino acids in the regions of D and con E are relatively conserved, while the 5’HVR and 3’HVR vary greatly, The immunogenicity of Asn-Gly-Thr sequence(aa439-aa441) in the region of5’HVR-E of vaccine strain F9 was very strong, and 1~3 amino acid variation occurred in 8 isolates. Some amino acids of the B cell epitope have mutated;the continuous amino acids mutation occurred between aa448 and aa450 in 8 isolated viruses. The above results indicate that FCV has a high degree of genetic variability, resulting in the failure of feline calicivirus vaccine immunity. In this study, FCV was isolated and identified in cats immunized with FCV vaccine for the first time, and the differences between isolates and vaccine strains were analyzed, which laid a foundation for the prevention and control of FCV and the further development of vaccine.