摘要：

为调查云南大理州一处奶牛养殖小区BEFV的感染情况及其流行毒株G基因的遗传多样性。对该大理州奶牛养殖小区的10份疑似病例,参考GenBank中牛流行热病毒的G基因序列,设计并合成2对特异性扩增G基因,测序并完成序列分析。同时,采60份血清样本进行抗体检测。结果显示,对10份疑似BEF病牛全血样的检测结果,6份呈BEFV核酸阳性。去除重复序列,获得4株牛流行热病毒云南流行株G基因扩增、测序,序列全长均为1 872 bp,编码623 aa。4株毒株之间组内核苷酸序列相似性为99.7%~99.9%,遗传进化分析与亚洲毒株聚为一类,并独自为一个细小分支。与泰国2017年分离TH-NP0065毒株和中国的毒株关系最近。与我国疫苗株JB76H的氨基酸同源性比较,发现7个氨基酸突变位点,为K⁵³→N⁵³、K⁶²→R⁶²、P¹⁸³→S¹⁸³、G²⁶³→E²⁶³、K⁴⁶¹→E⁴⁶¹、K⁴⁵⁷→R⁴⁵⁷、I⁴⁷⁵→M⁴⁷⁵。除中和抗原位点G1外,G2、G3、G4均出现变异位点。抗体检测试剂盒检测结果,抗体浓度≥52.5 ng/L临界值的样品有19份,阳性率为31.67%。综上所述,该奶牛养殖小区存在BEFV的感染,且流行株G基因存在多位点变异。

关键词: 牛流行热病毒, 云南流行毒株, G基因, 序列分析

Abstract:

To investigate the infection of BEFV and the genetic diversity of its prevalent strain G gene in a dairy farming community in Dali Prefecture,Yunnan Province.It designed and synthesized two pairs of specific amplified G genes for 10 suspected cases in dairy cattle breeding community of Dali Prefecture,with reference to the G gene sequence of bovine ephemeral fever virus in GenBank,sequenced and completed sequence analysis.At the same time,60 serum samples were collected for antibody detection.The results showed that 6 out of 10 whole blood samples of cattle suspected of BEF were positive for BEFV nucleic acid.The G gene of four bovine ephemeral fever virus Yunnan epidemic strains was amplified and sequenced by removing the repetitive sequence.The total sequence length was 1 872 bp,encoding 623 aa.The nucleotide sequence homology among the four strains was 99.7%-99.9%.Phylogenetic analysis showed that the four strains were grouped together with Asian strains and were a small branch alone.It had the closest relationship with TH-NP0065 strain isolated from Thailand in 2017 and Chinese strain.Compared with the amino acid homology of the vaccine strain JB76H in China,seven amino acid mutation sites were found:K⁵³ → N⁵³,K⁶² → R⁶²,P¹⁸³ → S¹⁸³,G²⁶³ → E²⁶³,K⁴⁶¹ → E⁴⁶¹,K⁴⁵⁷ → R⁴⁵⁷,I⁴⁷⁵ → M⁴⁷⁵.Except the neutralizing antigen site G1,mutation sites were present in G2,G3 and G4.According to the results of antibody test kit,19 samples with antibody concentration≥52.5 ng/L critical value were found,with a positive rate of 31.67%.In conclusion,BEFV infection was present in this dairy farming community and there were multi-locus variants in the G gene of the prevalent strain.

Key words: Bovine ephemeral fever virus, Yunnan prevalence strain, G gene, Sequence analysis