广西地区1株牛细小病毒1型分离鉴定及遗传进化分析

doi:10.16431/j.cnki.1671-7236.2024.10.034

摘要/Abstract

摘要： 【目的】了解广西地区牛细小病毒1型(Bovine parvovirus 1,BPV1)的生物学特性及遗传变异情况，为BPV1的防控提供理论依据和支撑。【方法】试验从广西地区养牛场采集409份犊牛腹泻粪便样本，通过BPV1特异引物进行PCR检测，使用牛鼻甲骨细胞(BT)对阳性样品进行病毒分离。收集产生细胞病变效应(cytopathic effect,CPE)的BT细胞上清液，通过PCR扩增、间接免疫荧光(indirect immunofluorescence assay,IFA)、电镜观察等方法进行病毒鉴定。测定BPV1分离株感染BT细胞后不同时间病毒半数组织培养感染剂量(median tissue culture infectious dose,TCID₅₀)，绘制病毒多步生长曲线。利用高通量测序获得病毒全基因组序列并对其进行遗传进化分析。【结果】 409份粪便样本检测到1例BPV1阳性，阳性率为0.24%。病料接种BT细胞盲传至第3代，可观察到细胞圆缩、弯曲、形成细胞团块、溶解等现象。PCR扩增结果显示，产生CPE细胞上清经PCR扩增可出现特异性条带；电镜可观察到圆形、无囊膜、直径约24 nm的病毒粒子；IFA结果显示，接种分离株的BT细胞内可观察到特异性绿色荧光，成功分离到1株BPV1，命名为GXBS2209。第6代分离株病毒滴度为10^5.75 TCID₅₀/mL，多步生长曲线显示分离株感染BT细胞后病毒滴度在120 h达到峰值，随后逐渐下降。测序结果显示，分离株基因组全长为5 515 bp(GenBank登录号: PP158225)，与美国Bovine parvovirus株全基因组序列相似性最高，为98.7%。遗传进化分析结果显示，分离株与Bovine parvovirus株处于同一分支，亲缘关系最近。分离株与参考毒株NS1、VP2基因核苷酸序列相似性分别为98.8%～99.4%、94.4%～98.1%，氨基酸序列相似性分别为98.8%～99.5%、90.3%～99.4%；NS1和VP2序列中各有2和3个氨基酸位点替换。【结论】本研究成功分离到1株BPV1,并对其全基因序列进行分析，为后续BPV1的致病机理、疫苗研究以及疾病防控奠定了基础。

关键词: 牛细小病毒1型(BPV1); 病毒分离; 序列分析

Abstract: 【Objective】 The aim of this study was to understand the biological characteristics and genetic variation of Bovine parvovirus 1 (BPV1) in Guangxi，and provide theoretical basis and support for the prevention and control of BPV1.【Method】 A total of 409 fecal samples of calf diarrhea were collected from cattle farms in Guangxi region，RCR was detected by BPV1 specific primer，and virus was isolated from positive samples by bovine turbinate bone cells (BT). The supernatant of BT cells with cytopathic effect (CPE) was collected，and the virus was identified by PCR amplification，indirect immunofluorescence assay (IFA) and electron microscope observation. The median tissue culture infectious dose (TCID₅₀) was measured at different times after BPV1 isolates infected BT cells，and the multi-step growth curve was drawn. The whole genome sequence of the virus was obtained by high throughput sequencing and its genetic evolution was analyzed.【Result】 One positive BPV1 was detected in 409 fecal samples，with a positive rate of 0.24%. After the blind transfer of BT cells to the third generation，the phenomenon of cell circle shrinkage，bending，formation of cell mass and dissolution was observed. PCR amplification results showed that specific bands could be produced by amplification of CPE supernatant. The round，membraneless virions with a diameter of about 24 nm were observed by electron microscopy. IFA results showed that specific green fluorescence could be observed in BT cells inoculated with the isolate，and a strain of BPV1 named GXBS2209 was successfully isolated. The titer of the 6th generation isolates was 10^5.75 TCID₅₀/mL，and the multi-step growth curve showed that the virus titer peaked at 120 h after the isolate infected BT cells，and then gradually decreased. The sequencing results showed that the full length of the isolated genome was 5 515 bp (GenBank accession No.: PP158225). The sequence similarity between the isolates and the American Bovine parvovirus strain was the highest (98.7%). The genetic and evolutionary analysis showed that the isolate and Bovine parvovirus strain were in the same branch and were closely related. The nucleotide sequence similarities of NS1 and VP2 genes were 98.8%-99.4% and 94.4%-98.1%，and the amino acid sequence similarities were 98.8%-99.5% and 90.3%-99.4%，respectively. There are 2 and 3 amino acid sites substitutions in NS1 and VP2 sequences，respectively.【Conclusion】 A strain of BPV1 was successfully isolated and its whole gene sequence was analyzed，which laid a foundation for the subsequent pathogenesis，vaccine research and prevention and control of BPV1.

Key words: Bovine parvovirus type 1 (BPV1); virus isolation; sequence analysis

中图分类号:

S852.65⁺3

吴奥祺, 罗宇航, 任同伟, 王豪, 董覃婷, 覃一峰, 韦祖樟, 欧阳康, 陈樱, 黄伟坚, 潘艳, 李凤梅, 谢江. 广西地区1株牛细小病毒1型分离鉴定及遗传进化分析[J]. 中国畜牧兽医, 2024, 51(10): 4540-4549.

WU Aoqi, LUO Yuhang, REN Tongwei, WANG Hao, DONG Qinting, QIN Yifeng, WEI Zuzhang, OUYANG Kang, CHEN Ying, HUANG Weijian, PAN Yan, LI Fengmei, XIE Jiang. Isolation，Identification and Genetic Evolution Analysis of a Strain of Bovine Parvovirus Type 1 in Guangxi[J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(10): 4540-4549.

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