一株牛病毒性腹泻病毒(BVDV)的分离鉴定与全基因序列分析

doi:10.7668/hbnxb.20194602

华北农学报 ›› 2024, Vol. 39 ›› Issue (6): 210-215. doi: 10.7668/hbnxb.20194602

所属专题： 植物保护；畜牧；

• 畜牧·水产·兽医 • 上一篇下一篇

一株牛病毒性腹泻病毒(BVDV)的分离鉴定与全基因序列分析

吕香玉¹, 温树波¹, 赵丽霞², 林浩³, 韩健健⁴, 杨芳⁴, 郭帅⁴, 翟景波⁵, 刘锴¹^,⁶

¹ 内蒙古民族大学动物科技学院,内蒙古通辽 028000
² 金宇保灵生物药品有限公司,内蒙古呼和浩特 010000
³ 内蒙古鹏莱农牧业有限公司,内蒙古库伦旗 028200
⁴ 内蒙古通辽市动物疫病预防控制中心,内蒙古通辽 028000
⁵ 内蒙古民族大学人兽共患病创新研究院,内蒙古通辽 028000
⁶ 内蒙古自治区肉牛疾病防控工程技术研究中心,内蒙古通辽 028000

收稿日期:2024-08-24 出版日期:2024-12-28
通讯作者:
刘锴(1972—),男,内蒙古多伦人,教授,博士,主要从事兽医微生物学研究。
作者简介:
吕香玉(1998—),女,内蒙古通辽人,在读硕士,主要从事预防兽医学研究。
基金资助:
内蒙古自治区科技重大专项(2021ZD001301); 内蒙古民族大学博士启动基金(BS584); 内蒙古科技支撑东北振兴研发项目(2022YFDZ0013); 内蒙古自治区中央引导地方发展基金(2022ZY0149); 内蒙古锡林郭勒盟科技计划(202213)

Isolation,Identification and Complete Genome Sequence Analysis of a BVDV Strain

LÜ Xiangyu¹, WEN Shubo¹, ZHAO Lixia², LIN Hao³, HAN Jianjian⁴, YANG Fang⁴, GUO Shuai⁴, ZHAI Jingbo⁵, LIU Kai¹^,⁶

¹ College of Animal Science and Technology,Inner Mongolia Minzu University,Tongliao 028000,China
² The Spirit Jinyu Biological Pharmaceutical Co.,Ltd.,Hohhot 010000,China
³ Inner Mongolia Penglai Agriculture and Animal Husbandry Co.,Ltd.,Hure Banner 028200,China
⁴ Animal Disease Prevention and Control Center of Tongliao of Inner Mongolia,Tongliao 028000,China
⁵ Institute of Innovative Research on Zoonoses,Inner Mongolia Minzu University,Tongliao 028000,China
⁶ Inner Mongolia Autonomous Region Beef Cattle Disease Prevention and Control Engineering Technology Research Center,Tongliao 028000,China

Received:2024-08-24 Published:2024-12-28

摘要/Abstract

摘要：

探究内蒙古自治区通辽市牛病毒性腹泻病毒(BVDV)感染毒株主要基因型,为BVDV流行病学及防控提供参考依据。试验前期在内蒙古通辽市某牛场采集腹泻犊牛粪便样品经PCR检测,将BVDV阳性的粪便样品处理后接种到牛肾细胞(MDBK)中进行分离,通过RT-PCR、间接免疫荧光染色对分离株进行鉴定,并对其基因组全长进行测序,根据5'UTR 、N^pro和E2基因序列进行遗传进化分析和基因分型鉴定。结果表明,试验成功分离到一株BVDV毒株,将其命名为NM-21,接种NM-21的MDBK未产生细胞病变,说明该毒株为非细胞病变型(NCP),RT-PCR、间接免疫荧光染色鉴定均为阳性,病毒滴度为10^-3 TCID₅₀/mL。基于基因组全长序列、5'UTR 、N^pro和E2基因序列的同源性和遗传进化分析,分离株NM-21与中国内蒙古自治区毒株NM2103(GenBank登录号ON337882.1)核苷酸同源性最高,为BVDV-1c亚型。

关键词: 牛病毒性腹泻病毒, BVDV-1c亚型, 分离, 鉴定, 遗传进化分析

Abstract:

Investigating the predominant genotypes of Bovine viral diarrhea virus(BVDV)infecting cattle in Tongliao,Inner Mongolia,to provide reference for the BVDV epidemiology and prevention and control.In the preliminary phase of the experiment,fecal samples from diarrheic calves were collected at a cattle farm in Tongliao,Inner Mongolia.These samples were tested using PCR to detect BVDV positivity.Positive fecal samples were then inoculated into madin-darby bovine kidney cells(MDBK)for isolation.The isolated strains were identified using RT-PCR and indirect immunofluorescence staining.Subsequently,the full-length genome of the isolates was sequenced,followed by genetic evolution analysis and genotype determination based on sequences of the 5'UTR,N^pro,and E2 genes.The results indicated that this experiment successfully isolated a strain of BVDV,designated as NM-21.Inoculation of NM-21 into MDBK did not induce cytopathic effects,indicating it was a non-cytopathic strain(NCP).Both RT-PCR and indirect immunofluorescence staining confirmed its positivity,with a virus titer of 10^-3 TCID₅₀/mL.Based on the full-length genomic sequence,and homology and genetic evolution analysis of the 5'UTR,N^pro,and E2 gene sequences,the isolate NM-21 showed the highest nucleotide homology with the BVDV-1c subtype strain NM2103(GenBank accession number ON337882.1)from Inner Mongolia,China.

Key words: Bovine viral diarrhea virus, BVDV-1c subtype, Isolation, Identification, Genetic evolution analysis

吕香玉, 温树波, 赵丽霞, 林浩, 韩健健, 杨芳, 郭帅, 翟景波, 刘锴. 一株牛病毒性腹泻病毒(BVDV)的分离鉴定与全基因序列分析[J]. 华北农学报, 2024, 39(6): 210-215. doi: 10.7668/hbnxb.20194602.

LÜ Xiangyu, WEN Shubo, ZHAO Lixia, LIN Hao, HAN Jianjian, YANG Fang, GUO Shuai, ZHAI Jingbo, LIU Kai. Isolation,Identification and Complete Genome Sequence Analysis of a BVDV Strain[J]. Acta Agriculturae Boreali-Sinica, 2024, 39(6): 210-215. doi: 10.7668/hbnxb.20194602.

http://www.hbnxb.net/CN/Y2024/V39/I6/210

图/表 7

表1 BVDV检测引物

Tab.1 Primers for BVDV detection

基因 Gene	引物名称 Primer name	引物序列(5'—3') Primer sequence(5'—3')	产物长度/bp Product length
5'UTR	BVDV-5'UTR-F	ATGCCCWTAGTAGGACTAGCA	288
	BVDV-5'UTR-R	TCAACTCCATGTGCCATGTAC
N^pro	N^pro-F	TCTCTGCTGTACATGGCACATG	428
	N^pro-R	CCATCTATRCACACATAAATGTGGT
E2	E2-F	GACCTCAGTTGTAAGCCTGAG	1 119
	E2-R	CCCCCTAGCTCCTTGTTCAGTT

图1 分离毒株E2、N^pro、5'UTR基因 M.2000 DNA Marker;1.E2 基因;2.N^pro 基因;3.5'UTR 基因。

Fig.1 E2,N^pro,5'UTR genes of isolation of strains M.2000 DNA Marker;1.E2 gene;2.N^pro gene;3.5'UTR gene.

图2 间接免疫荧光染色结果(400×) A.接种分离株的MDBK;B.未接种的MDBK。

Fig.2 Results of indirect immunofluore scence staining(400×) A.Vaccinate isolated MDBK;B.Unvaccinated MDBK.

图3 BVDV NM-21分离株的生物型鉴定(400×) A.接种NM-21毒株的MDBK;B.未接种NM-21毒株的MDBK。

Fig.3 Biotype identification of BVDV NM-21 isolates(400×) A.MDBK inoculated with NM-21 strain;B.MDBK without NM-21 strain inoculation.

图4 NM-21毒株核苷酸序列比对

Fig.4 Nucleotide sequences alignment of NM-21strain

图5 基于基因组全长序列构建的系统进化树

Fig.5 Phylogenetic tree constructed based on the complete genome sequence

图6 NM-21与参考毒株的遗传进化分析 A.5'UTR基因的系统进化树;B.N^pro基因的系统进化树;C.E2基因的系统进化树。

Fig.6 Phylogenetic analysis of NM-21 and reference strains A.The phylogenetic tree based on 5'UTR gene;B.The phylogenetic tree based on N^pro gene;C.The phylogenetic tree based on E2 gene.

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