胎牛血清中牛病毒性腹泻病毒2型毒株分离及脱脂奶粉中抗体的检测

doi:10.16431/j.cnki.1671-7236.2020.07.030

摘要/Abstract

摘要： 本研究旨在对进口胎牛血清中的牛病毒性腹泻病毒（BVDV）进行分离及鉴定。利用BVDV抗原和抗体检测试剂盒检测，提取胎牛血清中的病毒RNA，用5'-UTR巢式PCR进行扩增，PCR扩增产物连接pMD19-T进行测序分析。胎牛血清样品接种MDBK细胞，进行细胞传代培养，通过细胞分离培养、直接免疫荧光抗体检测对实验室进口胎牛血清样品进行病毒分离及鉴定，应用DNAStar对BVDV 5'-UTR、N^pro与GenBank中公布的瘟病毒参考株进行多序列比对，采用Mega 6.0进行遗传进化分析。同时通过包被脱脂奶粉进行间接ELISA检测其中的BVDV抗体。结果显示，胎牛血清中BVDV抗原和抗体均为阳性，并且从胎牛血清中成功分离到一株新的牛源BVDV，命名为BVDV-GC株，该病毒株在MDBK细胞上进行增殖培养时未能引起细胞病变；5'-UTR与N^pro PCR扩增为阳性，扩增产物大小均与预期相符；直接免疫荧光检测荧光信号为阳性；病毒滴度为10^-3.6TCID₅₀/0.1 mL；遗传进化分析表明，该分离株与USMARC-60779（BVDV-2）株有较近的亲缘关系，同属于BVDV-2型毒株；通过包被脱脂奶粉和商品化的ELISA试剂盒进行检测，结果表明脱脂奶粉中存在BVDV抗体。本研究从进口胎牛血清中分离出1株BVDV-2型非致细胞病变病毒，从脱脂奶粉中检测到BVDV抗体，表明进口胎牛血清和脱脂奶粉中都存在BVDV抗原和抗体污染，本研究为后续试验分析提供参考。

关键词: 牛病毒性腹泻病毒(BVDV); 胎牛血清; 分离; 鉴定; 脱脂奶粉; 抗体检测

Abstract: This study aimed to isolate and identify the bovine viral diarrhea virus (BVDV) in imported fetal bovine serum.The virus RNA in fetal bovine serum was extracted and amplified by 5'-UTR nested PCR.The PCR products were linked to pMD19-T for sequencing.The fetal bovine serum samples were inoculated with MDBK cells for cell subculture.The sample was identificated by cell culture,direct immunofluorescent assay,nucleotide sequencing and bioinformatics analysis.DNAStar was used to compare the multiple sequences of BVDV 5'-UTR,N^Pro and BLAST virus reference strains published in GenBank,and Mega 6.0 was used for genetic evolution analysis.At the same time,the antibody of BVDV was detected by indirect ELISA with skimmed milk powder.The results showed that BVDV antigen and antibody were both positive in fetal bovine serum,and a new bovine BVDV strain was successfully isolated from fetal bovine serum,and was named BVDV-GC strain,which failed to cause cytopathic changes in MDBK cell proliferation.The results showed that the virus strain did not cause cytopathic changes when it was cultured on MDBK cells.The PCR amplification of 5'-UTR and N^pro was positive,and the size of the amplification products was consistent with the expectation.The direct immunofluorescence was positive.The virus titer was 10^-3.6TCID₅₀/0.1 mL.Genetic evolution analysis showed that the isolate was closely related to USMARC-60779 (BVDV-2) strain,the same strain belonged to BVDV-2.The test was carried out with skimmed milk powder and commercial ELISA kit.The results showed that there was BVDV antibody in skimmed milk powder.In this study,a BVDV-2 virus was isolated from BVDV fetal bovine serum,which showed that BVDV antigen and antibody contamination existed in both imported fetal bovine serum and skimmed milk powder,providing a reference factor for the follow-up cell research.

Key words: bovine viral diarrhea virus (BVDV); FBS; isolation; identification; skim milk powder; antibody detection

中图分类号:

S852.65

张超, 何金科, 何延华, 马旭升, 陈创夫. 胎牛血清中牛病毒性腹泻病毒2型毒株分离及脱脂奶粉中抗体的检测[J]. 中国畜牧兽医, 2020, 47(7): 2239-2247.

ZHANG Chao, HE Jinke, HE Yanhua, MA Xusheng, CHEN Chuangfu. Isolation of Bovine Viral Diarrhea Virus Type 2 in Fetal Bovine Serum and Detection of Antibodies in Skimmed Milk Powder[J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(7): 2239-2247.

参考文献

[1] 刘占悝,李健友,刘泽余,等.牛病毒性腹泻病毒JY株分离鉴定[J].畜牧与兽医,2019,51(7):75-80. LIU Z L,LI J Y,LIU Z Y,et al.Isolation and identification of the bovine viral diarrhea virus JY strain[J].Animal Husbandry and Veterinary,2019,51(7):75-80.(in Chinese)
[2] 韩慧,郑源强,石艳春,等.牛病毒性腹泻病毒研究进展[J].畜禽业,2019,30(7):6-7. HAN H,ZHENG Y Q,SHI Y C,et al.Research progress on bovine viral diarrhea virus[J].Contemporary Animal Husbandry,2019,30(7):6-7.(in Chinese)
[3] 向文杰,林俊,宋文凤,等.3株牛病毒性腹泻病毒的分离鉴定及其基因分型[J].中国预防兽医学报,2019,41(4):422-425. XIANG W J,LIN J,SONG W F,et al.Identification and genotyping of three strains of bovine viral diarrhea virus[J].Chinese Journal of Preventive Veterinary Medicine,2019,41(4):422-425.(in Chinese)
[4] 任艳.牛病毒性腹泻病毒囊膜蛋白E2与牛胚胎滋养层细胞相互作用的分子机制研究[D].石河子:石河子大学,2010. REN Y.Molecular mechanism of bovine viral diarrhea virus envelope protein E2 interact with bovine trophoblast cells[D].Shihezi:Shihezi University,2010.(in Chinese)
[5] 马旭升,何延华,盛金良,等.牛病毒性腹泻病毒BVDV-LC株的分离与鉴定[J].中国预防兽医学报,2018,40(6):546-549. MA X S,HE Y H,SHENG J L,et al.Isolation and identification of a bovine viral diarrhea virus strain LC[J].Chinese Journal of Preventive Veterinary Medicine,2018,40(6):546-549.(in Chinese)
[6] RASMUSSEN T B,UTTENTHAL A,REIMANN I,et al.Virulence immunogenicity and vaccine properties of a novel chimeric pestivirus[J].Journal of General Virology,2007,88(2):481-486.
[7] BOLIN S R,RIDPATH J F.Prevalence of bovine viral diarrhea virus genotypes and antibody against those viral genotypes in fetal bovine serum[J].Journal of Veterinary Diagnostic Investigation,1998,10(2):135-139.
[8] DENG M,JI S,FEI W,et al.Prevalence study and genetic typing of bovine viral diarrhea virus (BVDV) in four bovine species in China[J].PLoS One,2015,10(4):e0121118.
[9] GIAMMARIOLI M,CEGLIE L,ROSSI E,et al.Increased genetic diversity of BVDV-1:Recent findings and implications thereof[J].Virus Genes,2015,50(1):147-151.
[10] XUE F,ZHU Y M,LI J,et al.Genotyping of bovine viral diarrhea viruses from cattle in China between 2005 and 2008[J].Veterinary Microbiology,2010,143(2-4):379-385.
[11] YEŞILBAǦ K,FÖRSTER C,OZYIIT M O,et al.Characterisation of bovine viral diarrhoea virus (BVDV) isolates from an outbreak with haemorrhagic enteritis and severe pneumonia[J].Veterinary Microbiology,2014,169(1-2):42-48.
[12] MAKOSCHEY B,VAN GELDER P T J A,KEIJSERS V,et al.Bovine viral diarrhoea virus antigen in foetal calf serum batches and consequences of such contamination for vaccine production[J].Biologicals:Journal of the International Association of Biological Standardization,2003,31(3):203-208.
[13] 张强,李树清,李健,等.进口胎牛血清中牛病毒性腹泻病毒污染的检测[J].中国动物检疫,2013,30(2):55-57. ZHANG Q,LI S Q,LI J,et al.Investigation for bovine viral diarrhea virus in imported commercial bovine serum[J].China Animal Health Inspection,2013,30(2):55-57.(in Chinese)
[14] 张淑琴,谭斌,刘泽余,等.猪源牛病毒性腹泻病毒JLS-01株的分离鉴定及致病性研究[J].中国畜牧兽医,2019,46(2):557-564. ZHANG S Q,TAN B,LIU Z Y,et al.Isolation,identification and pathogenicity analysis of bovine viral diarrhea virus JLS-01 strain from porcine[J].China Animal Husbandry & Veterinary Medicine,2019,46(2):557-564.(in Chinese)
[15] 郭燕.新疆北疆部分集约化奶牛场牛病毒性腹泻病毒分子流行病学调查[D].石河子:石河子大学,2007. GUO Y.Xinjiang northern border partial intensification cattle farm bovine viral diarrhea virus molecular epidemiology investigation[D].Shihezi:Shihezi University,2007.(in Chinese)
[16] 李娜,韩猛立,黄新,等.新疆石河子地区牛病毒性腹泻病毒的分子流行病学调查[J].石河子大学学报(自科版),2009,27(6):706-711. LI N,HAN M L,HUANG X,et al.Xinjiang Shihezi bovine viral diarrhea virus molecular epidemiology investigation[J].Journal of Shihezi University(Natural Science),2009,27(6):706-711.(in Chinese)
[17] 王龙,梁霄勇,季新成,等.新疆部分地区牛病毒性腹泻病毒的分离、鉴定及分子流行病学调查[J].新疆农业科学,2015,52(2):334-338. WANG L,LIANG X Y,JI X C,et al.Bovine viral diarrhea virus isolation identification and molecular epidemiology investigation in some areas in Xinjiang[J].Xinjiang Agricultural Sciences,2015,52(2):334-338.(in Chinese)
[18] RASMUSSEN T B,UTTENTHAL A,REIMANN I,et al.Virulence,immunogenicity and vaccine properties of a novel chimeric pestivirus[J].The Journal of General Virology,2007,88(2):481-486.
[19] 王竞晗,李素,何文瑞,等.进口胎牛血清中牛病毒性腹泻病毒的分离及鉴定[J].中国生物制品学杂志,2016,29(3):308-311. WANG J H,LI S,HE W R,et al.Isolation and identification of bovine viral diarrhea virus from imported fetal bovine serum[J].Chinese Journal of Biologicals,2016,29(3):308-311.(in Chinese)
[20] GIAMMARIOLI M,CEGLIE L,ROSSI E,et al.Increased genetic diversity of BVDV-1:Recent findings and implications thereof[J].Virus Genes,2015,50(1):147-151.
[21] POLAK M P,ZMUDZINSKI J F.Prevalence of bovine viral diarrhea virus infection in bulls in artificial insemination centers in Poland[J].Veterinary Microbiology,1999,64(2-3):253-257.