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

doi:10.7668/hbnxb.20194602

Abstract

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

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.

/ / Recommend / Download Citations

URL: http://www.hbnxb.net/EN/10.7668/hbnxb.20194602

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

Figures/Tables 7

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

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.

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

Fig.4 Nucleotide sequences alignment of NM-21strain

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

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.

References 20

[1]	Liu D X, Lu H J, Shi K, Su F Y, Li J M, Du R. Immunogenicity of recombinant BCGs expressing predicted antigenic epitopes of Bovine viral diarrhea virus E2 gene[J]. Research in Veterinary Science, 2014, 97(2):430-438.doi:10.1016/j.rvsc.2014.07.004.
[2]	李佑民, 刘振润, 武银莲. 牛病毒性腹泻-粘膜病病毒株(长春184)的分离与鉴定[J]. 兽医大学学报, 1983, 3(2):113-120.doi:10.16303/j.cnki.1005-4545.1983.02.001.
	Li Y M, Liu Z R, Wu Y L. Isolation and identification of Bovine viral diarrhea-mucosal disease virus strain Changchun 184[J]. Chinese Journal of Veterinary Science, 1983, 3(2):113-120.
[3]	Olafson P, MacCallum A D, Fox F H. An apparently new transmissible disease of cattle[J]. The Cornell Veterinarian, 1946,36:205-213.
[4]	Benavides B, Casal J, Diéguez J F, Yus E, Moya S J, Armengol R, Allepuz A. Development of a quantitative risk assessment of Bovine viral diarrhea virus and Bovine herpesvirus-1 introduction in dairy cattle herds to improve biosecurity[J]. Journal of Dairy Science, 2020, 103(7):6454-6472.doi:10.3168/jds.2019-17827. pmid: 32359990
[5]	Corapi W V, Donis R O, Dubovi E J. Monoclonal antibody analyses of cytopathic and noncytopathic viruses from Fatal bovine viral diarrhea virus infections[J]. Journal of Virology, 1988, 62(8):2823-2827.doi:10.1128/JVI.62.8.2823-2827.1988.
[6]	Camargo L J, Picoli T, Fischer G, de Freitas A C O, de Almeida R B, da Silva Pinto L.Antiviral activity of native banana lectin against Bovine viral diarrhea virus and Bovine alphaherpesvirus type 1[J]. International Journal of Biological Macromolecules, 2020, 157:569-576.doi:10.1016/j.ijbiomac.2020.04.125.
[7]	Ridpath J F. Practical significance of heterogeneity among BVDV strains:impact of biotype and genotype on U.S.control programs[J]. Preventive Veterinary Medicine, 2005, 72(1/2):17-30.doi:10.1016/j.prevetmed.2005.08.003.
[8]	Ridpath J. The contribution of infections with Bovine viral diarrhea viruses to bovine respiratory disease[J]. The Veterinary Clinics of North America Food Animal Practice, 2010, 26(2):335-348.doi:10.1016/j.cvfa.2010.04.003.
[9]	Kokkonos K G, Fossat N, Nielsen L, Holm C, Hepkema W M, Bukh J, Scheel T K H. Evolutionary selection of pestivirus variants with altered or no microRNA dependency[J]. Nucleic Acids Research, 2020, 48(10):5555-5571.doi:10.1093/nar/gkaa300. pmid: 32374844
[10]	Donis R O. Molecular biology of Bovine viral diarrhea virus and its interactions with the host[J]. The Veterinary Clinics of North America:Food Animal Practice, 1995, 11(3):393-423.doi:10.1016/S0749-0720(15)30459-X.
[11]	Deregt D, van Rijn P A, Wiens T Y, van den Hurk J. Monoclonal antibodies to the E2 protein of a new genotype(type 2)of Bovine viral diarrhea virus define three antigenic domains involved in neutralization[J]. Virus Research, 1998, 57(2):171-182.doi:10.1016/S0168-1702(98)00095-1. pmid: 9870585
[12]	胡俊英, 鲁海冰, 常晓冉, 李欣, 马振乾, 郭昌明, 张泽财, 王新平. 基于单抗捕获牛病毒性腹泻病毒(BVDV)抗原ELISA方法的建立及应用[J]. 中国兽医学报, 2019, 39(3):401-407.doi:10.16303/j.cnki.1005-4545.2019.03.05.
	Hu J Y, Lu H B, Chang X R, Li X, Ma Z Q, Guo C M, Zhang Z C, Wang X P. Development of a sandwich ELISA for capture of Bovine viral diarrhea virus antigen and epidemiological investigation[J]. Chinese Journal of Veterinary Science, 2019, 39(3):401-407.
[13]	温树波, 宋扬, 蒙小刚, 陈卓, 孟令浩, 王沐笙, 王云龙, 霍晓伟, 刘锴, 翟景波, 鲁会军, 金宁一. 一株进口胎牛血清源BVDV毒株的分离与鉴定[J]. 中国病原生物学杂志, 2021, 16(7):743-746.doi:10.13350/j.cjpb.210701.
	Wen S B, Song Y, Meng X G, Chen Z, Meng L H, Wang M S, Wang Y L, Huo X W, Liu K, Zhai J B, Lu H J, Jin N Y. Isolation and identification of a BVDV strain from imported fetal bovine serum[J]. Journal of Pathogen Biology, 2021, 16(7):743-746.
[14]	Fray M D, Paton D J, Alenius S. The effects of Bovine viral diarrhoea virus on cattle reproduction in relation to disease control[J]. Animal Reproduction Science, 2000,60:615-627.doi:10.1016/S0378-4320(00)00082-8.
[15]	Hou P L, Zhao G M, Wang H M, He H B. Prevalence of Bovine viral diarrhea virus in dairy cattle herds in Eastern China[J]. Tropical Animal Health and Production, 2019, 51(4):791-798.doi:10.1007/s11250-018-1751-z.
[16]	刘存, 邓永, 梁琳, 李金祥, 张彦明, 崔尚金. 牛病毒性腹泻病毒链特异性SYBR Green荧光定量PCR方法的建立及应用[J]. 畜牧兽医学报, 2019, 50(10):2079-2087.doi:10.11843/j.issn.0366-6964.2019.10.014.
	Liu C, Deng Y, Liang L, Li J X, Zhang Y M, Cui S J. Establishment and application of strand specific SYBR green real-time PCR for detection of Bovine viral diarrhea virus[J]. Chinese Journal of Animal and Veterinary Sciences, 2019, 50(10):2079-2087.
[17]	Baccili C C, Martin C C, Decaris N, Madureira K M, Chase C, Gomes V. Effects of 3 different commercial vaccines formulations against BVDV and BHV-1 on the inflammatory response of Holstein heifers[J]. Veterinary Sciences, 2019, 6(3):69.doi:10.3390/vetsci6030069.
[18]	GÓmez-Romero N, Velazquez-Salinas L, Ridpath J F, Verdugo-Rodríguez A, Basurto-Alcántara F J. Detection and genotyping of Bovine viral diarrhea virus found contaminating commercial veterinary vaccines,cell lines,and fetal bovine serum lots originating in Mexico[J]. Archives of Virology, 2021, 166(7):1999-2003.doi:10.1007/s00705-021-05089-9.
[19]	Nagayama K, Oguma K, Sentsui H. Survey on vertical infection of Bovine viral diarrhea virus from fetal bovine sera in the field[J]. Journal of Veterinary Medical Science, 2015, 77(11):1531-1534.doi:10.1292/jvms.14-0556. pmid: 26074250
[20]	Mody K T, Mahony D, Cavallaro A S, Zhang J, Zhang B, Mahony T J, Yu C Z, Mitter N. Silica vesicle nanovaccine formulations stimulate long-term immune responses to the Bovine viral diarrhoea virus E2 protein[J]. PLoS One, 2015, 10(12):e0143507.doi:10.1371/journal.pone.0143507.

Please choose a citation manager

Content to export