牦牛源牛呼吸道合胞体病毒的分子流行病学调查

doi:10.11843/j.issn.0366-6964.2023.05.024

摘要/Abstract

摘要： 牛呼吸道合胞体病毒(bovine respiratory syncytial virus,BRSV)是引起牛呼吸道疾病综合征(bovine respiratory diseases complex, BRDC)的重要病原之一,本试验旨在调查BRSV是否在青藏高原牦牛中存在及其在患呼吸道疾病的牦牛中的分子流行情况。2021年3月―2022年7月,在四川省甘孜藏族自治州(甘孜州)和阿坝藏族羌族自治州(阿坝州)的10个牦牛场中收集了122份患有呼吸道疾病牦牛的深部鼻腔棉拭子,其中,91份样本来自甘孜州,31份样本来自阿坝州,采用反转录恒温隔绝式PCR(reverse transcription insulated isothermal PCR, RT-iiPCR)方法对临床样本进行了BRSV的检测。结果显示,BRSV的平均阳性率为64.75%,其中,甘孜州的样本阳性率为73.63%,阿坝州的样本阳性率为38.71%;场阳性率为100%。从阳性样本中扩增到了10条完整的G基因序列(均为Ⅲ亚群)和9条完整的F基因序列,与GenBank中所有国外的BRSV毒株相比,牦牛源G和F蛋白与本实验室最近上传的BRSV毒株之间存在多个相同的氨基酸位点突变。此外,还获得了1条牦牛源的Ⅲ亚群BRSV全基因组,与本实验室最近上传的国内肉牛源BRSV毒株(GenBank登录号:OP137030~OP137034)遗传关系最近。本研究首次证实了BRSV在牦牛中的存在与流行,获得了1条牦牛源的BRSV Ⅲ亚群基因组序列,丰富了牦牛的呼吸道疾病的病原谱,为牦牛呼吸道疾病的防控提供了参考。

关键词: 牦牛, 牛呼吸道合胞体病毒, 分子流行病学, 基因特征

Abstract: Bovine respiratory syncytial virus (BRSV) is an important pathogen of bovine respiratory disease complex (BRDC). This experiment aims to investigate whether BRSV were present in Tibetan plateau yaks and its molecular prevalence in yaks with respiratory diseases. One hundred and twenty-two nasal swabs from Ganzi (Garzê) Tibetan Autonomous Prefecture and Aba (Ngawa) Tibetan and Qiang Autonomous Prefecture were collected from Yak with BRDC in 10 farms across Sichuan provinces from March 2021 to July 2022, among them, 91 samples were from Ganzi Prefecture and 31 samples were from Aba Prefecture. Using reverse transcription insulated isothermal PCR (RT-iiPCR), 64.75% of samples tested positive for BRSV. Among them, the positive rate in Ganzi Prefecture and Aba Prefecture was 73.63% and 38.71% respectively, and the farm positive rate was 100%. Further, 10 complete G genes were identified as subgroup Ⅲ strains, and 9 complete F genes were amplified from positive samples. Compared to known BRSV strains in GenBank, G proteins and F proteins from yaks with BRSV strains previously amplified by our laboratory shared several identical amino acid mutations. Moreover, complete genomes from Yaks were obtained, which was closest to the BRSV strain (GenBank accession number:OP137030-OP137034) uploaded recently by our laboratory. In conclusion, this study confirmed the existence and prevalence of BRSV in yaks for the first time, obtained a genome sequence of BRSV in the Ⅲ subgroup from yaks, enriched the pathogenic spectrum of respiratory diseases in yaks, and provided a reference for the prevention and control of respiratory diseases in yaks.

Key words: yaks, bovine respiratory syncytial virus, molecular epidemiology, genetic characteristics

中图分类号:

常益铭, 汤承, 岳华. 牦牛源牛呼吸道合胞体病毒的分子流行病学调查[J]. 畜牧兽医学报, 2023, 54(5): 2030-2041.

CHANG Yiming, TANG Cheng, YUE Hua. Molecular Epidemiological Investigation of Bovine Respiratory Syncytial Virus in Yaks[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(5): 2030-2041.

参考文献 42

[1]	STOTT E J, TAYLOR G. Respiratory syncytial virus. Brief review[J]. Arch Virol, 1985, 84(1-2):1-52.
[2]	VAN DER POEL W H M, LANGEDIJK J P M, KRAMPS J A, et al. Bovine respiratory syncytial virus antibodies in non-bovine species[J]. Arch Virol, 1995, 140(9):1549-1555.
[3]	BRYSON D G, MCCONNELL S, MCALISKEY M, et al. Ultrastructural features of alveolar lesions in induced respiratory syncytial virus pneumonia of calves[J]. Vet Pathol, 1991, 28(4):286-292.
[4]	LERCH R A, ANDERSON K, AMANN V L, et al. Nucleotide sequence analysis of the bovine respiratory syncytial virus fusion protein mRNA and expression from a recombinant vaccinia virus[J]. Virology, 1991, 181(1):118-131.
[5]	MITRA N, CERNICCHIARO N, TORRES S, et al. Metagenomic characterization of the virome associated with bovine respiratory disease in feedlot cattle identified novel viruses and suggests an etiologic role for influenza D virus[J]. J Gen Virol, 2016, 97(8):1771-1784.
[6]	BERTOLOTTI L, GIAMMARIOLI M, ROSATI S. Genetic characterization of bovine respiratory syncytial virus strains isolated in Italy:evidence for the circulation of new divergent clades[J]. J Vet Diagn Invest, 2018, 30(2):300-304.
[7]	欧阳忠明. 牛呼吸道合胞体病毒感染[J]. 中国畜禽传染病, 1995(1):62-63.OUYANG Z M. Bovine respiratory syncytial virus infection[J]. Chinese Journal of Preventive Veterinary Medicine, 1995(1):62-63. (in Chinese)
[8]	JIA S, YAO X, YANG Y Q, et al. Isolation, identification, and phylogenetic analysis of subgroup III strain of bovine respiratory syncytial virus contributed to outbreak of acute respiratory disease among cattle in Northeast China[J]. Virulence, 2021, 12(1):404-414.
[9]	TAYLOR G, STOTT E J, FURZE J, et al. Protective epitopes on the fusion protein of respiratory syncytial virus recognized by murine and bovine monoclonal antibodies[J]. J Gen Virol, 1992, 73(Pt 9):2217-2223.
[10]	THOMAS L H, COOK R S, WYLD S G, et al. Passive protection of gnotobiotic calves using monoclonal antibodies directed at different epitopes on the fusion protein of bovine respiratory syncytial virus[J]. J Infect Dis, 1998, 177(4):874-880.
[11]	TAYLOR G, BRUCE C, BARBET A F, et al. DNA vaccination against respiratory syncytial virus in young calves[J]. Vaccine, 2005, 23(10):1242-1250.
[12]	LEVINE S, KLAIBER-FRANCO R, PARADISO P R. Demonstration that glycoprotein G is the attachment protein of respiratory syncytial virus[J]. J Gen Virol, 1987, 68(Pt 9):2521-2524.
[13]	LARSEN L E, TJϕRNEHϕJ K, VIUFF B. Extensive sequence divergence among bovine respiratory syncytial viruses isolated during recurrent outbreaks in closed herds[J]. J Clin Microbiol, 2000, 38(11):4222-4227.
[14]	VALARCHER J F, SCHELCHER F, BOURHY H. Evolution of bovine respiratory syncytial virus[J]. J Virol, 2000, 74(22):10714-10728.
[15]	KLEM T B, RIMSTAD E, STOKSTAD M. Occurrence and phylogenetic analysis of bovine respiratory syncytial virus in outbreaks of respiratory disease in Norway[J]. BMC Vet Res, 2014, 10:15.
[16]	LERCH R A, STOTT E J, WERTZ G W. Characterization of bovine respiratory syncytial virus proteins and mRNAs and generation of cDNA clones to the viral mRNAs[J]. J Virol, 1989, 63(2):833-840.
[17]	SARMIENTO-SILVA R E, NAKAMURA-LOPEZ Y, VAUGHAN G. Epidemiology, molecular epidemiology and evolution of bovine respiratory syncytial virus[J]. Viruses, 2012, 4(12):3452-3467.
[18]	FURZE J M, ROBERTS S R, WERTZ G W, et al. Antigenically distinct G glycoproteins of BRSV strains share a high degree of genetic homogeneity[J]. Virology, 1997, 231(1):48-58.
[19]	ELVANDER M, VILCEK S, BAULE C, et al. Genetic and antigenic analysis of the G attachment protein of bovine respiratory syncytial virus strains[J]. J Gen Virol, 1998, 79(Pt 12):2939-2946.
[20]	LEME R A, DALL AGNOL A M, BALBO L C, et al. Molecular characterization of Brazilian wild-type strains of bovine respiratory syncytial virus reveals genetic diversity and a putative new subgroup of the virus[J]. Vet Quart, 2020, 40(1):83-96.
[21]	KUMAGAI A, KAWAUCHI K, ANDOH K, et al. Sequence and unique phylogeny of G genes of bovine respiratory syncytial viruses circulating in Japan[J]. J Vet Diagn Invest, 2021, 33(1):162-166.
[22]	王红, 侯喜林, 朴范泽. 牛呼吸道合胞体病毒的分离鉴定[J]. 畜牧兽医学报, 2009, 40(9):1414-1419.WANG H, HOU X L, PIAO F Z. Isolation and identification of bovine respiratory syncytial virus[J]. Acta Veterinaria et Zootechnica Sinica, 2009, 40(9):1414-1419. (in Chinese)
[23]	王炜. 牛主要呼吸道病毒病血清学调查、牛病毒性腹泻病毒分离株鉴定及疫苗研究[D]. 北京:中国农业科学院, 2014.WANG W. Serosurvey of major bovine resporitary viruses and identification of BVDV isolates and vaccine development[D]. Beijing:Chinese Academy of Agricultural Sciences, 2014. (in Chinese)
[24]	黄张玲. 广西部分地区牛场主要疫病血清流行病学调查[D]. 南宁:广西大学, 2016.HUANG Z L. The serosurvey of major diseases in cattle farms in parts of Guangxi Province[D]. Nanning:Guangxi University, 2016. (in Chinese)
[25]	王旭. 新疆集约化牛场犊牛IBR、BPI3、BRS流行病学调查[D]. 阿拉尔:塔里木大学, 2022.WANG X. Epidemiological investigation on IBR, BPI3 and BRS of calves in intensive cattle farms in Xinjiang[D]. Alaer:Tarim University, 2022. (in Chinese)
[26]	LIU Z K, LI J Y, LIU Z Y, et al. Development of a nanoparticle-assisted PCR assay for detection of bovine respiratory syncytial virus[J]. BMC Vet Res, 2019, 15(1):110.
[27]	GUO T, ZHANG J H, CHEN X D, et al. Investigation of viral pathogens in cattle with bovine respiratory disease complex in Inner Mongolia, China[J]. Microb Pathog, 2021, 153:104594.
[28]	魏硕佟. 牛呼吸道综合征多重PCR检测方法的建立及牛呼吸道合胞体病毒的分离鉴定[D]. 银川:宁夏大学, 2021.WEI S T. Establishment of a multiplex PCR detection of bovine respiratory disease complex and isolation of bovine respiratory syncytial virus[D]. Yinchuan:Ningxia University, 2021. (in Chinese)
[29]	孟庆辉, 陈永杏, 董红敏, 等. 牦牛分布特点及其种群数量[J]. 家畜生态学报, 2017, 38(3):80-85.MENG Q H, CHEN Y X, DONG H M, et al. The distribution characteristics and populations of yak[J]. J Livest Ecol, 2017, 38(3):80-85. (in Chinese)
[30]	何琪富, 汤承, 郭紫晶, 等. 牦牛源牛冠状病毒部分基因的扩增、序列分析及病毒分离鉴定[J]. 畜牧兽医学报, 2019, 50(2):343-353.HE Q F, TANG C, GUO Z J, et al. Amplification, sequence analysis of bovine coronavirus genes and isolation of the viruses from yak[J]. Acta Veterinaria et Zootechnica Sinica, 2019, 50(2):343-353. (in Chinese)
[31]	王生梅. 青海省海北州牦牛血清BVDV、IBRV流行病学调查[J]. 中国兽医杂志, 2021, 57(10):67-71.WANG S M. Epidemiological investigation of BVDV and IBRV in yak sera from Haibei Prefecture, Qinghai Province[J]. Chinese Journal of Veterinary Medicine, 2021, 57(10):67-71. (in Chinese)
[32]	REN Y X, CHEN X, TANG C, et al. First isolation and characteristics of bovine parainfluenza virus type 3 from yaks[J]. Pathogens, 2022, 11(9):962.
[33]	CHANG Y, YUE H, TANG C. Prevalence and molecular characteristics of bovine respiratory syncytial virus in beef cattle in China[J]. Animals (Basel), 2022, 12(24):3511.
[34]	VALENTOVA V, ANTONIS A F G, KOVARCIK K. Restriction enzyme analysis of RT-PCR amplicons as a rapid method for detection of genetic diversity among bovine respiratory syncytial virus isolates[J]. Vet Microbiol, 2005, 108(1-2):1-12.
[35]	VALENTOVA V. The antigenic and genetic variability of bovine respiratory syncytial virus with emphasis on the G protein[J]. Vet Med-Czech, 2003, 48(9):254-266.
[36]	DORELEIJERS J F, LANGEDIJK J P M, HÅRD K, et al. Solution structure of the immunodominant region of protein G of bovine respiratory syncytial virus[J]. Biochemistry, 1996, 35(47):14684-14688.
[37]	FURZE J, WERTZ G, LERCH R, et al. Antigenic heterogeneity of the attachment protein of bovine respiratory syncytial virus[J]. J Gen Virol, 1994, 75(Pt 2):363-370.
[38]	LANGEDIJK J P, MELOEN R H, TAYLOR G, et al. Antigenic structure of the central conserved region of protein G of bovine respiratory syncytial virus[J]. J Virol, 1997, 71(5):4055-4061.
[39]	SUN J F, AI H, CHEN L N, et al. Surveillance of class I Newcastle disease virus at live bird markets in China and identification of variants with increased virulence and replication capacity[J]. J Virol, 2022, 96(10):e0024122.
[40]	KARGER A, SCHMIDT U, BUCHHOLZ U J. Recombinant bovine respiratory syncytial virus with deletions of the G or SH genes:G and F proteins bind heparin[J]. J Gen Virol, 2001, 82(Pt 3):631-640.
[41]	AMANN V L, LERCH R A, ANDERSON K, et al. Bovine respiratory syncytial virus nucleocapsid protein:mRNA sequence analysis and expression from recombinant vaccinia virus vectors[J]. J Gen Virol, 1992, 73(4):999-1003.
[42]	QIU Q, ZHANG G J, MA T, et al. The yak genome and adaptation to life at high altitude[J]. Nat Genet, 2012, 44(8):946-949.