蓝舌病病毒血清9型毒株在我国的首次分离

doi:10.11843/j.issn.0366-6964.2019.02.013

畜牧兽医学报 ›› 2019, Vol. 50 ›› Issue (2): 354-363.doi: 10.11843/j.issn.0366-6964.2019.02.013

蓝舌病病毒血清9型毒株在我国的首次分离

李占鸿, 王金萍, 杨恒, 廖德芳, 宋建领, 高林, 何于雯, 李华春*

云南省畜牧兽医科学院热带亚热带动物病毒重点实验室, 昆明 650224

收稿日期:2018-07-06 出版日期:2019-02-23 发布日期:2019-02-23
通讯作者: 李华春,主要从事口蹄疫、蓝舌病等重大动物疫病和虫媒病毒病研究工作以及跨境动物疫病防控研究,Tel:0871-65015606,E-mail:Li_huachun@hotmail.com
作者简介:李占鸿(1989-),男,云南人,硕士,研究实习员,主要从事牛羊虫媒病毒研究,E-mail:dy081lzh@163.com;王金萍(1973-),女,辽宁人,硕士,副研究员,主要从事蓝舌病病毒研究,E-mail:ynzyfeed@163.com。
基金资助:
国家自然科学基金（31360621；31760744）；公益性行业（农业）科研专项（201303035）；云南省中青年学术和技术带头人后备人才培养项目（2017HB055）

Isolation of Bluetongue Virus Serotype 9 Strain in China for the First Time

LI Zhanhong, WANG Jinping, YANG Heng, LIAO Defang, SONG Jianling, GAO Lin, HE Yuwen, LI Huachun*

Yunnan Tropical and Subtropical Animal Virus Diseases Laboratory, Yunnan Animal Science and Veterinary Institute, Kunming 650224, China

Received:2018-07-06 Online:2019-02-23 Published:2019-02-23

摘要/Abstract

摘要：

旨在分离流行于我国云南省的蓝舌病病毒（BTV），掌握分离BTV的遗传特征与感染特性。采用"鸡胚-C6/36细胞-BHK-21细胞"接种的方式，采集哨兵牛的BTV阳性血液进行病毒分离；采用血清中和试验以及Segment 2与Segment 6 ORF区的克隆测序确定分离病毒的血清型；通过病毒噬斑形成和增殖曲线的测定，分析病毒在BHK-21细胞的增殖特性；通过qRT-PCR与血清中和试验分析BTV感染动物血液中病毒含量与中和抗体动态变化情况。结果显示：2013年8月，在云南芒市设定的哨兵牛中分离出一株BTV（毒株号V013/YN/2013），血清中和试验显示V013/YN/2013为BTV-9型病毒，Segment 2与Segment 6序列分析表明分离的病毒属BTV-9 Eastern型，与日本毒株和澳大利亚BTV-9型毒株具有最近的亲缘关系。病毒噬斑与增殖曲线测定结果显示V013/YN/2013在BHK-21细胞上增殖能力明显强于BTV-9型参考毒株。自然感染V013/YN/2013的牛在连续5个月的监控期内未出现临床症状，感染动物虽产生了特异性中和抗体，但血液中始终能持续检测到病毒核酸。本研究首次报道了BTV-9 Eastern型毒株V013/YN/2013在我国的分离，为进一步开展中国BTV-9型病毒的全基因组测序、诊断方法的建立、流行病学调查与致病性研究奠定了基础。

Abstract:

The study aims to isolate bluetongue virus (BTV) endemic in Yunnan Province of China and to understand the genetic and infection characteristics of the isolated BTV. Virus isolation was performed by inoculation method of "Embryos-C6/36 cells-BHK-21 cells" with BTV nucleic acid positive blood samples collected from sentinel cattle. Serotype of the isolated virus was decided by micro-neutralization test and sequencing the ORF regions of Segment 2 and Segment 6. The proliferation characteristics of isolated virus in BHK-21 cells were determined by the tests of growth curve and plaque formation. The viral load and neutralizing antibody of infected animal were analyzed by qRT-PCR and neutralization test. Results were as follows:In August 2013, BTV strain of V013/YN/2013 was isolated form sentinel cattle in Mangshi, Yunnan Province. Micro-neutralization test showed that V013/YN/2013 belonging to serotype of BTV-9. Further sequence analysis confirmed the identity of isolated virus by correlation with other isolated BTV-9 strains belonging to Eastern topotype and showed closest relationship with BTV-9 strains isolated from Japan and Australia. The results of growth curve and plaque formation showed that the proliferation capacity of V013/YN/2013 is significantly higher than BTV-9 reference strain. Although specific neutralizing antibodies are produced in infected animals, the presence of the virus in the blood can be continuously detected, suggesting that the virus can cause a long-lasting infection in cattle. This study reported for the first time the isolation of an BTV-9 strain V013/YN/2013 belonging to Eastern topotype in China, these results will provide a basis for further whole-genome sequencing, establishment of diagnostic methods, epidemiological investigation and pathogenicity study of BTV-9 in China.

中图分类号:

S852.659.4

李占鸿, 王金萍, 杨恒, 廖德芳, 宋建领, 高林, 何于雯, 李华春. 蓝舌病病毒血清9型毒株在我国的首次分离[J]. 畜牧兽医学报, 2019, 50(2): 354-363.

LI Zhanhong, WANG Jinping, YANG Heng, LIAO Defang, SONG Jianling, GAO Lin, HE Yuwen, LI Huachun. Isolation of Bluetongue Virus Serotype 9 Strain in China for the First Time[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2019, 50(2): 354-363.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.xmsyxb.com/CN/10.11843/j.issn.0366-6964.2019.02.013

https://www.xmsyxb.com/CN/Y2019/V50/I2/354

参考文献

[1] MACLACHLAN N J, OSBURN B I. Teratogenic bluetongue and related orbivirus infections in pregnant ruminant livestock:timing and pathogen genetics are critical[J]. Curr Opin Virol, 2017, 27:31-35.
[2] ZIENTARA S, SÁNCHEZ-VIZCAÍNO J M. Control of bluetongue in Europe[J]. Vet Microbiol, 2013, 165(1-2):33-37.
[3] MAAN S, MAAN N S, ROSS-SMITH N, et al. Sequence analysis of bluetongue virus serotype 8 from the Netherlands 2006 and comparison to other European strains[J]. Virology, 2008, 377(2):308-318.
[4] CARPENTER S, WILSON A, MELLOR P S. Culicoides and the emergence of bluetongue virus in northern Europe[J]. Trends Microbiol, 2009, 17(4):172-178.
[5] BENELLI G, BUTTAZZONI L, CANALE A, et al. Bluetongue outbreaks:Looking for effective control strategies against Culicoides vectors[J]. Res Vet Sci, 2017, 115:263-270.
[6] ROY P. Bluetongue virus structure and assembly[J]. Curr Opin Virol, 2017, 24:115-123.
[7] KOCHINGER S, RENEVEY N, HOFMANN M A, et al. Vesicular stomatitis virus replicon expressing the VP2 outer capsid protein of bluetongue virus serotype 8 induces complete protection of sheep against challenge infection[J]. Vet Res, 2014, 45:64.
[8] XIE S Y, SHI Y M, GONG R Y, et al. Identification of antigenic epitopes of monoclonal antibodies against the VP2 protein of the 25 serotype of bluetongue virus[J]. Vet Microbiol, 2018, 219:136-143.
[9] MAAN S, MAAN N S, SAMUEL A R, et al. Analysis and phylogenetic comparisons of full-length VP2 genes of the 24 bluetongue virus serotypes[J]. J Gen Virol, 2007, 88(Pt 2):621-630.
[10] ZHANG X, BOYCE M, BHATTACHARYA B, et al. Bluetongue virus coat protein VP2 contains sialic acid-binding domains, and VP5 resembles enveloped virus fusion proteins[J]. Proc Natl Acad Sci U S A, 2010, 107(14):6292-6297.
[11] RUSSELL B L, PARBHOO N, GILDENHUYS S. Analysis of conserved, computationally predicted epitope regions for VP5 and VP7 across three orbiviruses[J/OL]. Bioinform Biol Insights, 2018, doi:10. 1177/1177932218755348[2017-07-10]. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5802602/.
[12] HOFMANN M A, RENZULLO S, MADER M, et al. Genetic characterization of toggenburg orbivirus, a new bluetongue virus, from goats, Switzerland[J]. Emerg Infect Dis, 2008, 14(12):1855-1861.
[13] MAAN S, MAAN N S, NOMIKOU K, et al. Complete genome characterisation of a novel 26th bluetongue virus serotype from Kuwait[J]. PLoS One, 2011, 6(10):e26147.
[14] SCHULZ C, BRÉARD E, SAILLEAU C, et al. Bluetongue virus serotype 27:Detection and characterization of two novel variants in Corsica, France[J]. J Gen Virol, 2016, 97(9):2073-2083.
[15] MCLACHLANA N J, DREW C P, DARPEL K E, et al. The pathology and pathogenesis of bluetongue[J]. J Comp Pathol, 2009, 141(1):1-16.
[16] KIRKLANDA P D, ZHANG N, HAWKES R A, et al. Studies on the epidemiology of bluetongue virus in China[J]. Epidemiol Infect, 2002, 128(2):257-263.
[17] CLAVIJOA A, HECKERT R A, DULAC G C, et al. Isolation and identification of bluetongue virus[J]. J Virol Methods, 2000, 87(1-2):13-23.
[18] 李华春,朱建波,花群义,等. GB/T 18636-2017蓝舌病诊断技术[S]. 北京:中国标准出版社, 2017.
LI H C, ZHU J B, HUA Q Y, et al.GB/T 18636-2017 Diagnostic techniques for bluetongue[S]. Beijing:China Standard Press, 2017. (in Chinese)
[19] YANG H, ZHU J B, LI H C, et al. Full genome sequence of bluetongue virus serotype 4 from China[J]. J Virol, 2012, 86(23):13122-13123.
[20] TAMURA K L, STECHER G, PETERSON D, et al. MEGA6:molecular evolutionary genetics analysis version 6. 0[J]. Mol Biol Evol, 2013, 30(12):2725-2729.
[21] SHIRAFUJI H, YANASE T, KATO T, et al. Genetic and phylogenetic characterization of genome segments 2 and 6 of bluetongue virus isolates in Japan from 1985 to 2008[J]. J Gen Virol, 2012, 93(Pt 7):1465-1473.
[22] RAO P P, REDDY Y V, MEENA K, et al. Genetic characterization of bluetongue virus serotype 9 isolates from India[J]. Virus Genes, 2012, 44(2):286-294.
[23] FIRTH C, BLASDELL K R, AMOS-RITCHIE R, et al. Genomic analysis of bluetongue virus episystems in Australia and Indonesia[J]. Vet Res, 2017, 48(1):82.
[24] BOYLE D B, BULACH D M, AMOS-RITCHIE R, et al. Genomic sequences of Australian bluetongue virus prototype serotypes reveal global relationships and possible routes of entry into Australia[J]. J Virol, 2012, 86(12):6724-6731.
[25] NOMIKOU K, HUGHES J, WASH R, et al. Widespread reassortment shapes the evolution and epidemiology of bluetongue virus following European invasion[J]. PLoS Pathog, 2015, 11(8):e1005056.
[26] ATTOUI H, MAAN S, SIMON J, et al. Bluetongue virus, other orbiviruses and other reoviruses:Their relationships and taxonomy[M]//MELLOR P S, BAYLIS M, MERTENS P P C. Bluetongue. London, UK:Elsevier Academic Press, 2009.
[27] MAAN N S, MAAN S, BELAGANAHALLI M N, et al. Identification and differentiation of the twenty six bluetongue virus serotypes by RT-PCR amplification of the serotype-specific genome segment 2[J]. PloS One, 2012, 7(2):e32601.
[28] SHAFIQ M, MINAKSHI P, BHATEJA A, et al. Evidence of genetic reassortment between Indian isolate of bluetongue virus serotype 21(BTV-21) and bluetongue virus serotype 16(BTV-16)[J]. Virus Res, 2013, 173(2):336-343.
[29] COETZEE P, VAN VUUREN M, VENTER E H, et al. A review of experimental infections with bluetongue virus in the mammalian host[J]. Virus Res, 2014, 182(4):21-34.
[30] WILSON A, DARPEL K, MELLOR S. Where does bluetongue virus sleep in the winter?[J]. PLoS Biol, 2008, 6(8):e210.

蓝舌病病毒血清9型毒株在我国的首次分离

Isolation of Bluetongue Virus Serotype 9 Strain in China for the First Time

RichHTML

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 15

编辑推荐

Metrics

本文评价

[1]	陈姝宇, 朱雪蛟, 周金柱, 陶然, 张雪寒, 索朗斯珠, 贡嘎, 李彬. 鸟苷酸结合蛋白GBP1和GBP2抑制猪轮状病毒体外复制[J]. 畜牧兽医学报, 2024, 55(1): 245-257.
[2]	李惠惠, 董可儿, 刘馨博, 张春晓, 马超, 陈利苹, 钟旗, 姚刚, 马雪连. 牛诺瓦病毒和牛轮状病毒双重RAA-LFD快速检测方法的建立及应用[J]. 畜牧兽医学报, 2024, 55(1): 406-412.
[3]	李晓涵, 杨伏春, 刘芮, 高立, 崔红玉, 张艳萍, 刘长军, 祁小乐, 王笑梅, 高玉龙, 李凯. 表达鸡传染性法氏囊病病毒VP2基因的重组火鸡疱疹病毒的构建及生物学特性分析[J]. 畜牧兽医学报, 2023, 54(2): 656-662.
[4]	李占鸿, 朱沛, 宋子昂, 李卓然, 杨振兴, 李华春, 杨恒, 廖德芳. 蓝舌病病毒群特异性RT-LAMP检测方法的建立与初步应用[J]. 畜牧兽医学报, 2021, 52(8): 2244-2253.
[5]	吕珽, 陈虹吟, 汤承, 岳华. 牦牛源正呼肠孤病毒2型的检测和分离鉴定[J]. 畜牧兽医学报, 2021, 52(8): 2361-2368.
[6]	李占鸿, 宋子昂, 廖德芳, 杨振兴, 谢佳芮, 高翔, 胡忠燕, 李卓然, 李华春, 杨恒. 云南省蓝舌病病毒血清7型毒株的分离与基因组序列分析[J]. 畜牧兽医学报, 2021, 52(5): 1337-1348.
[7]	李茂林, 张七斤, 徐青元. 蓝舌病在全球的分布概况[J]. 畜牧兽医学报, 2021, 52(4): 881-890.
[8]	易华山, 赵瑶, 马鲜平, 李欢, 夏宇, 朱文青, 刘倩如, 陈思倩, 曹慧贞, 姚婷, 高丽旭, 张金阳, 杨发挥, 魏晓蓉, 李前勇, 谢远兵. 基于NS4蛋白的蓝舌病病毒间接ELISA抗体检测方法的建立及初步应用[J]. 畜牧兽医学报, 2020, 51(12): 3133-3140.
[9]	于可响, 刘存霞, 宫晓, 路晓, 胡峰, 郭效珍, 马秀丽, 李玉峰, 黄兵, 宋敏训. 一株肉鸡呼肠孤病毒变异株的分离鉴定[J]. 畜牧兽医学报, 2019, 50(5): 1039-1047.
[10]	李然, 汤承, 罗雪, 岳华. 一株牦牛源G6P[11]型牛轮状病毒的分离鉴定及部分基因的序列分析[J]. 畜牧兽医学报, 2019, 50(3): 592-601.
[11]	杨振兴, 孟锦昕, 肖雷, 朱建波, 廖德芳, 高林, 李占鸿, 杨恒, 李华春. 流行性出血病病毒(EHDV)血清7型毒株在中国的首次分离与鉴定[J]. 畜牧兽医学报, 2019, 50(3): 602-610.
[12]	杨恒, 肖雷, 李占鸿, 孟锦昕, 杨振兴, 吕敏娜, 林栩慧, 廖德芳, 牛保生, 李华春. 2012—2016年中国南方地区帕利亚姆血清群病毒的分离与序列特征分析[J]. 畜牧兽医学报, 2018, 49(4): 761-770.
[13]	池晓娟, 邵文涵, 陈吉龙, 陈月香, 王松. 禽源呼肠孤病毒诱导宿主免疫应答研究进展[J]. 畜牧兽医学报, 2017, 48(12): 2249-2257.
[14]	黄莉, 谢芝勋, 谢丽基, 黄娇玲, 谢志勤, 范晴, 邓显文, 罗思思, 张艳芳, 曾婷婷, 王盛, 张民秀. 禽呼肠孤病毒强毒株和弱毒疫苗株感染鸡肝癌细胞的转录组学分析[J]. 畜牧兽医学报, 2017, 48(11): 2135-2147.
[15]	朱果真, 黄梅清, 程晓霞, 郑敏, 陈仕龙, 陈少莺, 俞伏松. 番鸭呼肠孤病毒人工感染雏番鸭肝的蛋白质组学分析[J]. 畜牧兽医学报, 2017, 48(10): 1949-1957.