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

doi:10.11843/j.issn.0366-6964.2021.05.019

畜牧兽医学报 ›› 2021, Vol. 52 ›› Issue (5): 1337-1348.doi: 10.11843/j.issn.0366-6964.2021.05.019

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

李占鸿¹, 宋子昂², 廖德芳¹, 杨振兴¹, 谢佳芮¹, 高翔³, 胡忠燕³, 李卓然¹, 李华春^1*, 杨恒^1*

1. 云南省畜牧兽医科学院, 云南省热带亚热带动物病毒病重点实验室, 昆明 650224;
2. 云南农业大学动物医学院, 昆明 650201;
3. 云南省景洪市动物疫病预防控制中心, 景洪 666100

收稿日期:2020-10-06 出版日期:2021-05-23 发布日期:2021-05-22
通讯作者: 杨恒,主要从事动物虫媒病毒研究,E-mail:yangheng2008.cool@163.com;李华春,主要从事动物虫媒病毒研究,E-mail:Li_huachun@hotmail.com
作者简介:李占鸿(1989-),男,云南大理人,助理研究员,硕士,主要从事牛羊虫媒病毒研究,E-mail:dy081lzh@163.com;宋子昂(1992-),男,河北沧州人,硕士,主要从事牛羊虫媒病毒研究,E-mail:786722311@qq.com。
基金资助:
国家自然科学基金（31760744）；国家重点研发计划（2017YFC1200505）；国家公益性行业（农业）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）

Whole Genome Sequence Analyses of Bluetongue Virus Serotype 7 Strain Isolated from Yunnan Province

LI Zhanhong¹, SONG Ziang², LIAO Defang¹, YANG Zhenxing¹, XIE Jiarui¹, GAO Xiang³, HU Zhongyan³, LI Zhuoran¹, LI Huachun^1*, YANG Heng^1*

1. Yunnan Tropical and Subtropical Animal Virus Disease Laboratory, Yunnan Veterinary and Animal Science Institute, Kunming 650224, China;
2. College of Veterinary Medicine, Yunnan Agricultural University, Kunming 650201, China;
3. Animal Disease Control Center of Jinghong, Jinghong 666100, China

Received:2020-10-06 Online:2021-05-23 Published:2021-05-22

摘要/Abstract

摘要： 2014年，我国广东省的哨兵牛上分离出蓝舌病病毒血清7型（BTV-7）毒株，但该血清型病毒在我国的流行情况尚不清楚，本研究旨在分离我国流行的BTV-7型毒株并掌握其遗传特征。作者在云南省景洪市勐罕镇设立哨兵牛，每周定时采血，并接种C6/36、BHK-21细胞分离虫媒病毒，通过病毒蚀斑试验与增殖曲线分析病毒在细胞上的感染特性，采用高通量测序获取分离毒株的全基因组序列，通过qRT-PCR与血清中和试验对哨兵牛血液中的病毒核酸与中和抗体变化进行回溯分析。结果表明：2020年5月，分离到1株能引起BHK-21细胞发生细胞病变的病毒（毒株号V303/YNJH/2020），经鉴定为BTV-7型。分离毒株的基因组全长19 154 bp （GenBank收录号MW046280至MW046289），与广东省2014年分离的BTV-7型GDST008毒株具有最近的亲缘关系，基因节段1至6，基因节段9与10的核酸与编码蛋白氨基酸序列（nt/aa）相似度分别大于98%、99%；V303/YNJH/2020毒株的基因的节段7和基因节段8属Western地域型，与广东GDST008毒株对应基因节段的nt/aa序列相似度仅为71.5%/81.6%、79.6/%84.4%。病毒蚀斑与增殖曲线比较显示，V303/YNJH/2020在BHK-21细胞的增殖能力明显强于GDST008。回溯分析显示，感染V303/YNJH/2020的哨兵牛未出现临床症状，血液中的病毒核酸持续存在长达12周；在病毒感染后第4~9周，血液中的中和抗体滴度水平维持在1∶256。云南省2020年分离的BTV-7型V303/YNJH/2020毒株与广东省分离的BTV-7型GDST008毒株具有最近的亲缘关系，在BHK-21细胞上的增殖能力强于GDST008毒株；V303/YNJH/2020虽未引起感染动物的临床症状，但病毒核酸与中和抗体在感染动物血液中长时间存在。研究结果为开展BTV-7型在我国的演化规律、病毒变异以及致病性等方面的研究奠定了基础。

关键词: 蓝舌病病毒, 血清7型, 全基因组测序, 基因重配, 感染特性

Abstract: Bluetongue virus serotype 7 (BTV-7) was firstly isolated from sentinel cattle in Guangdong Province in 2014; however, the epidemic situation of this serotype was still not clear in China. The objective of the present study is to isolate and analyze the genomic characterization of BTV-7 epideictic in China. Sentinel cattle were placed in Jinghong County, Yunnan Province, and blood samples were collected weekly for arbovirus isolation by inoculating cells. Complete genome sequence of the isolated virus was obtained by high-throughput sequencing. The infection feature of the isolated virus on cells were analyzed by virus plaque assay and proliferation curve analysis. The dynamics of viral nucleic acids and antibodies in the blood of infected sentinel cattle were monitored by serum neutralization test (SNT) and qRT-PCR. In May 2020, a virus strain (V303/YNJH/2020) causing cytopathic effects (CPE) on BHK-21 cells was isolated from the blood sample collected from sentinel cattle, which was serotyped as BTV-7. The genome of V303/YNJH/2020 was 19 154 bp in length (GenBank accession numbers: MW046280 to MW046289), which showed the closest relationship to the BTV-7 GDST008 strain isolated in Guangdong Province in 2014, with nucleic acid (nt) and encoding protein amino acid (aa) sequence identities of segment 1 to 6, segment 9 and 10 higher than 98% and 99%. However, the segment 7 and 8 of V303/YNJH/2020 belonged to the Western topotype, shared nt/aa sequence identities of 71.5%/81.6% and 79.6%/84.4% with the corresponding segment of GSDT008. Results of virus plaque assay and proliferation curve analysis showed that the proliferation ability of V303/YNJH/2020 in BHK-21 cells was significantly robust than that of GDST008. Sentinel cattle infected with V303/YNJH/2020 did not show observed clinical symptoms, but viral nucleic acids in the blood persisted up to 12 weeks, and neutralization antibodies in blood remained at titers of 1:256 during 4-9 weeks after virus infection. The BTV-7 strain V303/YNJH/2020 isolated in Yunnan Province in 2020 has the closest relationship with BTV-7 strain of GDST008, and possessed robust proliferation ability on cells than GDST008. The infection of V303/YNJH/2020 did not cause clinical symptoms in cattle, but viral nucleic acids and neutralization antibodies existed in the blood of infected cattle for a long time. The results of this study laid the foundation for the research on evolution, viral mutation caused by reassortment and pathogenicity of BTV-7 strains.

Key words: bluetongue virus, serotype 7, whole genome sequencing, gene reassortment, infection characteristics

中图分类号:

S852.659.4

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.

LI Zhanhong, SONG Ziang, LIAO Defang, YANG Zhenxing, XIE Jiarui, GAO Xiang, HU Zhongyan, LI Zhuoran, LI Huachun, YANG Heng. Whole Genome Sequence Analyses of Bluetongue Virus Serotype 7 Strain Isolated from Yunnan Province[J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(5): 1337-1348.

参考文献 35

[1]	MACLACHLAN N J. Bluetongue:history, global epidemiology, and pathogenesis[J]. Prev Vet Med, 2011, 102(2):107-111.
[2]	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.
[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]	ROY P. Bluetongue virus proteins[J]. J Gen Virol, 1992, 73(Pt 12):3051-3064.
[5]	BELHOUCHET M, MOHD JAAFAR F, FIRTH A E, et al. Detection of a fourth Orbivirus non-structural protein[J]. PLoS One, 2011, 6(10):e25697.
[6]	NOMIKOU K, DOVAS C I, MAAN S, et al. Evolution and phylogenetic analysis of full-length VP3 genes of Eastern Mediterranean bluetongue virus isolates[J]. PLoS One, 2009, 4(7):e6437.
[7]	BELBIS G, ZIENTARA S, BRÉARD E, et al. Chapter Seven-bluetongue virus:from BTV-1 to BTV-27[J]. Adv Virus Res, 2017, 99:161-197.
[8]	BUMBAROV V, GOLENDER N, JENCKEL M, et al. Characterization of bluetongue virus serotype 28[J]. Transbound Emerg Dis, 2020, 67(1):171-182.
[9]	吕敏娜, 杨恒, 孙铭飞, 等. 蓝舌病病毒7型毒株的分离与分子鉴定[J]. 畜牧兽医学报, 2017, 48(7):1281-1287.LÜ M N, YANG H, SUN M F, et al. Isolation and identification of bluetongue virus serotype 7 field isolate[J]. Acta Veterinaria et Zootechnica Sinica, 2017, 48(7):1281-1287. (in Chinese)
[10]	YANG H, LV M N, SUN M F, et al. Complete genome sequence of the first bluetongue virus serotype 7 isolate from China:evidence for entry of African-lineage strains and reassortment between the introduced and native strains[J]. Arch Virol, 2016, 161(1):223-227.
[11]	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.
[12]	苗海生, 李乐, 朱建波, 等. 蓝舌病病毒阻断ELISA抗体检测方法的建立[J]. 中国预防兽医学报, 2014, 36(2):111-115.MIAO H S, LI L, ZHU J B, et al. Establishment of the blocking ELISA method for detection of the antibody titer against bluetongue virus[J]. Chinese Journal of Preventive Veterinary Medicine, 2014, 36(2):111-115. (in Chinese)
[13]	杨振兴, 朱建波, 李占鸿, 等. 蓝舌病病毒和流行性出血病病毒双重荧光定量RT-PCR检测方法的建立及应用[J]. 中国兽医科学, 2019, 49(9):1104-1111.YANG Z X, ZHU J B, LI Z H, et al. Establishment and application of a duplex real-time RT-PCR assay for simultaneous detection of bluetongue virus and epidemic haemorrhagic disease virus[J]. Chinese Veterinary Science, 2019, 49(9):1104-1111. (in Chinese)
[14]	LEE F, LIN Y L, TSAI H J. Comparison of primer sets and one-step reverse transcription polymerase chain reaction kits for the detection of bluetongue viral RNA[J]. J Virol Methods, 2014, 200:6-9.
[15]	杨振兴, 朱建波, 廖德芳, 等. 流行性出血病病毒一步法RT-PCR检测方法的建立及应用[J]. 中国兽医科学, 2019, 49(3):280-286.YANG Z X, ZHU J B, LIAO D F, et al. Development and application of one-step RT-PCR method for the detection of epizootic haemorrhagic disease virus[J]. Chinese Veterinary Science, 2019, 49(3):280-286. (in Chinese)
[16]	李占鸿, 廖德芳, 杨振兴, 等. 帕利亚姆血清群病毒一步法RT-PCR检测技术的建立[J]. 中国预防兽医学报, 2020, 42(1):40-45.LI Z H, LIAO D F, YANG Z X, et al. The development of a one-step RT-PCR assay for the detection of PALV[J]. Chinese Journal of Preventive Veterinary Medicine, 2020, 42(1):40-45. (in Chinese)
[17]	李占鸿, 宋子昂, 杨振兴, 等. 中国流行的十二种血清型蓝舌病病毒一步法RT-PCR定型方法的建立与应用[J]. 中国兽医科学, 2020, 50(12):1486-1493.LI Z H, SONG Z A, YANG Z X， et al. Establishment and application of one-step RT-PCR method to identify the serotype of bluetongue virus epidemic in China[J]. Chinese Veterinary Science, 2020, 50(12):1486-1493. (in Chinese)
[18]	杨恒, 李占鸿, 张怡轩, 等. 牛血液中一株新型环状病毒的分离与全基因组序列分析[J]. 病毒学报, 2018, 34(1):75-84.YANG H, LI Z H, ZHANG Y X, et al. Isolation and genomic characterization of a novel Orbivirus strain from the blood of cattle[J]. Chinese Journal of Virology, 2018, 34(1):75-84. (in Chinese)
[19]	MAAN S, RAO S J, MAAN N S, et al. Rapid cDNA synthesis and sequencing techniques for the genetic study of bluetongue and other dsRNA viruses[J]. J Virol Methods, 2007, 143(2):132-139.
[20]	BOLGER A M, LOHSE M, USADEL B. Trimmomatic:a flexible trimmer for Illumina sequence data[J]. Bioinformatics, 2014, 30(15):2114-2120.
[21]	HERNANDEZ D, FRANÇOIS P, FARINELLI L, et al. De novo bacterial genome sequencing:millions of very short reads assembled on a desktop computer[J]. Genome Res, 2008, 18(5):802-809.
[22]	LUO R D, LIU B H, XIE Y L, et al. SOAP denovo2:an empirically improved memory-efficient short-read de novo assembler[J]. Gigascience, 2012, 1(1):18.
[23]	KATOH K, ASIMENOS G, TOH H. Multiple alignment of DNA sequences with MAFFT[J]. Methods Mol Biol, 2009, 537:39-64.
[24]	KUMAR S, STECHER G, LI M, et al. MEGA X:molecular evolutionary genetics analysis across computing platforms[J]. Mol Biol Evol, 2018, 35(6):1547-1549.
[25]	ALIU Y O, NWUDE N. Veterinary pharmacology and toxicology experiments[M]. Zaria: ABU Press, 1982:104-110.
[26]	李占鸿, 肖雷, 孟锦昕, 等. 中国蓝舌病病毒血清5型毒株的分离与鉴定[J]. 中国兽医科学, 2019, 49(1):36-43.LI Z H, XIAO L, MENG J X, et al. Isolation and identification of bluetongue virus serotype 5 strain in China[J]. Chinese Veterinary Science, 2019, 49(1):36-43. (in Chinese)
[27]	杨恒, 王金萍, 李乐, 等. 我国蓝舌病病毒血清24型毒株的分离与遗传特征分析[J]. 中国兽医学报, 2019, 39(1):31-37.YANG H, WANG J P, LI L, et al. Genetic characteristics of bluetongue virus serotype 24 strains isolated in China[J]. Chinese Journal of Veterinary Science, 2019, 39(1):31-37. (in Chinese)
[28]	李占鸿, 王金萍, 杨恒, 等. 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 Z H, WANG J P, YANG H, et al. Isolation of bluetongue virus serotype 9 strain in China for the first time[J]. Acta Veterinaria et Zootechnica Sinica, 2019, 50(2):354-363. (in Chinese)
[29]	YANG H, XIAO L, WANG J P, et al. Phylogenetic characterization genome segment 2 of Bluetongue virus strains belonging to serotypes 5, 7 and 24 isolated for the first time in China during 2012 to 2014[J]. Transbound Emerg Dis, 2017, 64(4):1317-1321.
[30]	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.
[31]	NELSON M I, VIBOUD C, SIMONSEN L, et al. Multiple reassortment events in the evolutionary history of H1N1 influenza A virus since 1918[J]. PLoS Pathog, 2008, 4(2):e1000012.
[32]	GARTEN R J, DAVIS C T, RUSSELL C A, et al. Antigenic and genetic characteristics of swine-origin 2009 A(H1N1) influenza viruses circulating in humans[J]. Science, 2009, 325(5937):197-201.
[33]	JANOWICZ A, CAPORALE M, SHAW A, et al. Multiple genome segments determine virulence of bluetongue virus serotype 8[J]. J Virol, 2015, 89(10):5238-5249.
[34]	KAR A K, BHATTACHARYA B, ROY P. Bluetongue virus RNA binding protein NS2 is a modulator of viral replication and assembly[J]. BMC Mol Biol, 2007, 8:4.
[35]	BOYCE M, CELMA C C P, ROY P. Development of reverse genetics systems for bluetongue virus:recovery of infectious virus from synthetic RNA transcripts[J]. J Virol, 2008, 82(17):8339-8348.

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

Whole Genome Sequence Analyses of Bluetongue Virus Serotype 7 Strain Isolated from Yunnan Province

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