我国蓝舌病病毒血清24型毒株的分离与遗传特征分析
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摘要
掌握云南省蓝舌病病毒(BTV)的活动情况与流行毒株的遗传特征。2012—2015年,在云南省的师宗县、江城县与芒市分别设立3个监控点,进行BTV的分离。自监控动物采集的BTV核酸阳性血液通过"鸡胚-C6/36细胞-BHK细胞"接种的方式进行病毒分离;采用RT-PCR与中和试验进行分离病毒的血清型鉴定;设计特异性引物对分离病毒的Seg-2、Seg-3、Seg-7与Seg-6基因节段进行RT-PCR扩增与克隆测序。2012—2015年,从云南省的师宗县、江城县与芒市分别分离获得3株BTV-24型毒株。分离病毒的Seg-2、Seg-3、Seg-7与Seg-6序列系统发育分析显示:我国毒株的Seg-2与BTV-24型参考毒株聚为一簇,而Seg-6与BTV-10型毒株聚为一簇;我国毒株的Seg-3在系统发生树上划分为"东方型"地域型,Seg-7在系统发生树上形成一个独立于其他地域型的分支。本试验报道了我国BTV-24型毒株的分离与Seg-2、Seg-3、Seg-6与Seg-7序列特征,结果表明,我国BTV-24型毒株的Seg-6基因节段与BTV-10型毒株发生了基因重配;Seg-7具有独特的遗传特征,形成了一个新的Seg-7地域型,暂定为"Chinese topotype"。本试验为进一步开展BTV-24型的流行病学、感染特性与疫苗的研究奠定基础。
To study the prevalence of bluetongue virus(BTV)in Yunnan province and their genetic characteristics.Three BTV monitoring points were set at Shizong,Jiangcheng and Mangshi of Yunnan province.Virus isolation was carried out through the inoculating method of"chicken embryoC6/36cells-BHK cells"from BTV nucleic acid positive blood samples collected from the sentinel herds.The serotypes of the isolated BTV strains were identified by RT-PCR amplification and neutralization assay.Segment-2,-3,-6and-7of isolated viruses were amplified with specific primers,then cloned and sequenced.From 2012 to 2015,three strains of BTV-24 were isolated from Shizong,Jiangcheng and Mangshi in Yunnan province.Phylogenetic analysis of segment-2,-3,-6and-7of Chinese BTV-24 strains showed that segment 2of Chinese strains clustered with BTV-24 reference strain,however,the segment 6Chinese strains clustered with BTV-10 reference strain.Segment 3of Chinese BTV-24 strain was divided into"Eastern topotype",while segment 7formed an independent branch in the phylogenetic tree.This is the first report on isolation and sequence characterization of BTV-24 strain in China.The results showed that there existed reassortment between segment 6 of China BTV-24 strain and BTV-10 strain.Segment 7 of Chinese BTV-24 strains has unique genetic feature,forms a new topotype,tentatively designated as"Chinese topotype".The results will lay foundation for further research on epidemiology,infection characteristics and vaccine of Chinese BTV-24.
To study the prevalence of bluetongue virus(BTV)in Yunnan province and their genetic characteristics.Three BTV monitoring points were set at Shizong,Jiangcheng and Mangshi of Yunnan province.Virus isolation was carried out through the inoculating method of"chicken embryoC6/36cells-BHK cells"from BTV nucleic acid positive blood samples collected from the sentinel herds.The serotypes of the isolated BTV strains were identified by RT-PCR amplification and neutralization assay.Segment-2,-3,-6and-7of isolated viruses were amplified with specific primers,then cloned and sequenced.From 2012 to 2015,three strains of BTV-24 were isolated from Shizong,Jiangcheng and Mangshi in Yunnan province.Phylogenetic analysis of segment-2,-3,-6and-7of Chinese BTV-24 strains showed that segment 2of Chinese strains clustered with BTV-24 reference strain,however,the segment 6Chinese strains clustered with BTV-10 reference strain.Segment 3of Chinese BTV-24 strain was divided into"Eastern topotype",while segment 7formed an independent branch in the phylogenetic tree.This is the first report on isolation and sequence characterization of BTV-24 strain in China.The results showed that there existed reassortment between segment 6 of China BTV-24 strain and BTV-10 strain.Segment 7 of Chinese BTV-24 strains has unique genetic feature,forms a new topotype,tentatively designated as"Chinese topotype".The results will lay foundation for further research on epidemiology,infection characteristics and vaccine of Chinese BTV-24.
引文
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[23]MAAN S,MAAN N S,BELAGANAHALLI M N,et al.Full-genome sequencing as a basis for molecular epidemiology studies of bluetongue virus in India[J].PLoS One,2015,10(6):e0131257.
[24]BHATTACHARYA B,ROY P.Bluetongue virus outer capsid protein VP5interacts with membrane lipid rafts via a SNARE domain[J].Virol J,2008,82(21):10600-10612.
[25]ZHANG X,SHATKIN A J.Bluetongue virus coat protein VP2contains sialic acid-binding domains,and VP5resembles enveloped virus fusion proteins[J].Proc Natl Acad Sci USA,2010,107(14):6292-6297.
[2]SAEGERMAN C,BERKVENS D,MELLOR P S.Bluetongue epidemiology in the European Union[J].Emerg Infect Dis,2008,14(4):539-544.
[3]MAAN S,MAAN N S,ROSS-SMITH N,et al.Sequence analysis of bluetongue virus serotype 8from the Netherlands 2006and comparison to other European strains[J].Virology,2008,377(2):308-318.
[4]ROY P.Orbivirus structure and assembly[J].Virology,1996,216(1):1-11.
[5]ROY P.Bluetongue virus proteins[J].J Gen Virol,1992,73(Pt 12):3051-3064.
[6]MAAN S,MAAN N S,VAN RIJN P A,et al.Full genome characterisation of bluetongue virus serotype 6from the netherlands 2008and comparison to other field and vaccine strains[J].PLoS One,2010,5(4):e10323.
[7]NOMIKOU K,DOVAS C I,MAAN S,et al.Evolution and phylogenetic analysis of full-length VP3genes of eastern Mediterranean bluetongue virus isolates[J].PLoS One,2009,4(7):e6437.
[8]MAAN S,MAAN N S,SAMUEL A R,et al.Analysis and phylogenetic comparisons of full-length VP2genes of the 24bluetongue virus serotypes[J].J Gen Virol,2007,88(Pt 2):621-630.
[9]SHIRAFUJI H,YANASE T,KATO T,et al.Genetic and phylogenetic characterization of genome segments2and 6of bluetongue virus isolates in Japan from1985to 2008[J].J Gen Virol,2012,93(Pt7):1465-1473.
[10]HOFMANN M A,SANDRA R,MARKUS M,et al.Genetic characterization of toggenburg orbivirus,a new bluetongue virus,from goats,switzerland[J].Emerg Infect Dis,2008,14(12):1855-1861.
[11]MAAN S,MAAN N S,NOMIKOU K,et al.Complete genome characterization of a novel 26th bluetongue virus serotype from Kuwait[J].PLoS One,2011,6(11):e26147.
[12]ZIENTARA S,SAILLEAU C,VIAROUGE C,et al.Novel bluetongue virus in goats,Corsica,France[J].Emerg Infect Dis,2014,20(12):2123-2125.
[13]SUN E C,HUANG L P,XU Q Y,et al.Emergence of a novel bluetongue virus serotype,China 2014[J].Transbound Emerg Dis,2016,63(6):585-589.
[14]SAVINI G,PUGGIONI G,MELONI G,et al.Novel putative bluetongue virus in healthy goats from Sardinia,Italy[J].Infect Genet Evol,2017,51:108-117.
[15]SCHWARTZCORNIL I,MERTENS P P,CONTR-ERAS V,et al.Bluetongue virus:virology,pathogenesis and immunity[J].Vet Res,2008,39(5):39-46.
[16]ZHU J,YANG H,LI H,et al.Full-genome sequence of bluetongue virus serotype 1(BTV-1)strain Y863,the first BTV-1isolate of eastern origin found in China[J].Genome Announc,2013,1(4):pii:e00403-13.
[17]ZHANG N,MACLACHLAN N J,BONNEAU K R,et al.Identification of seven serotypes of bluetongue virus from the People’s Republic of China[J].Vet Rec,1999,145(15):427-429.
[18]KIRKLAND 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.
[19]YANG H,LV M,SUN M,et al.Complete genome sequence of the first bluetongue virus serotype 7isolate 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.
[20]全国动物检疫标准化技术委员会.GBT18636-2002蓝舌病诊断技术国家标准[S].北京:中国标准出版社,2002.
[21]TAMURA K,STECHER G,PETERSON D,et al.MEGA6:molecular evolutionary genetics analysis version 6.0[J].Mol Biol Evol,2013,30:2725-2729.
[22]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].PLoSOne,2012,7(2):e32601.
[23]MAAN S,MAAN N S,BELAGANAHALLI M N,et al.Full-genome sequencing as a basis for molecular epidemiology studies of bluetongue virus in India[J].PLoS One,2015,10(6):e0131257.
[24]BHATTACHARYA B,ROY P.Bluetongue virus outer capsid protein VP5interacts with membrane lipid rafts via a SNARE domain[J].Virol J,2008,82(21):10600-10612.
[25]ZHANG X,SHATKIN A J.Bluetongue virus coat protein VP2contains sialic acid-binding domains,and VP5resembles enveloped virus fusion proteins[J].Proc Natl Acad Sci USA,2010,107(14):6292-6297.