新疆蓝舌病病毒的分离及初步鉴定

针对新疆蓝舌病流行现状,本试验以蓝舌病血清学检测为阴性的动物建立监控群,于2014年5月至9月每周采集1次血样,血清学检测出现转阳动物后,取其转阳前后2周血样,接种10日龄鸡胚,收获死亡鸡胚肝脏,研磨浸毒后接种C6/36细胞和BHK-21细胞,出现细胞病变(cytopathic effect,CPE)样本进行PCR鉴定和电镜观察,并用细胞微量中和试验进行血清定型。经RT-PCR扩增蓝舌病血清型群特异VP7片段和NS1片段,鉴定在1 156,272,101bp处有特异性条带。电镜观察到疑似的病毒颗粒,中和试验结果显示该毒株为BTV-1型。上述结果显示,本研究分离到蓝舌病病毒1株,中和试验定型为BTV-1型。     

我国蓝舌病病毒血清24型毒株的分离与遗传特征分析

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。     

蓝舌病病毒7型毒株的分离与分子鉴定

为监测广东省蓝舌病流行状况,2014年从广东省某一奶牛场采集血液样品,检测虫媒病病原,对经RTPCR检测蓝舌病病毒核酸阳性的样品进行病毒分离并鉴定。经鸡胚静脉接种后取肝,研磨后离心取上清转接C6/36细胞和BHK-21细胞,出现CPE,RT-PCR检测蓝舌病病毒的VP7基因并进行序列比对,电镜观察病毒粒子,证实分离到蓝舌病病毒,命名为GD/ST2014。进一步扩增VP2基因,进行系统发育树分析,以及血清中和试验,表明此分离株为血清型7型。这是国内首次报道牛源蓝舌病病毒血清型7型的分离鉴定。     

动物蓝舌病监控系统的建立及其流行病学研究

观测绵羊蓝舌病自然发病区蓝舌病的传播规律及病毒血清型的分布，为本病的防治提供科学依据。于1995年7月起，在师宗县建立了动物蓝舌病监控动物群，经3年的观测，对19头牛监控，共采血667份，分离获得蓝舌病病毒10株，经血清学鉴定为3、4、5、15型血清型，该3个血清型均为首次分离到的国内新毒株。     

云南江城蓝舌病病毒16型毒株分离鉴定及其L2基因序列分析

为了解近年来云南江城县蓝舌病病毒16型(Bluetongue virus type 16,BTV-16)毒株的流行情况及其L2基因与国外流行株的遗传进化关系,本研究将江城县送检的300份牛肝素钠抗凝血提取红细胞后静脉接种10日龄鸡胚,将收集的鸡胚肝脏捣碎离心,上清液接种于C6/36和BHK21细胞传代。针对出现细胞病变的样品,应用群特异性VP7片段引物进行RT-PCR检测,应用BTV-16 L2基因特异性引物对检测出的BTV核酸阳性样品进行RT-PCR扩增和测序,采用DNAStar和Mega 6.0软件对获得的L2基因编码区序列进行核苷酸、氨基酸同源性比对及遗传进化分析,同时利用BTV-16标准阳性血清对分离到的病毒进行中和试验鉴定。结果显示,江城县发现30个可致细胞病变的样品,其中17个样品经RT-PCR初步确认为BTV;经L2基因序列分析和中和试验鉴定,确定其中6株为BTV-16型毒株;核苷酸、氨基酸同源性比对分析结果显示,6个毒株核苷酸和氨基酸同源性分别在93.4%～98.0%和94.2%～99.1%之间;遗传进化分析发现,其中5株与2001—2008年日本及1982—2011年印度分离的BTV-16毒株亲缘关系较近;1株与1985—1990年日本分离的BTV-16毒株亲缘关系较近。本研究发现,云南江城县BTV-16毒株呈现新旧毒株交叉持续流行态势,但在自然进化中遗传变异不大,有一定的稳定性,本研究在分子水平阐明了云南江城县地方流行BTV-16 L2基因间的遗传和差异,为进一步开展BTV分子流行病学及检测研究提供科学依据。     

云南江城蓝舌病病毒16型毒株分离鉴定及其L2基因序列分析

为了解近年来云南江城县蓝舌病病毒16型（Bluetongue virus type 16,BTV-16）毒株的流行情况及其L2基因与国外流行株的遗传进化关系,本研究将江城县送检的300份牛肝素钠抗凝血提取红细胞后静脉接种10日龄鸡胚,将收集的鸡胚肝脏捣碎离心,上清液接种于C6/36和BHK21细胞传代。针对出现细胞病变的样品,应用群特异性VP7片段引物进行RT-PCR检测,应用BTV-16 L2基因特异性引物对检测出的BTV核酸阳性样品进行RT-PCR扩增和测序,采用DNAStar和Mega 6.0软件对获得的L2基因编码区序列进行核苷酸、氨基酸同源性比对及遗传进化分析,同时利用BTV-16标准阳性血清对分离到的病毒进行中和试验鉴定。结果显示,江城县发现30个可致细胞病变的样品,其中17个样品经RT-PCR初步确认为BTV;经L2基因序列分析和中和试验鉴定,确定其中6株为BTV-16型毒株;核苷酸、氨基酸同源性比对分析结果显示,6个毒株核苷酸和氨基酸同源性分别在93.4%~98.0%和94.2%~99.1%之间;遗传进化分析发现,其中5株与2001-2008年日本及1982-2011年印度分离的BTV-16毒株亲缘关系较近;1株与1985-1990年日本分离的BTV-16毒株亲缘关系较近。本研究发现,云南江城县BTV-16毒株呈现新旧毒株交叉持续流行态势,但在自然进化中遗传变异不大,有一定的稳定性,本研究在分子水平阐明了云南江城县地方流行BTV-16 L2基因间的遗传和差异,为进一步开展BTV分子流行病学及检测研究提供科学依据。     

中国蓝舌病病毒血清2型病毒株的分离与鉴定

为了解我国云南省蓝舌病病毒(BTV)的流行情况和基因分型,本研究在云南师宗、江城、芒市分别设立监控点,通过对哨兵动物定期采血监控其血清学转阳情况,采用"鸡胚-C6/36细胞-BHK-21细胞"接种方式分离病毒,利用qRT-PCR及中和试验鉴定病毒,通过特异性引物对分离株Seg-2及Seg-7序列的ORF区进行RT-PCR扩增与克隆测序。结果显示,从2017年6月师宗县牛的抗凝血中分离到一株BTV,鉴定为BTV-2型,TCID_(50)为10~(-4.88)/50μL。分离株Seg-2及Seg-7序列的系统发生树显示其Seg-2序列和日本以及台湾BTV-2株的Seg-2序列相似度较高,同为BTV-2型,而Seg-7序列和台湾BTV-2株以及日本BTV-21株亲缘关系最近,同为Eastern 2型。本研究分离鉴定了BTV-2型病毒株YN/SZ/22457,并掌握了分离株Seg-2及Seg-7序列的遗传特性,明确了云南省存在BTV-2的流行。本研究为进一步开展BTV-2型的流行病学、感染特性研究奠定了基础。     

四川省美姑县羊蓝舌病病原分离鉴定

对采自四川省美姑县的疑似蓝舌绵羊、山羊血清１４份,全血１３份,进行蓝舌病抗原、抗体检测和病毒分离鉴定。结果在１０份血清样品中检测到蓝舌病抗体,在１３份全血样品中分离到２株病毒,经蓝舌病病毒群、血清型鉴定,分离毒株的血清型为ＢＴＶ－１型。     

人工感染BTV的绵羊血液中进行病毒分离的研究

为了指导蓝舌病(BT)研究中病毒分离工作,确定最佳病毒分离时间,比较鸡胚分离方法和BHK21分离方法的优缺点。本研究通过绵羊人工感染云南优势蓝舌病毒株BTV-1和BTV-16,采集不同时间的血液样品,用鸡胚和BHK21进行病毒分离。结果表明:用鸡胚的分离时间为接种后4~14 d,而BHK21细胞分离时间为2~8 d。本研究结果有助于BTV分离。     

蓝舌病16型病毒核心样颗粒的获取与鉴定

为了制备一种新型基因工程疫苗BTV-16 VLP疫苗,将蓝舌病病毒16型VP3与VP7基因片段插入杆状病毒双表达载体pFastBac Dual质粒中,通过转染Sf9细胞,同时表达VP3与VP7蛋白。经负染后电镜观察到有与蓝舌病病毒形态相似的核心样颗粒,表明蓝舌病VP3与VP7蛋白能自主组装形成核心样颗粒,并通过Western Blot试验验证该核心样颗粒与蓝舌病病毒具有相同的抗原性。因此本研究成功构建出16型蓝舌病病毒核心样颗粒,为病毒样颗粒装配打下了基础,为制备16血清型BTV VLP疫苗做好前期准备。     

Bluetongue sentinel surveillance program and cross-sectional serological survey in cattle in Belgium in 2010-2011

Bluetongue virus serotype 8 (BTV-8) emerged in Central Western Europe in 2006 causing a large scale epidemic in 2007 that involved several European Union (EU) countries including Belgium. As in several other EU member states, vaccination against BTV-8 with inactivated vaccines was initiated in Belgium in spring 2008 and appeared to be successful. Since 2009, no clinical cases of Bluetongue (BT) have been reported in Belgium and BTV-8 circulation seemed to have completely disappeared by spring 2010. Therefore, a series of repeated cross-sectional surveys, the BT sentinel surveillance program, based on virus detection in blood samples by means of real-time RT-PCR (RT-qPCR) were carried out in dairy cattle from the end of 2010 onwards with the aim to demonstrate the absence of BTV circulation in Belgium. This paper describes the results of the first two sampling rounds of this BT sentinel surveillance program carried out in October-November 2010 and January-February 2011. In addition, the level of BTV-specific maternal antibodies in young non-vaccinated animals was monitored and the level of herd immunity against BTV-8 after 3 consecutive years of compulsory BTV-8 vaccination was measured by ELISA. During the 1st sampling round of the BT sentinel surveillance program, 15 animals tested positive and 2 animals tested doubtful for BTV RNA by RT-qPCR. During the 2nd round, 17 animals tested positive and 5 animals tested doubtful. The positive/doubtful animals in both rounds were re-sampled 2-4 weeks after the original sampling and then all tested negative by RT-qPCR. These results demonstrate the absence of BTV circulation in Belgium in 2010 at a minimum expected prevalence of 2% and 95% confidence level. The study of the maternal antibodies in non-vaccinated animals showed that by the age of 7 months maternal antibodies against BTV had disappeared in most animals. The BTV seroprevalence at herd level after 3 years of compulsory BTV-8 vaccination was very high (97.4% [95% CI: 96.2-98.2]). The overall true within-herd BTV seroprevalence in 6-24 month old Belgian cattle in early 2011 was estimated at 73.4% (95% CI: 71.3-75.4).     

Incursion of bluetongue virus into the Okanagan Valley, British Columbia

Bluetongue virus was isolated from a sentinel herd in British Columbia. Virus isolation was by intravenous inoculation of embryonated chicken eggs and subculture in BHK-21 cells. The cytopathic agent was identified as bluetongue virus by electron microscopy and the immunoperoxidase test. The serotype was identified as serotype 11 by virus neutralization.     

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

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。     

上海市三株H9N2鸭禽流感病毒全基因遗传进化分析

为了解上海市鸭群中H9N2亚型禽流感病毒（Avian influenza virus,AIV）的遗传变异特征,以及与疫苗株A/Chicken/Shan dong/6/1996和A/Chicken/Shanghai/F/1998之间的遗传距离,对2007年和2009年分离自上海市鸭气管和泄殖腔样品采用荧光RT-PCR检测,将H9亚型禽流感病毒核酸阳性样品处理后,经鸡胚尿囊腔接种分离病毒,HI进一步确定血凝素（haemagglutin,HA）亚型,随后进行了全基因测序,并结合GenBank中的相关序列进行遗传进化分析。结果表明：3株分离毒株为H9N2亚型鸭禽流感病毒,HA蛋白裂解位点的氨基酸组成为PARSSRGLF,符合低致病性禽流感病毒特征,均属于经典的H9N2 Ck/Bei群系;NA基因均属于Y280系;NP、PA基因和A/Goose/Guangdong/1/1996（H5亚型）归为一群;PB2和M基因属于Qa/HK/G1/97系;NS基因仍为Ck/Bei系;2007年的分离株和2009年的分离株在PB1基因上分属不同亚群。3株病毒的HA1基因与疫苗株A/Chicken/Shandong/6/1996和A/Chicken/Shanghai/F/1998之间的遗传距离均大于7%。由此可见,3株鸭H9N2亚型毒株可能是由不同禽流感病毒基因亚群间发生自然重排的产物,现有疫苗对分离株的保护性需要进一步评估。     

1型鸭甲肝病毒LY0801株VP1基因在鸭胚传代中的变异规律研究

为了解1型鸭甲肝病毒（Duck hepatitis A virus type 1）VP1基因在鸭胚传代中的变异规律,本研究将LY0801株DHAV-1在鸭胚体内传代至30代,分别对1~5、10、15、20、25、30代病毒进行VP1基因的克隆测序。各代次病毒分别以108copies/胚接种9日龄健康鸭胚,测定各组鸭胚的平均死亡时间和病毒在鸭胚尿囊液中的增殖拷贝数。结果表明：DHAV-1在鸭胚传代过程中,不同代次之间出现12个氨基酸的反复变异和同步变异,分别为R43M（K）、T48A、T101S、L169F、T180I、S181L、R183Q、E184A（K）、G187D、D193N、M213R、H219Y;在传代过程中,病毒致死鸭胚的时间逐渐延迟,但病毒在鸭胚内的增殖拷贝数未呈现稳定增长的趋势。     

Evidence for BTV-4 circulation in free-ranging red deer (Cervus elaphus) in Cabañeros National Park, Spain

Bluetongue (BT) is an infectious disease of wild and domestic ruminants caused by bluetongue virus (BTV). BTV-4 spread through southern Spain from 2004 to 2006, whereas a BTV-1 outbreak that started in southern Spain in 2007 is still ongoing. Vaccination and movement restriction regulations are applied to domestic ruminants to control BT, but the potential reservoir role of wild European ungulates has not been clarified so far. The aim of this study was to describe the epidemiology of BTV in the wild free-ranging red deer (Cervus elaphus) population of Caba?eros National Park (CNP) in central Spain during the BTV-4 and BTV-1 epizootics, assessing the potential role of this deer population as a BTV reservoir. Blood samples from 2885 (2542 adults, 208 calves and 135 undetermined) wild red deer were collected from 2005 to 2010 in CNP and surrounding hunting estates. All sera were tested for antibodies against the BTV VP7 protein by ELISA. Ninety-four of the ELISA-positive samples were analysed by serum neutralization to detect BTV-4 and BTV-1 specific antibodies, and 142 blood samples were analysed by RT-PCR for BTV RNA. A total of 371 (12.9%) out of the 2,885 deer (35/208 calves, 307/2,542 adults, and 29/135 undetermined) were positive for antibodies against BTV. Prevalence increased in adult deer from 2005-2006 to 2008-2009, declining thereafter. No positive samples for BTV-1 were found by serum neutralization, whereas 43 deer (38 adults, four calves and one undetermined) were positive for BTV-4 specific antibodies. No BTV RNA positive deer were found by RT-PCR. Antibody detection throughout the study period suggests a maintained circulation of BTV in red deer. However, the lack of BTV RNA detection suggests a minor transmission risk to livestock.     

VP2-segment sequence analysis of some isolates of bluetongue virus recovered in the Mediterranean basin during the 1998-2003 outbreak

The complete nucleotide sequences of the VP2 segments of bluetongue virus (BTV) isolates recovered from Italy, Greece and Israel, from 1998 to 2003, were determined. Phylogenetic analysis of these sequences, those from related viruses and the South African vaccine strains, were used to determine the probable geographic origin of BTV incursions into Italy. Results indicated that viruses from each of the four serotypes isolated in Italy (2, 4, 9 and 16) possibly had a different origin. Analysis of the bluetongue virus serotype 2 (BTV-2) isolates gave evidence that this serotype probably moved from Tunisia. BTV-4 results showed probable incursion from the southwest and not from Greece or Israel. BTV-9 isolates clearly have an eastern origin (most probably Greece), whereas BTV-16 isolates are indistinguishable from the BTV-16 live attenuated vaccine strain. The phylogenetic findings were supported by polyacrylamide gel electrophoresis (PAGE) analysis of the complete amplified genome of each isolate except for BTV-16 Italian field isolate, which showed a slightly different PAGE profile. A combination of the complete VP2 sequencing and PAGE analysis of complete genomes, allowed not only phylogenetic analysis, but also vaccine detection and assessment of reassortment events.