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

流行性出血病(EHD)是由流行性出血病病毒(EHDV)感染反刍动物引起的一种虫媒病毒病,为了解中国云南EHDV的感染情况和病毒遗传特征,笔者课题组在云南省师宗县以EHDV血清学和核酸阴性的牛、羊为哨兵动物,拟对流行于云南省的EHDV进行分离与鉴定。将采集于哨兵动物的EHDV核酸阳性血液接种BHK-21细胞进行病毒分离;通过血清中和试验与病毒Seg-2、Seg-3 ORF区的序列分析,确定病毒的血清型与遗传特征;采用C-ELISA和qRT-PCR方法对EHDV感染动物血液中的抗体水平与病毒核酸进行监测。结果如下:从2013年8月采集自云南师宗县哨兵牛的血样中分离出一株EHDV(毒株号YNSZ/V269/2013),血清中和试验结果表明分离的病毒为血清型7型(EHDV-7);Seg-2、Seg-3序列分析表明分离的病毒属EHDV-7 Eastern型,与日本毒株和澳大利亚EHDV-7型毒株具有最近的亲缘关系。哨兵动物病毒核酸转阳后,血清抗体迅速上升,3周后达最高点并能在该水平持续较长时间,而病毒核酸含量却迅速下降,7周后已经检测不到。本研究首次报道了EHDV-7型毒株在中国的分离、毒株序列特征以及在动物上的感染特性。研究结果可为进一步开展中国EHDV-7型病毒的全基因组测序、流行病学调查、诊断方法的建立和致病性等相关研究提供基础。     

蓝舌病病毒血清9型毒株在我国的首次分离

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 6ORF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-9Eastern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-9Eastern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。     

云南省蓝舌病病毒“历史毒株”的遗传特征

20世纪90年代,本实验室从云南省哨兵动物采集的血液中分离到7种血清型蓝舌病病毒(bluetongue virus,BTV)。但这些毒株的遗传特征至今仍不清楚,因而阻碍了我国的BTV演化历史分析。为掌握云南省早期流行BTV毒株的遗传特征,对1995—1997年云南省分离的25株BTV毒株的基因组节段2、3、7和10(Seg-2、-3、-7、-10)进行一步法RT-PCR扩增、测序以及序列分析。Seg-2序列分析显示,1995—1997年云南省分离到的7种不同血清型(BTV-1、-2、-3、-4、-12、-15、-16)BTV毒株,分属A、B、G、H、I和J等6种基因型;BTV-12型毒株的Seg-2为西方地域型,其余毒株的Seg-2则属于东方地域型;Seg-3和Seg-10序列分析显示,25株BTV毒株均为东方地域型;Seg-7序列分析显示,1株BTV-2型、2株BTV-12型和1株BTV-16型毒株属于西方地域亚型,并与南非和荷兰BTV毒株的Seg-7具有最近的亲缘关系,其余毒株则均为东方地域型。上述结果表明,云南省存在多种血清型BTV的流行,而Seg-2和Seg-7基因重配毒株的发现,提示国外的西方地域型毒株已侵入云南省,并在传播过程中与我国毒株发生了基因重配。本研究为我国BTV的演化分析与溯源研究提供了数据参考。     

2013—2016年云南省江城县牛羊蓝舌病血清流行病学调查

为了解近年来云南省江城县蓝舌病病毒（BTV）的感染和流行情况,从2013年5月至2016年4月连续3年在江城县设立监控动物群,每年选择BTV病原及抗体检测阴性牛10头、羊5只作为哨兵动物,用于BTV抗体监测和病毒分离。应用BTV C-ELISA抗体检测试剂盒,对哨兵动物进行血清抗体检测,应用鸡胚、C6/36细胞和BHK-21细胞进行病毒分离。结果显示：连续3年的哨兵动物BTV抗体阳性率分别为40.35%、44.08%、50.65%; 3年内共分离到45株BTV,包括1~5型、15~16型、21型和24型共9种血清型。结果表明,江城县蓝舌病流行有逐年加重趋势,且呈现多种血清型毒株交叉感染状况,亟需加大BTV监测力度,及时发现毒株变化。     

2019年云南省景洪市哨兵动物多种虫媒病毒的分离鉴定

本研究旨在了解云南省景洪市虫媒病毒的流行情况。2019年在景洪市勐罕镇设立了3头哨兵动物牛,定期采血进行虫媒病毒的分离与鉴定,共获得7株病毒分离物。经病毒核酸的RT-PCR鉴定,分离到2株血清型分别为6型和7型流行性出血热病毒（epizootic haemorrhagic disease virus,EHDV）,2株血清型分别为4型和5型的蓝舌病病毒（bluetongue virus,BTV）,1株帕利亚血清群病毒（palyam serogroup virus,PALV）中的D’Aguilar virus（DAV）血清型病毒和2株未鉴定出的环状病毒。经病毒Seg-2、Seg-3序列ORF区的比对和进化分析显示,7株病毒的地域型均为Eastern型,与日本、澳大利亚和印度毒株具有最近的亲缘关系。3头哨兵动物的血液和血清,经病毒核酸及血清中和试验检测,证明3头动物均被相应的病毒感染。动物感染病毒后,血清中的特异性抗体迅速上升,3~4周后达最高点并能够在该水平维持较长时间,而血液中病毒核酸含量2~4周到达最高点后则呈迅速下降趋势。本研究报道了景洪虫媒病毒的分离、毒株序列特征以及在动物上的感染特性,研究结果为进一步了解当地的牛虫媒病毒提供数据支撑,同时3头牛分离获得7株病毒,提示当地可能还存在更多种类的虫媒病毒。     

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。     

猪圆环病毒2型田间分离株的全基因组克隆与基因亚型分析

为了解我国猪圆环病毒2型(PCV2)的流行态势和毒株的基因变异情况,从北京、江苏、湖北等地疑似断奶仔猪多系统综合征(PMWS)患病死亡猪中采集肺脏、脾脏和淋巴结等组织,将PCR鉴定为阳性的病料在PK-15细胞上进行增殖培养,成功分离到6株猪圆环病毒2型毒株,分别命名为B07、B12、HB、LHW、LPC和NJ。对其全基因组进行克隆和序列分析的结果显示,LPC基因组全长为1766 bp,其余均为1767 bp;6个毒株序列之间同源性高达96.0%~99.9%,6个毒株与参考毒株序列的同源性为94.4%~99.8%。PCV2全基因组的进化分析结果表明,B12、HB、LHW、LPC和NJ为PCV2b亚型,B07为PCV2d亚型,从而证实PCV2b亚型毒株成为我国猪群中分离率较高的流行毒株,并且我国猪群存在PCV2d亚型毒株。     

2006年夏江西蓝耳病流行毒株的分离及初步鉴定

2006年9月,江西省某猪场发生疫病,发病率为95%以上,死亡率高达70%。通过Marc-145细胞盲传,从该场病死猪脑和肺脏中分离到美洲型蓝耳病病毒,该蓝耳病分离毒株的ORF7核苷酸序列与其他6个发病猪场的蓝耳病病毒的同源性为99%以上,与美洲型蓝耳病标准毒株VR-2332的同源性为93.3%。根据流行病学调查、临床症状、病理变化和病原学检测结果,可以推断该场本次疫病的病原为美洲型蓝耳病病毒变异毒株,且与其他猪场流行的蓝耳病病毒应为同一流行毒株。     

云南边境地区流行性出血热病毒血清学监测及血清型鉴定

为了解近几年云南边境地区牛、羊流行性出血热病毒（EHDV）的感染和流行情况,本研究从2014年起连续3年在与老挝、越南接壤的江城县设置EHDV监测点,每年选择投放EHDV抗体阴性的10头牛和5只山羊作为哨兵动物进行跟踪监测。每年5~10月份对哨兵动物采血,每周1次,11、12月每月采集一次,进行EHDV抗体、抗原监测和病毒分离。针对致细胞病变的样品,采用EHDV群特异性S7基因片段引物进行RT-PCR方法检测,同时利用EHDV-1、-5、-6、-7、-10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,2014－2016年江城县牛EHDV抗体阳性率分别为41.9%、58.6%和75.4%;3年期间共监测到15头EHDV抗体阳性黄牛,并从中分离到20个可致细胞病变样品,经RT-PCR确认为EHDV,遗传进化分析发现有11个毒株与1997和2003年日本分离的EHDV毒株亲缘关系较近,9个毒株与1977和1981年澳大利亚分离的EHDV毒株亲缘关系较近,5个毒株与2015年广西分离株的亲缘关系较近;3年期间在山羊体内未检测出抗体,未发现抗原阳性动物;经中和试验血清型鉴定,确定20株毒株包括EHDV-5、-6、-7、-10型4种血清型,感染时间均在5~9月之间。本研究发现,江城县长期存在多种血清型EHDV同时流行,2014－2016年EHDV抗体阳性率逐年增加,亟需加强对EHDV感染情况及活动规律的持续研究,提高流行性出血热的防控效率。     

云南边境地区流行性出血热病毒血清学监测及血清型鉴定

为了解近几年云南边境地区牛、羊流行性出血热病毒(EHDV)的感染和流行情况,本研究从2014年起连续3年在与老挝、越南接壤的江城县设置EHDV监测点,每年选择投放EHDV抗体阴性的10头牛和5只山羊作为哨兵动物进行跟踪监测。每年5～10月份对哨兵动物采血,每周1次,11、12月每月采集一次,进行EHDV抗体、抗原监测和病毒分离。针对致细胞病变的样品,采用EHDV群特异性S7基因片段引物进行RT-PCR方法检测,同时利用EHDV-1、-5、-6、-7、-10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,2014-2016年江城县牛EHDV抗体阳性率分别为41.9%、58.6%和75.4%;3年期间共监测到15头EHDV抗体阳性黄牛,并从中分离到20个可致细胞病变样品,经RT-PCR确认为EHDV,遗传进化分析发现有11个毒株与1997和2003年日本分离的EHDV毒株亲缘关系较近,9个毒株与1977和1981年澳大利亚分离的EHDV毒株亲缘关系较近,5个毒株与2015年广西分离株的亲缘关系较近;3年期间在山羊体内未检测出抗体,未发现抗原阳性动物;经中和试验血清型鉴定,确定20株毒株包括EHDV-5、-6、-7、-10型4种血清型,感染时间均在5～9月之间。本研究发现,江城县长期存在多种血清型EHDV同时流行,2014-2016年EHDV抗体阳性率逐年增加,亟需加强对EHDV感染情况及活动规律的持续研究,提高流行性出血热的防控效率。     

云南省师宗县蓝舌病病毒的分离及鉴定

为了解近年来云南省师宗县蓝舌病病毒流行情况,2012年在师宗县五龙乡建立了10头蓝舌病血清学阴性黄牛的监控动物群。从2012年5~10月,每周采血1次,11~12月,每月采血1次,采用C-ELISA进行血清学监测。8月开始动物血清学检测结果转阳性,至11月,监控动物全部转为阳性。用转阳前1周、转阳本周、转阳后2~13周的经处理的红细胞静脉接种鸡胚,收获鸡胚肝脏,用PBS悬浮捣碎的鸡胚肝脏,上清接种于C6/36细胞一代、BHK-21三代后,出现细胞病变(cytopathic effect,CPE)。采用RT-PCR方法,针对蓝舌病较为保守的血清型群特异片段VP7设计了2对引物,扩增其相应片段。结果显示,共分离到86份疑似分离物,其中67份疑似分离物细胞培养液上清经RT-PCR扩增,均扩增出1156 bp片段,初步确认为蓝舌病病毒。采用国际24个蓝舌病标准毒及24个标准阳性血清对86份疑似分离物及其对应血清进行细胞微量中和试验,67份毒株为蓝舌病病毒,与RT-PCR结果一致。通过对2份经中和试验定型为BTV-1、BTV-16分离株的VP2基因测序分析发现,BTV-1株序列与同型Y863(登录号:KC879616)参考毒株的同源性为92%,BTV-16株序列与登录号为AB686221的毒株同源性为99%。结果表明共分离到67株蓝舌病毒株,分离株主要为BTV-1、BTV-9、BTV-16三个血清型。     

Isolation and Identification of Arbovirus from the Sentinel Herds in Jinghong City,Yunnan Province,China in 2019

The purpose of this study was to understand the prevalence of arboviruses in Jinghong city,Yunnan province.Three sentinel cattle were set up in Menghan township of Jinghong city in 2019,blood samples were collected regularly for isolation and identification of arboviruses,a total of 7 virus isolates were obtained.Viral nucleic acid were identification by RT-PCR,and the results showed that two strains of epidemic hemorrhagic fever virus (EHDV) with serotypes of 6 and 7,two strains of bluetongue virus (BTV) with serotypes 4 and 5,D’Aguilar virus (DAV) serotype in one strain of PALV and two unidentified circoviruses were isolated,respectively.The ORF region of virus Seg-2 and Seg-3 sequences was compared and analyzed,all the 7 regional strains of the virus were Eastern,which was most closely related to strains in Japan,Australia and India.The blood and serum of three sentinel animals were tested by viral nucleic acid and serum neutralization test,which proved that all three animals were infected with the corresponding virus.When animals were infected with the virus,specific antibodies in the serum rise rapidly,it reached peaks 3 to 4 weeks later and could remain at this level for a long time.However,the content of viral nucleic acid in the blood decreased rapidly after reaching the peak from 2 to 4 weeks.The isolation,sequence characteristics and infection characteristics of Jinghong arboviruses in animals were reported in this study,the results provided data support for further understanding of local bovine arboviruses.At the same time,7 strains of virus were isolated from 3 cattle,suggesting that there might be more arboviruses in the area.     

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。     

1株分离自云南省师宗县库蠓的新型西藏环状病毒

西藏环状病毒(Tibet orbivirus,TIBOV)为环状病毒属的新成员,病毒于2009年首次从我国西藏墨脱县采集的圆斑按蚊中分离,目前,对该病毒的认识仍十分有限.本研究对1株分离自我国云南省师宗县库蠓的新型TIBOV毒株YNSZ/V290/2019进行了鉴定,结果表明,病毒可在C6/36与BHK-21细胞上引起...     

Variation amongst the neutralizing epitopes of bluetongue viruses isolated in the United States in 1979-1981.

Neutralizing epitopes present on field isolates of bluetongue virus (BTV) serotypes 10, 11, 13 and 17 were evaluated with a panel of polyclonal and neutralizing monoclonal antibodies (MAbs). A total of 91 field isolates were evaluated, including 15 isolates of BTV-10, 29 isolates of BTV-11, 26 isolates of BTV-13, and 21 isolates of BTV-17. The viruses were isolated from cattle, goats, sheep, elk and deer in Idaho, Louisiana, Nebraska and, predominantly, California, in the years 1979, 1980 and 1981. The isolates were analyzed and compared using a panel of neutralizing MAbs which included five MAbs raised against BTV-2, seven against BTV-10, five against BTV-13, and six against BTV-17. Neutralization patterns obtained with the MAb panel and individual field isolates were compared to those obtained with prototype viruses of each serotype. All field isolates were neutralized by at least some of the MAbs raised against the prototype virus of the same serotype. All field isolates of BTV-10 were neutralized by the seven MAbs raised to BTV-10, whereas the field isolates of BTV-11, BTV-13 and BTV-17 were not consistently neutralized by all of the MAbs raised against the prototype virus of the same serotype. Variation in neutralizing epitopes recognized by the MAb panel was most pronounced amongst the field isolates of BTV-17. A one-way cross neutralization was evident between BTV-10 and BTV-17 as all field isolates of BTV-17 were neutralized by four of the MAbs raised against BTV-10. In contrast, no BTV-10 isolates were neutralized by the MAbs raised against BTV-17. Differences in the MAb neutralization patterns of field isolates of BTV-11, BTV-13 and BTV-17 suggest that the immunogenic domain responsible for their neutralization is plastic, such that individual epitopes within the domain may vary in their significance to the neutralization of different viruses, even of the same serotype. The apparent conservation of neutralizing epitopes on field isolates of BTV-10 suggests that the field isolates may be derived from the modified-live vaccine strain of BTV-10.     

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).     

Molecular epidemiology of bluetongue virus in Portugal during 2004-2006 outbreak

After 44 years of epidemiological silence, bluetongue virus (BTV) was reintroduced in Portugal in the autumn of 2004. The first clinical cases of bluetongue disease (BT) were notified in sheep farms located in the South of Portugal, close to the Spanish border. A total of six BTV, five of serotype 4 and one of serotype 2 were isolated from sheep and cattle during the 2004-2006 epizootics. The nucleotide sequence of gene segments L2, S7 and S10 of BTV-4 prototype strain (BTV4/22045/PT04) obtained from the initial outbreak and of BTV-2 (BTV2/26629/PT05) was fully determined and compared with those from other parts of the world. The phylogenetic analysis revealed that BTV4/22045/PT04 is related to other BTV-4 strains that circulate in the Mediterranean basin since 1998, showing the highest identity (99%) with BTV-4 isolates of 2003 from Sardinia and Corsica, whereas BTV2/26629/PT05 is almost indistinguishable from the Onderstepoort BTV-2 live-attenuated vaccine strain and its related field strain isolated in Italy. Since live-attenuated BTV-2 vaccine was never used in Portugal, the isolation of this strain may represent a natural circulation of the vaccine virus used in other countries in Mediterranean Europe.     

牛病毒性腹泻病毒JN株的分离鉴定及E2基因的序列分析

本研究从疑似牛病毒性腹泻病毒(bovine viral diarrhea virus,BVDV)感染牛的分泌物与排泄物中分离鉴定1株牛病毒性腹泻病毒,并进行E2基因序列分析。结果表明,分离株病毒命名为JN株;Reed-Muench法测定分离株病毒TCID₅₀为10^-7.5/0.1 mL;病毒中和试验结果表明,BVDV JN分离株可被BVDV阳性血清特异性中和,而不能被BVDV阴性血清中和;分离株病毒E2基因序列测序结果表明,该分离毒株属于BVDVⅠa亚型。