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(B T V-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BT V-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、B H K-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...     

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。     

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。     

蓝舌病病毒血清8型定性传入风险评估

2006年8月荷兰暴发的蓝舌病8型(BTV-8)疫情已蔓延至欧洲大陆,而国内目前尚未发现BTV-8感染的报道.为评估BTV-8传入国内的风险,采用定性评估方法,通过收集BTV-8疫源地国家的疫情信息数据,结合其病原学特征、传播媒介、分布及流行情况、传染源、传播途径和易感动物等方面的研究内容进行传入风险分析.鉴于我国与欧...     

用蓝舌病病毒血清11型实验感染牛

用血清１１型蓝舌病病毒（ＢＴＶ－１１）接种１６头处于繁殖期或妊娠阶段的易感牛。这些牛不含蓝舌病病毒（ＢＴＶ）和流行性出血热病病毒（ＥＨＤＶ），病毒接种前无ＢＴＶ抗体。一组４头牛与１头牛可从精液中排ＢＴＶ－１１（ＣＯ７５Ｂ３００株）的公牛自然交配。以推测该公牛是否能通过交配使母牛感染ＢＴＶ－１１，继而经胎盘感染胎儿，并持续感染，产出先天性畸胎儿。有４头妊娠牛的两个组，接种昆虫介导的ＢＴＶ－１１（ＣＯ     

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。     

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。     

云南蓝舌病病毒1型毒株M6基因序列分析

为了解云南蓝舌病病毒(Bluetongue virus,BTV) 1型M6基因流行株的遗传变异及其与国内外流行病毒的遗传进化关系,试验从细胞培养物中分别提取4株云南分离株BTV-1 (Y863、SZ120169、6-12和7-12) RNA,用M6基因特异引物进行RT-PCR扩增和测序,采用生物信息学软件对获得的M6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.结果表明,分别获得4株云南分离株BTV-1 M6基因1 763 bp序列;4株云南分离株BTV-1核苷酸同源性在95.2%~99.9%之间,氨基酸同源性在97.6%~99.8%之间,1979年师宗分离的Y863病毒毒株与2012年师宗(SZ120169)、2013年江城(6-12、7-12)分离的3株病毒毒株核苷酸同源性分别为95.5%、95.2%和95.2%,氨基酸同源性分别为97.6%、98.4%和98.2%,而近两年(2012、2013)分离病毒核苷酸和氨基酸同源性较高,分别在96.9%~99.9%和99.1%~99.8%之间;遗传进化分析发现,4株云南分离株BTV-1为Eastern基因群病毒,它们之间核苷酸和氨基酸同源性分别为95.2%~99.9%和97.6%~99.8%;进一步分析发现4株云南分离株BTV-1与希腊及澳大利亚 BTV-1型毒株亲缘关系较近,核苷酸和氨基酸同源性分别为90.4%~95.6%和95.1%~99.1%,而与地中海国家(意大利、法国、阿尔及利亚、摩洛哥和突尼斯)和南非毒株关系较远,核苷酸和氨基酸同源性分别在83.8%和95.7%以下.4株云南分离株BTV-1属于Eastern基因群病毒,云南分离株BTV-1 M6基因在自然进化中发生遗传变异缓慢,该基因可以用来进行BTV-1基因群分布及毒株的地理区域来源相关的研究.     

A new bluetongue virus serotype isolated in Kenya.

An apparently new strain of bluetongue virus was first isolated in Kenya in 1965 and since, has been obtained on 7 further occasions from diseased sheep during clinical outbreaks of disease. It proved to be serologically different from the 16 bluetongue virus strains then held at this laboratory. The virus was modified by passage in embryonated hens eggs to produce a live virus strain suitable for inclusion in a polyvalent vaccine. Recent neutralisation tests, carried out with 24 guinea pig immune sera prepared at Pirbright against the currently known World serotypes, have confirmed the earlier results and show that it is different from any of the existing serotypes.     

Molecular epidemiology of bluetongue virus serotype 1 isolated in 2006 from Algeria

This study reports on an outbreak of disease that occurred in central Algeria during July 2006. Sheep in the affected area presented clinical signs typical of bluetongue (BT) disease. A total of 5245 sheep in the affected region were considered to be susceptible, with 263 cases and thirty-six deaths. Bluetongue virus (BTV) serotype 1 was isolated and identified as the causative agent. Segments 2, 7 and 10 of this virus were sequenced and compared with other isolates from Morocco, Italy, Portugal and France showing that they all belong to a ‘western’ BTV group/topotype and collectively represent a western Mediterranean lineage of BTV-1.     

新型鸭肝炎病毒全基因组序列分析

为了解新型鸭肝炎病毒(DHV)基因组变异情况,本研究将广东地区分离到的1株,山东地区分离到的2株与1型DHV(DHV-1)无抗原交叉性的新型DHV(N-DHV)全基因组序列测定的结果进行了分析。结果表明,N-DHV基因组全长7786nt～7788nt,仅有一个ORF,其结构具有典型的微RNA病毒科病毒基因组特征。ORF编码一个2251aa的聚合蛋白,该蛋白经3Cpro切割产生3个结构蛋白(VP0、VP3、VP1)和8个非结构蛋白(2A1、2A2、2B、2C、3A、3B、3C、3D)。与其他N-DHV毒株及DHV-1的抗原性相关VP1蛋白氨基酸序列比对表明,3株病毒之间及与国内分离的N-DHVG株的同源性均在98%以上,与韩国N-DHV同源性均大于92%,与中国台湾地区N-DHV同源性为80.1%～80.9%,与DHV-1的同源性为76.9%～77.3%。3株病毒与韩国N-DHVVP1氨基酸差异位点主要集中在C-端的高变异区。依据微RNA病毒科人肠病毒属血清型分型标准,GD株、SD01株、SD02株与韩国N-DHV和G株属于同一血清型,而与DHV-1和中国台湾地区新型DHV属于不同的血清型。     

Neurovirulence of the UC-2 and UC-8 strains of bluetongue virus serotype 11 in newborn mice

In vivo and in vitro experiments were done to investigate whether the difference in neurovirulence between the two strains of bluetongue virus 11, UC-2 and UC-8, is based on a different capability to gain access to the brain from the subcutaneous inoculation site or on a different tropism for neural cells. In newborn Balb/c mice subcutaneous inoculation of UC-8 at doses between 10(-0.2) plaque forming units (PFU) and 10(4.8) PFU caused a severe necrotizing encephalitis whereas UC-2 at doses of up to 10(4.4) PFU did not affect newborn Balb/c mice. However, intracranial inoculation of 10(2.4) PFU of either virus strain produced severe necrotizing encephalitis. In vitro both virus strains infected dissociated brain cell cultures similarly. Double labelling immunofluorescent staining with markers specific for neural cells did not reveal differences in the target cells for the two viruses. The difference in neurovirulence between UC-2 and UC-8, therefore, appears to be determined by the ability of UC-8 to infect the brain from a subcutaneous inoculation site.     

Genetic variation of bluetongue virus serotype 11 isolated from host (sheep) and vector (Culicoides variipennis) at the same site

Genetic relatedness of 2 strains of bluetongue virus (BTV) serotype 11 that were isolated from the same geographic site--one from host (sheep) and the other from the vector Culicoides variipennis during an enzootic of bluetongue at Bruneau, Idaho, in August 1973--was determined by comparing the oligonucleotide fingerprint analyses of the individual double-stranded RNA segments of the genomes. It was observed that the 2 strains of BTV-11 exhibit considerable differences in their genotypes, the percentage of diversity being different for each of the corresponding RNA species of the 2 strains of BTV-11. These results indicate that more than one genotype of BTV can circulate in juxtaposition in a given geographic site. The observed genotypic diversity might be due to the accumulation of point mutations on specific RNA species or antecedent reassortment of RNA segments between different BTV in nature or both.     

Trajectory analysis and bluetongue virus serotype 2 in Florida 1982.

Examination of Northern Hemisphere synoptic charts and computation of backward trajectories indicated that Culicoides infected with bluetongue virus serotype 2 could have been carried on the wind and brought the virus to Florida on the afternoon of August 19, 1982 after leaving northern Cuba the previous evening. Flight would have occurred at a height of 1-1.5 km at temperatures of 15-17 degrees C. The distance of 500 km from northern Cuba to Ona would have been covered in 20 h at an average speed of 25 km h-1. Computation of trajectories indicated that a second electropherotype, Ona B, was unlikely to have been introduced by infected Culicoides.     

Epidemiology of bluetongue virus serotype 8 outbreaks in the Netherlands in 2006

In August 2006 a major epidemic of Bluetongue (BT) occurred in north-western Europe, affecting The Netherlands, Belgium, Germany, Luxemburg, and the north of France. It was caused by Br virus serotype 8 (BTV-8), a serotype previously unknown to the EU. Although clinical disease is usually restricted to sheep, this virus also caused clinical disease in a small proportion of cattle. The last clinical outbreak of BT in The Netherlands occurred mid-December 2006. The delay between observation of the first clinical signs by the owner and reporting of a clinically suspect BT situation to the veterinary authorities was approximately 2 weeks. BTV-8-associated clinical signs were more prominent in sheep than in cattle, and the relative frequency of specific clinical signs was different in cattle and sheep. Morbidity and mortality rates were significantly higher among sheep than among cattle, and a higher proportion of cattle than sheep recovered from clinical disease.