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。     

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。     

广西首例牛源鹿流行性出血热病毒的分离鉴定

为了解广西反刍动物鹿流行性出血热病毒(EHDV)的感染情况,本研究在广西马山县设立牛羊虫媒病监控点,筛选EHDV抗体阴性的10头牛和5只羊作为哨兵动物,分别混养在EHDV抗体阳性的牛群及羊群中,采取白天放牧,夜间赶回栏舍的方式饲养。采用竞争性ELISA全年监测其抗体转阳情况,取抗体阳转动物的红细胞,接种BHK-21细胞分离EHDV。以RT-PCR、病毒中和试验等对分离株进行鉴定,结果在3头哨兵牛EHDV抗体转阳前后的7份抗凝血中分离到3株病毒,TCID50分别为102.5/0.1 m L、102.83/0.1 m L和102.5/0.1 m L,血清型均为EHDV-5型。该结果为首次在广西牛群中分离到EHDV,表明广西反刍动物存在EHDV感染。本研究为国家监测反刍动物重要虫媒病毒病流行情况和疫病风险分析提供了有价值的参考资料。     

云南省2株鹿流行性出血热病毒的分离鉴定

为了监测云南省反刍动物鹿流行性出血热病毒(epizootic hemorrhagic disease virus,EHDV)的感染情况,本试验在云南省师宗县设立了监控点,筛选出EHDV抗体阴性的10头牛和5只羊作为哨兵动物,每年的5～10月份每周采血1次,11月到次年4月份每月采血1次,通过酶联免疫吸附试验监测其抗体转阳情况,用转阳牛的红细胞接种BHK以分离病毒,用病毒RT-PCR和中和试验鉴定病毒。结果显示,从2头转阳牛的抗凝血中分离到2株EHDV,其TCID₅₀分别为10^-2.5/0.1 mL和10^-3.44/0.1 mL,血清型均为EHDV-5型。本试验在云南省分离到EHDV,明确了云南省反刍动物存在EHDV感染,为我国监控EHDV流行情况及疫病风险防范提供了重要借鉴意义。     

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。     

云南省牛流行性出血病血清学调查

为了解牛流行性出血病(epidemic hemorrhagic disease,EHD)在云南省的流行和分布情况,采用竞争ELISA方法,2019年对11个县市的1 199份牛血清进行流行性出血病病毒(epidemic hemorrhagic disease virus,EHDV)抗体检测,同时从各县市检测出的阳性血清中,随机选取18～50份通过微量血清中和试验进行血清型鉴定。结果显示:云南省11个县市均检测出EHDV抗体阳性样品,阳性率介于49.3%～91.3%,平均为81.8%;各地普遍存在3个及以上血清型,其中EHDV-7、-10、-6、-5血清型检出率较高,EHDV-8、-2血清型检出率较低,未检出EHDV-1型。结果表明,EHD在云南省流行广泛且较严重,流行血清型复杂。结果提示,需持续开展EHDV血清学监测,重视对该病的防控。此次血清学调查为我国西南地区EHD防控提供了数据参考。     

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。     

广东一株牛源流行性出血病病毒的分离鉴定

为了解广东反刍动物流行性出血病(EHD)的感染情况,本研究对采集的牛血液样品进行EHD病毒(EHDV)的RT-PCR检测,对核酸检测为阳性的样品进行病毒分离鉴定。将阳性样品接种BHK-21细胞连续传3代时出现细胞病变,扩增出了VP7基因,进行BLAST序列比对,结果表明该分离株与国内外EHDV分离株的同源性达98%~100%,TCID_(50)为10~(-6.5)/50μL,不能凝集鸡红细胞,表明该分离株为EHDV,命名为GD-N133。根据VP2基因序列的分析和微量中和试验结果,确定该病毒株为EHDV-1型。     

流行性出血病多克隆抗体C-ELISA检测方法的建立

为能简便、快速地进行流行性出血病病毒(epizootic haemorrhagic disease virus,EHDV)抗体监测,采用纯化的6型EHDV抗原包被ELISA板,以豚鼠抗EHDV-2型多克隆抗体作为竞争抗体,建立了检测EHDV群特异性抗体的竞争ELISA(C-ELISA)方法。结果显示:抗原最佳包被浓度为5.12μg·mL-1(1∶10 000),竞争抗体最佳稀释倍数为1∶10 000;通过对各270份阴性和阳性的牛、羊血清检测,确定该检测方法临界值为50%;特异性试验表明该ELISA方法仅能检测出不同血清型EHDV抗体,具有较好的群特异性;对已知抗体效价的阳性血清检测表明,建立的C-ELISA方法敏感性好于血清中和试验(SNT)和琼脂扩散试验(AGID);分别用C-ELISA、SNT、RTPCR和病毒分离试验对监控动物采集的血清和抗凝血样品进行检测,ELISA结果与其他3种方法检测结果对应一致。结果表明本研究建立的C-ELISA为EHDV抗体的检测提供了一种快速、敏感、稳定的方法。     

广西鹿流行性出血热病毒血清型调查及分布影响分析

2014年采集广西5市7县7个养殖场313份牛血清,经cELISA检测随机筛选出100份鹿流行性出血热病毒(EHDV)血清强阳性样品(cut-off值70%)进行微量细胞中和试验,以调查广西EHDV血清型的存在情况,以及分析其地理分布的影响因素,为丰富EHDV流行病学数据,进一步做好EHDV的综合防制提供依据。     

Development of an enzyme-linked immunosorbent assay for the detection of antibody to epizootic hemorrhagic disease of deer virus.

An enzyme-linked immunosorbent assay has been developed to detect antibodies to epizootic hemorrhagic disease of deer virus (EHDV). The assay incorporates a monoclonal antibody to EHDV serotype 2 (EHDV-2) that demonstrates specificity for the viral structural protein, VP7. The assay was evaluated with sequential sera collected from cattle experimentally infected with EHDV serotype 1 (EHDV-1) and EHDV-2, as well as the four serotypes of bluetongue virus (BTV), BTV-10, BTV-11, BTV-13, and BTV-17, that currently circulate in the US. A competitive and a blocking format as well as the use of antigen produced from both EHDV-1- and EHDV-2-infected cells were evaluated. The assay was able to detect specific antibody as early as 7 days after infection and could differentiate animals experimentally infected with EHDV from those experimentally infected with BTV. The diagnostic potential of this assay was demonstrated with field-collected serum samples from cattle, deer, and buffalo.     

Replication of bluetongue virus and epizootic hemorrhagic disease virus in pulmonary artery endothelial cells obtained from cattle,sheep, and deer

OBJECTIVE: To compare replication of bluetongue virus (BTV) and epizootic hemorrhagic disease virus (EHDV) in pulmonary artery endothelial cells (ECs) obtained from juvenile cattle, sheep, white-tailed deer (WTD; Odocoileus virginianus), and black-tailed deer (BTD; O hemionus columbianus). SAMPLE POPULATION: Cultures of pulmonary artery ECs obtained from 3 cattle, 3 sheep, 3 WTD, and 1 BTD. PROCEDURE: Purified cultures of pulmonary artery ECs were established. Replication, incidence of infection, and cytopathic effects of prototype strains of BTV serotype 17 (BTV-17) and 2 serotypes of EHDV (EHDV-1), and (EHDV-2) were compared in replicate cultures of ECs from each of the 4 ruminant species by use of virus titration and flow cytometric analysis. RESULTS: All 3 viruses replicated in ECs from the 4 ruminant species; however, BTV-17 replicated more rapidly than did either serotype of EHDV. Each virus replicated to a high titer in all ECs, although titers of EHDV-1 were significantly lower in sheep ECs than in ECs of other species. Furthermore, all viruses caused extensive cytopathic effects and a high incidence of cellular infection; however, incidence of cellular infection and cytopathic effects were significantly lower in EHDV-1-infected sheep ECs and EHDV-2-infected BTD ECs. CONCLUSIONS AND CLINICAL RELEVANCE: There were only minor differences in replication, incidence of infection, and cytopathic effects for BTV-17, EHDV-1, or EHDV-2 in ECs of cattle, sheep, BTD, and WTD. It is not likely that differences in expression of disease in BTV- and EHDV-infected ruminants are attributable only to species-specific differences in the susceptibility of ECs to infection with the 2 orbiviruses.     

No evidence for involvement of sheep in the epidemiology of cattle virulent epizootic hemorrhagic disease virus

Epizootic hemorrhagic disease virus (EHDV) is an Orbivirus. While not previously considered as an important disease in cattle, several EHDV serotypes (EHDV-6 and 7) have recently been implicated in disease outbreaks. The involvement of sheep in the epidemiology of EHDV is still not understood. In this study we compared the prevalence of antibodies to EHDV and bluetongue virus (BTV) in sheep to their prevalence in cattle after an outbreak of EHDV that occurred in Israel during 2006. Sixty-six sheep and lambs scattered in seven herds were compared to 114 cows and calves scattered in 13 dairy cattle herds, matched to the sheep herds by location. While antibody prevalence to EHDV was high in cattle (35.2% within the outbreak zone) no evidence of exposure to EHDV was found in sheep (p<0.0001). Antibodies to BTV were apparent in both cattle and sheep though in the former it was significantly higher (63.2%, 16.7% respectively, p<0.0001), suggesting higher exposure of cattle to biting Culicoides midges. Taken together, these results imply that sheep have a negligible role in the epidemiology of EHDV.     

Epizootic hemorrhagic disease virus serotype 7 in European cattle and sheep: Diagnostic considerations and effect of previous BTV exposure

Epizootic hemorrhagic disease virus (EHDV), an arthropod-borne orbivirus (family Reoviridae), is an emerging pathogen of wild and domestic ruminants that is closely related to bluetongue virus (BTV). The present study examines the outcome of an experimental EHDV-7 infection of Holstein cattle and East Frisian sheep. Apart from na?ve animals that had not been exposed to BTV, it included animals that had been experimentally infected with either BTV-6 or BTV-8 two months earlier. In addition, EHDV-infected cattle were subsequently challenged with BTV-8. Samples were tested with commercially available ELISA and real-time RT-PCR kits and a custom NS3-specific real-time RT-PCR assay. Virus isolation was attempted in Vero, C6/36 and KC cells (from Culicoides variipennis), embryonated chicken eggs and type I interferon receptor-deficient IFNAR(-/-) mice. EHDV-7 productively infected Holstein cattle, but caused no clinical signs. The inoculation of East Frisian sheep, on the other hand, apparently did not lead to a productive infection. The commercial diagnostic kits performed adequately. KC cells proved to be the most sensitive means of virus isolation, but viremia was shorter than 2 weeks in most animals. No interference between EHDV and BTV infection was observed; therefore the pre-existing immunity to some BTV serotypes in Europe is not expected to protect against a possible introduction of EHDV, in spite of the close relation between the viruses.     

A survey of cattle for antibodies against bluetongue and epizootic hemorrhagic disease of deer viruses in British Columbia and southwestern Alberta in 1987.

In 1987 a serological survey of cattle for antibodies (Ab) to bluetongue virus (BTV) and epizootic hemorrhagic disease virus (EHDV) was undertaken in British Columbia and southwestern Alberta after infection with the viruses was diagnosed in wild and domestic ruminants in the Okanagan Valley. Of 4610 cattle tested, five had Ab only to BTV, 125 had antibodies only to EHDV and 16 had Ab to both viruses. The Ab were identified as specific for BTV type 11 (BT-11) or EHDV type 2 (EHDV-2). All but one of the seropositive cattle originated in the Okanagan Valley of British Columbia. The remaining one seropositive animal which had Ab to EHDV-2 was pastured with a bull purchased from the Okanagan Valley.     

Diagnostic complementary DNA probes for genome segments 2 and 3 of epizootic hemorrhagic disease virus serotype 1

Potential diagnostic complementary DNA (cDNA) clones of gene segments 2 and 3 from epizootic hemorrhagic disease virus serotype 1 (EHDV-1) have been produced. Individual segments of EHDV-1 were isolated, denatured with methylmercury hydroxide, and polyadenylated. The polyadenylated RNA was reverse-transcribed and self-hybridized into duplex structures, and the incomplete ends were repaired. The resulting product was then cloned into the plasmid vector pBR322, using the complementary tailing method. Two clones, 1 from segment 2 (E1-2-10) and 1 from segment 3 (E1-3-16) were isolated, colony-purified, and characterized by cDNA/RNA blot hybridization and endonuclease restriction analysis. The cDNA clones of RNA segment 3 of EHDV-1 cross hybridized with the corresponding segment of EHDV serotype 2 by results of cDNA/RNA blot hybridization, but not with RNA of bluetongue virus serotypes isolated in the United States. After cDNA/RNA dot-blot hybridization analysis of 17 EHDV field strains, the segment-2 clone was found to be serotype-specific, whereas the segment-3 clone was serogroup-specific.     

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。     

Molecular and genetic comparisons of two serotypes of epizootic hemorrhagic disease of deer virus

The virus-specific double-stranded genome RNA of 2 serotypes of epizootic hemorrhagic disease of deer virus (EHDV) was evaluated by use of coelectrophoresis in polyacrylamide and agarose gel systems. The molecular weights of virion RNA segments were 0.32 to 2.57 X 10(6) for EHDV-1 and 0.33 to 2.54 X 10(6) for EHDV-2. Seven of 10 double-stranded RNA segments of the 2 serotypes had different electrophoretic mobilities in the polyacrylamide-gel electrophoresis system. Although the individual RNA segments of each serotype contained unique RNA sequences determined on the basis of 2-dimensional polyacrylamide-gel electrophoresis analysis of oligonucleotides, the corresponding segments of the 2 serotypes were found to be comparable and at least 1 pair of RNA segment was almost identical. Virus-specific polypeptides for the 2 serotypes were compared by use of gel electrophoresis. Eleven polypeptides were detected for EHDV-1 and 10 for EHDV-2. Six corresponding polypeptides of these 2 serotypes had different electrophoretic mobilities, indicating that these corresponding polypeptides differ in their molecular weights. A genetic relationship was not determined between the 2 EHDV serogroups and the blue-tongue serogroup viruses, using oligonucleotides mapping.