蓝舌病病毒分离株的定型研究

用国际标准蓝舌病病毒(BTV)型特异性血清和新制备的BTV型特异性血清,按OIE推荐方法进行蚀斑抑制试验,并试用“悬浮法”蚀斑抑制试验,对BTV分离株(L001)作了血清型鉴定,结果表明该分离株为BTV16型。与标准毒株相对照,两者试验结果完全一致;L001株的RNA的PAGE电泳带谱与BTV16型标准株的带谱相同。     

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。     

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。     

蓝舌病病毒单克隆抗体的制备及生物学特性鉴定

以纯化的蓝舌病病毒（ＢＴＶ）１１型ＶＰ７免疫ＢＡＬＢ／ｃ小鼠,动用淋巴细胞杂交瘤技术,借助间接ＥＬＩＳＡ筛选,获得了３株分泌抗ＢＴＶ１１ ＶＰ７的群特异性单克隆抗体（ＭｃＡｂ）的杂交瘤细胞株,命名为Ｃ１２Ｄ９、Ｂ４Ｆ３、Ｅ１Ａ７。这３株杂交瘤细胞株连续传代３个月再经液氮冻存６个月后复苏培养,仍能稳定分泌特异性ＭｃＡｂ。３株ＭｃＡｂ均具有ＥＬＩＳＡ特性和免疫沉淀特性,其中Ｃ１２Ｄ９株上清液的ＥＬＩＳ     

Experimental infection of camels with bluetongue virus

Three camels aged 4–5 years were experimentally infected with Bluetongue virus serotype 1 (BTV-1) and were observed for 75 days. No clinical signs of disease were observed throughout the experiment, however all three animals seroconverted and developed BTV-1 specific neutralising antibodies after challenge. All three camels developed a viraemia from 7 days post infection albeit at a lower level than that usually observed in experimental infections of sheep and cattle. Virus was isolated from the blood of all three animals suggesting that camels may act as a reservoir for BTV and play an important role in its transmission.     

Dynamics of viral spread in bluetongue virus infected calves

The kinetics of viremia and sites of viral replication in bluetongue virus (BTV) infected calves were characterized by virus isolation, serology and immunofluorescence staining procedures. In addition, the role of the regional lymph node and lymphatics draining inoculated skin in the pathogenesis of BTV infection was determined by analyzing efferent lymph collected from indwelling cannulas. Viremia persisted for 35 to 42 days after inoculation (DAI) and virus co-circulated with neutralizing antibodies for 23 to 26 days. Virus was first isolated from peripheral blood mononuclear (PBM) cells at 3 DAI, after stimulation of PBM cells with interleukin 2 and mitogen. BTV was frequently isolated from erythrocytes, platelets and stimulated PBM cells but never from granulocytes and rarely from plasma during viremia. Virus was consistently isolated from erythrocytes late in the course of veremia. Interruption of efferent lymph flow by cannulation delayed the onset of viremia to 7 DAI. BTV was infrequently isolated from lymph cells, and few fluorescence positive cells were observed after lymph and PBM cells were labelled with a BTV-specific monoclonal antibody. Virus was isolated from spleen by 4 DAI and most tissues by 6 DAI, whereas virus was isolated from bone marrow only at 10 DAI. Virus was not isolated from any tissue after termination of viremia. It is concluded that primary viral replication occurred in the local lymph node and BTV then was transported in low titer to secondary sites of replication via infected lymph and PBM cells. We speculate that virus replication in spleen resulted in release of virus into the circulation and non-selective infection of blood cells which disseminated BTV to other tissues. Virus association with erythrocytes likely was responsible for prolonged viremia, although infected erythrocytes eventually were cleared from the circulation and persistent BTV infection of calves did not occur.     

Probability of introduction of exotic strains of bluetongue virus into the US and into California through importation of infected cattle

Strategies designed to minimize the probability of bluetongue virus (BTV) introduction to new areas should be based on a quantitative assessment of the probability of actually establishing the virus once it is introduced. The risk of introducing a new strain of bluetongue virus into a region depends on the number of viremic animals that enter and the competency of local vectors to transmit the virus. We used Monte Carlo simulation to model the probability of introducing BTV into California, USA, and the US through importation of cattle. Records of cattle and calf imports into California and the US were obtained, as was seroprevalence information from the exporting countries. A simulation model was constructed to evaluate the probability of importing either a viremic PCR-negative animal after 14-day quarantine, a c-ELISA BTV-antibody-negative animal after 28-day quarantine, or an untested viremic animal after 100-day quarantine into California and into the US. We found that for animals imported to the US, the simulated (best to worst scenarios) median percentage that tested positive for BTV-antibody ranged from 5.4 to 7.2%, while for the subset imported to California, the simulated median percentage that tested positive for BTV-antibody ranged from 20.9 to 78.9%. Using PCR, for animals imported to the US these values were 71.8–85.3%, and for those imported to California, the simulated median that test positive ranged from 74.3 to 92.4%. The probability that an imported animal was BTV-viremic is very low regardless of the scenario selected (median probability = 0.0%). The probability of introducing an exotic strain of BTV into California or the US by importing infected cattle was remote, and the current Office International des Epizooties (OIE) recommendation of either a final PCR test performed 14 days after entry into quarantine, a c-ELISA performed 28 days after entry into quarantine or a 100-day quarantine with no testing requirement was adequate to protect cattle in the US and California from an exotic strain of BTV.     

Active circulation of bluetongue vaccine virus serotype-2 among unvaccinated cattle in central Italy

Several seroconversions occurring in 2002 among sentinel cattle during the bluetongue-vaccination campaign in Lazio and Tuscany (central Italy) led to the suspicion of vaccine-virus circulation. Therefore in 2003, 17 seroconverting sentinel herds were investigated for the characteristics of the virus involved. From these farms, 91 unvaccinated animals and 57 Culicoides pools were tested for the presence of the bluetongue vaccine virus (serotype-2) or other strains. The presence of vaccine virus serotype-2 was confirmed by PCR followed by restriction analysis in the whole blood of 17 unvaccinated sentinel cattle and 12 pools of Culicoides imicola or C. obsoletus. Of the 17 herds, five were positive only for vaccine virus serotype-2, four were positive for other strains and two for both the vaccine and other strains; the remaining premises were virologicaly negative. The vaccine virus serotype-2 also was detected in areas not included in the vaccination campaign.     

Validation and standardization of virus neutralizing test using indirect immunoperoxidase technique for the quantification of antibodies to rabies virus

A virus neutralizing test using an indirect immunoperoxidase technique (VNT-IIP) for rabies has been developed for the titration of dog and cat serum samples in Japan. The VNT-IIP has the advantage that results obtained can be viewed by the naked eye. The purpose of this study was to validate the VNT-IIP and compare it with one of the international standard methods, the fluorescent antibody virus neutralization test (FAVNT). The VNT-IIP showed satisfactory repeatability, high analytical specificity and good accuracy. Regarding the comparison between the VNT-IIP and the FAVNT, the VNT-IIP showed good agreement (91.9%), high sensitivity (92.8%) as well as specificity (87.0%) and good correlation (r = 0.92). As described above, the validation of the VNT-IIP was satisfactory and the performances of the test proved to be equivalent to those of an international standard method.     

The impact of bluetongue virus on reproduction

Bluetongue virus has been recognized as an important noncontagious, arthropodborne infectious viral disease of ruminants. 24 different serotypes of virus have been recognized world-wide. The most severe clinical disease has been associated with severe clinical disease in sheep and some free ranging wild ruminants. A number of reports have implicated the viruses as causing reproductive disorders in both males and females. The bluetongue related reproductive disorders include early embryonic deaths, abortions, malformed fetal calves or lambs, transient infertility in bulls and rams, and shedding of virus in semen. Recently, bluetongue virus contamination of modified live commercial canine vaccine was associated with abortion and acute death of pregnant bitches. The pathogenesis of these various aspects of reproductive failure are discussed herein.     

马病毒性动脉炎血凝及血凝抑制试验研究与应用

在RK-13细胞上培的马动脉炎病毒在4℃、室温和37℃条件下，用各种动物的红细胞进行血凝试验，证实病毒在不同温度下均可凝集小鼠和鸡的红细胞，但不能凝集牛、绵羊、山羊、马、猪、豚鼠、仓鼠、兔及鹅红细胞，病毒用吐温-80和乙醚处理后，血凝活力会显著增强，其血凝滴度比未经处理的病毒高4-8倍，且血凝像更加清晰，最适处理条件是病毒在冰、浴状态下用终浓度0.06%-0.125%(V/V)二温-80振荡处理15min-60min，再加50%（V/V）乙醚振荡作用5min-15min。用EAV免疫SPF豚鼠，4周后采血做HI和NT试验，显示HI抗体和NT抗体产物成正相关，对550份来自新西兰、吉尔吉斯、内蒙古、新疆的马血清做EAV的抗体检测，比较两个试验，二者符合率为97.8%，血凝抑制抗体与中和抗体效价呈显著的正相关，但抗体效价略低于后者。     

1株牛传染性鼻气管炎病毒的分离鉴定

在对进口种用奶牛隔离检疫期间,从1头IBRV中和抗体阳性奶牛中分离出1株病毒。该分离株表现类似于IBRV特征的细胞病变,细胞圆缩,聚集成葡萄串样群落,在单层细胞上形成空洞。用特异性抗IBRV阳性血清与其进行中和试验,发现IBRV标准阳性血清对分离株的中和抗体滴度为27,与IBRV标准株的中和抗体滴度相差不到1个滴度。用OIEV推荐的IBR特异性引物对分离病毒进行PCR扩增,获得与设计基因片段大小一致的特异性条带,表明分离到的病毒为IBRV。     

Modelling local dispersal of bluetongue virus serotype 8 using random walk

The knowledge of the place where a disease is first introduced and from where it later spreads is a key element for the understanding of an epizootic. For a contagious disease, the main method is back tracing. For a vector-borne disease such as the Bluetongue virus serotype 8 epizootic that occurred in 2006 in North-Western Europe, the efficiency of tracing is limited because many infected animals are not showing clinical signs. In the present study, we propose to use a statistical approach, random walk, to model local spread in order to derive the Area of First Infection (AFI) and spread rate. Local spread is basically described by the random movements of infected insect vectors. Our model localised the AFI centre, origin of the infection, in the Netherlands, South of Maastricht. This location is consistent with the location of the farms where the disease was first notified in the three countries (Netherlands, Belgium, and Germany) and the farm where retrospectively the earliest clinical signs were found. The derived rate of spread of 10-15km/week is consistent with the rates observed in other Bluetongue epizootics. In another article Mintiens (2008), the AFI definition has then been used to investigate possible ways of introduction (upstream tracing) and to study the effect of animal movements from this area (downstream tracing).