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。     

Comparison of visual microscopic and computer-automated fluorescence detection of rabies virus neutralizing antibodies.

The rapid fluorescent focus inhibition test (RFFIT) and the fluorescent antibody virus neutralization test (FAVNT) are both diagnostic tests for determining levels of rabies neutralizing antibodies. An automated method for determining fluorescence has been implemented to reduce the work time required for fluorescent visual microscopic observations. The automated method offers several advantages over conventional visual observation, such as the ability to rapidly test many samples. The antibody titers obtained with automated techniques were similar to those obtained with both the RFFIT (n = 165, r = 0.93, P < 0.001) and the FAVNT (n = 52, r = 0.99, P < 0.001).     

三种检测猪伪狂犬病抗体的方法比较

应用血清中和试验(SNT)、乳胶凝集试验(LAT)和PRVgpI抗体鉴别ELISA三种方法检测了42份猪血清中的伪狂犬病抗体效价并进行了比较。结果表明SNT与LAT二种方法的阳性符合率为87.1%(27/31),阴性符合率为90.1%(10/11),总符合率为88.1%(37/42),且SNT的敏感性高于LAT,但PRVgpI抗体与SN和LA抗体无相关性。     

West Nile virus infection in horses in Saudi Arabia (in 2013–2015)

West Nile virus (WNV) is an important emerging zoonotic arbovirus giving rise to clinical syndromes of varying severity in humans and horses. Culex mosquitoes are the main vector. Although WNV has been reported in many countries in the Middle East and Asia, little is known about its prevalence in equine populations in the Arabian Peninsula. We have carried out a serological study on 200 horses to assess WNV infection in the Eastern and Central regions of Saudi Arabia in 2013–2015. Sera were tested for the presence of WNV antibodies in parallel using a commercial enzyme‐linked immunosorbent assay (ELISA) kit and microneutralization (MN) tests. In comparison with the MN assay used as “gold standard,” we find the ELISA had a sensitivity of 94.7% and specificity of 80.1%. The prevalence of WNV neutralizing antibody ranged from 5 (17.3%) of 29 sera collected in Riyadh up to 15 (55.6%) of 27 sera collected from Al‐Qateef. These findings highlight the need to be aware of the possibility of WNV disease in humans and horses presenting with central nervous system disease in the Kingdom of Saudi Arabia.     

鸡传染性喉气管炎病毒PCR诊断方法的建立与应用

根据GenBank登录的传染性喉气管炎病毒(ILTV)的TK基因序列设计并合成1对特异性引物,以ILTV疫苗株DNA为模板,建立了检测ILTV TK基因的PCR方法。应用该方法能从临床分离毒株和疫苗株中扩增到长为427 bp的目的片段;但不能从新城疫病毒(NDV)、传染性法氏囊病毒(IBDV)、禽呼肠孤病毒(ARV)、减蛋综合征病毒(EDSV)、H9亚型禽流感病毒(H9-AIV)、传染性支气管炎病毒(IBV)、大肠杆菌以及金黄色葡萄球菌等病原中扩增出阳性条带;敏感性试验表明其DNA最小检出量为4.9 ng;应用该方法和病毒分离法对2份临床病例和人工感染鸡的检测,两者符合率为100%。上述结果表明该PCR方法具有良好的特异性和敏感性,可用于传染性喉气管炎病毒鉴定和临床诊断。     

Development and evaluation of an indirect enzyme-linked immunosorbent assay for serological detection of Schmallenberg virus antibodies in ruminants using whole virus antigen

Background

In late 2011, a new Orthobunyavirus of the Simbu serogroup named Schmallenberg virus (SBV) emerged in continental Europe. The virus is transmitted by hematophagous arthropods, with the Culicoides species as, so far known, main vectors. Infection with the virus can cause clinical signs in adult ruminants including diarrhea, fever and reduced milk production. Transplacental infection of the developing fetus can lead to malformations of varying severity. To assess seroprevalence of SBV in Sweden an indirect enzyme-linked immunosorbent assay (ELISA) was established in connection with the surveys. Here, we describe the development and evaluation of the indirect ELISA, based on whole virus as the coating antigen and a monoclonal antibody for the detection of antibodies to SBV in ruminant sera. The evaluation includes comparison between the in-house ELISA, virus neutralization test and an indirect commercial ELISA.

Results

The optimal working dilutions of antigens and conjugate were estimated with checkerboard titrations. Comparative studies, including ROC analyses, were used for the selection of an optimal cut-off (S/P value = sample value as percentage of positive control value). With an estimated S/P value of 15% the whole virus ELISA showed a specificity of 100% and a sensitivity of 99.19% compared to virus neutralization test (VNT) and with a good consistency as shown in reproducibility and variability experiments. Furthermore, the comparison of our whole virus indirect ELISA to an indirect ELISA with a SBV nucleoprotein antigen, demonstrated a higher sensitivity of our test.

Conclusion

The indirect whole virus ELISA described in this paper is a readily available test for serological analysis of SBV antibodies. Since this in-house ELISA demonstrates a specificity and sensitivity comparable to virus neutralization test and also shows a higher sensitivity compared to commercially available indirect ELISA, it is a useful alternative for surveillance and screening purposes of SBV.     

Serosurveillance for Japanese encephalitis virus infection among equines in India

The seroprevalence of Japanese encephalitis virus (JEV) among equines was evaluated from January 2006 to December 2009 in 13 different states of India by hemagglutination inhibition (HI) test and virus neutralization test (VNT). Antibodies against JEV were detected in 327 out of 3,286 (10%) equines with a maximum prevalence reported in the state of Manipur (91.7%) followed by Gujarat (18.5%), Madhya Pradesh (14.4%), and Uttar Pradesh (11.6%). Evidence of JEV infection was observed in equines in Indore (Madhya Pradesh) where a 4-fold or higher rise in antibody titer was observed in 21 out of 34 horses in November 2007 to October 2006. In March 2008, seven of these horses had a subsequent 4-fold rise in JEV antibody titers while this titer decreased in nine animals. JEV-positive horse sera had a JEV/WNV (West Nile virus) ratio over 2.0 according to the HI and/or VNT. These results indicated that JEV is endemic among equines in India.     

牛传染性鼻气管炎病毒单克隆抗体的制备及阻断ELISA方法的建立

为建立检测牛传染性鼻气管炎病毒(IBRV)血清抗体的阻断ELISA方法,本研究以经蔗糖密度梯度离心法纯化的IBRV作为免疫原制备1株单克隆抗体(MAb),命名为cp-1-1。经间接ELISA、IFA和western blot鉴定,该MAb与IBRV呈阳性反应,与牛病毒性腹泻病毒(BVDV)及牛副流感病毒3型(BPIV3)呈阴性反应,具有较强的特异性。质谱分析结果显示MAb cp-1-1识别的表位位于IBRV VP8蛋白。以纯化的IBRV作为包被抗原、MAb cp-1-1作为检测抗体,建立检测IBRV血清抗体的阻断ELISA方法。该检测方法的抗原包被量为0.89μg/孔,样品稀释度为12,检测抗体MAb量为1.3μg/孔,二抗稀释度为15000。利用50份IBRV抗体呈弱阳性的牛血清(中和抗体效价为14~116)作为标准参考血清,确定该检测方法的阻断率Cut Off值为52.06%,即阻断率高于52.06%时判为阳性,低于52.06%时判为阴性。阻断ELISA方法特异性试验显示仅IBRV阳性血清检测为阳性,而BVDV、BPIV3、牛腺病毒3型(BADV-3)和O型口蹄疫病毒(O-FMDV)阳性牛血清均检测为阴性,表明该方法具有较强的特异性;该方法可检测的最低中和抗体效价为14,与病毒中和试验的敏感性一致,表明该方法具有较高的敏感性;重复性试验显示该方法批内、批间变异系数均小于10%,显示较好的重复性。对130份现地牛血清检测结果显示,该方法与病毒中和试验的符合率为98.46%。用该方法对某牛场接种IBRV灭活疫苗的牛血清进行检测,抗体阳性率为99.51%(205/206)。另外,采用该方法对我国8个省(市、自治区)的801份牛血清进行检测,IBRV的抗体阳性率为41.6%(333/801)。本研究建立的阻断ELISA方法可以用于IBRV疫苗免疫监测和血清流行病学调查,为我国IBR的防控提供技术支持。     

猪伪狂犬病毒PCR检测方法的优化

猪伪狂犬病是由伪狂犬病毒(Pseudorabies virus,PRV)引起的一种疱疹病毒性疾病,在世界大部分地区都是地方性家畜流行病。而且猪伪狂犬病与其他猪传染病不易区分,因此,建立一个特异性强的检测方法对于其诊断具有重要意义。实验是在实验室已经建立伪狂犬病毒单项PCR检测方法的基础上,对扩增条件进行优化后对其特异性进行了验证。结果发现,优化后的方法特异性好,只有猪伪狂犬病毒扩增为阳性,其余DNA病毒:猪细小病毒、猪圆环病毒Ⅰ型(PCV1)、猪圆环病毒Ⅱ型(PCV2)均呈现阴性。因此,本研究方法可以为今后实验室检测猪伪狂犬病毒提供参考。     

蓝舌病病毒甘肃分离株的定型研究

继利用蚀斑抑制中和试验对蓝舌病病毒山东分离株（L001）进行血清型鉴定后，又对蓝舌病病毒甘肃分离株进行了血清型鉴定，证实甘肃分离株为蓝舌病病毒11型。     

Evaluation of two enzyme-linked immunosorbent assays for serodiagnosis of Aleutian mink disease virus infection in mink

Background

Aleutian disease in mink is caused by infection with Aleutian mink disease virus (AMDV). In Sweden, the infection most commonly causes classical Aleutian disease in which the immune system fails to neutralize the virus and the infection becomes persistent. Diagnosis of AMDV infection is based on serological methods that detect virus-specific antibodies. Traditionally counterimmunoelectrophoresis (CIEP) has been the preferred method, but in order to enable automation interest has been paid to other antibody detecting systems. Recently, at least two different ELISA systems that detect antibodies to AMDV have been manufactured; one is based on an in vitro grown AMDV as antigen, and the other system is based on the AMDV capsid protein VP2 as antigen. The aim of this study was to evaluate the two ELISA systems for detection of antibodies to AMDV using CIEP as the gold standard.

Results

When employing the mean optical density of the samples from CIEP negative mink plus three standard deviations as cut-off value, the ELISA with the VP2 antigen had a sensitivity of 99.7% and a specificity of 98.3% compared to CIEP (n = 364). Analysis of samples with the AMDV-G antigen based ELISA employing an assay cut-off value based on the negative control samples, as suggested by the manufacturer, resulted in a sensitivity of 54.3% and a specificity of 93.2% with reference to CIEP as the gold standard (n = 359). When employing the mean optical density of the samples from CIEP negative mink plus three standard deviations as cut-off value, the AMDV-G ELISA had a sensitivity of 37.6% and a specificity of 98.3%.

Conclusions

The ELISA system based on VP2 antigen had high sensitivity and specificity, and was concluded to be an alternative to the CIEP as a diagnostic tool for AMDV antibodies. In contrast, the AMDV-G ELISA suffered from low sensitivity when compared to CIEP.     

H3亚型犬流感病毒的荧光定量PCR方法建立及其增殖特性研究

旨在建立H3亚型犬流感病毒(canine influenza virus,CIV)荧光定量PCR方法,利用该方法对病毒在犬肾细胞(MDCK细胞)上增殖特性进行研究.根据CIV的血凝素(HA)片段上的HA2基因保守区设计并合成特异性引物,构建CIV-HA重组质粒作为标准品,经过条件优化,建立H3亚型CIV荧光定量检测方法...     

新城疫病毒和禽流感病毒复合型胶体金免疫层析试纸条的制备

为了建立一种简便、快速而且能同时检测新城疫病毒(NDV)和禽流感病毒(AIV)的方法,本试验采用柠檬酸三钠还原法制备胶体金颗粒,分别标记NDV单克隆抗体6C4和AIV单克隆抗体制备免疫检测探针。在硝酸纤维素膜上,用微定量喷头喷好2条病毒检测线(T线)和1条羊抗鼠抗体质控线(C线),制备复合型免疫层析检测试纸条。结果在10 min内,可同时检测出两种病毒。试纸条检测NDV的灵敏度比HA试验结果提高8倍;AIV重组抗原的检测灵敏度为1.7μg/mL。两种病毒互相测试,未出现交叉反应。用缓冲液对照测试结果为阴性。在密封、干燥、低温的条件下,试纸条的灵敏性与特异性没有明显变化。说明本研究建立的NDV和AIV免疫层析检测法具有特异、灵敏、稳定、操作简单等特点,符合现场快速检测的要求。     

两种消毒剂对流感病毒灭活效果的对比研究

采用Klein-Defors悬浮杀灭与感染试验方法，研究“卫可”、“卫康”两种消毒剂在不同浓度下，分别与流感H51N1和H9N2亚型病毒作用5min和10min，对流感病毒的灭活作用。结果表明：这两种消毒剂在相同或不同的浓度范围内。对病毒的杀灭率是有区别的。尽管每种消毒剂在本身稀释浓度范围内都有较好的杀毒效果，但在应用中也应考虑其他因素的影响。因此，我们推荐使用该消毒剂的工作浓度为100％杀灭病毒的稀释度。这将为其临床应用提供依据，对环境消毒和防止流感爆发具有参考价值。     

Evaluation of a bulk-milk ELISA test for the classification of herd-level bovine leukemia virus status

The results of a commercial bulk-milk enzyme-linked immunosorbent assay (ELISA) test for herd-level bovine leukemia virus (BLV) status were compared to results obtained from individual agar-gel immunodiffussion (AGID) testing on sampled cattle. A positive herd was defined as a herd having one or more AGID-positive animals. The estimated true herd status was based on the sensitivity and specificity of the AGID test and the number of cattle sampled per herd. Ninety-seven herds were used, with a mean of 13 cows sampled per herd. The AGID test indicated an apparent herd prevalence of 70.1%. After accounting for the number of cows sampled and the sensitivity and specificity of the AGID test, the estimated true herd prevalence of BLV was 52.3%. The ELISA test identified 79.4% of herds as positive for BLV, and had an apparent sensitivity and specificity of 0.97 and 0.62, respectively. However, after accounting for the sensitivity and specificity of the AGID test in individual animals, the specificity of the ELISA test was 0.44. The ELISA test was useful for identifying BLV-negative herds (i.e., ruling out the presence of BLV infection in test negative herds). With the moderately low specificity, herds identified as positive by the ELISA test would require further testing at the individual or herd level to definitively establish their BLV status.     

间接ELISA检测鸭病毒性肝炎抗体的研究

本试验以氯仿去脂-滤膜过滤-浓缩层析方法纯化病毒作包被抗原，成功地建立了检测鸡抗鸭肝炎病毒(DHV)血清抗体的间接ELISA方法，经交叉试验和阻断试验表明，该方法具有很高的敏感性和特异性，应用于DHV抗体检测比较有效，为鸭抗DHV血清抗体检测以及进行DHV单克隆抗体筛选提供了依据。     

The P 37/24 modification of the infectious bovine rhinotracheitis virus-serum neutralization test.

The applicability of a modified infectious bovine rhino-tracheitis constant-virus/varying-serum neutralization test, with preincubation of virus-serum mixtures at 37°C for 24 hrs. ( test) as against 1 hr. in the conventional () test, was elucidated by examination of about 5000 bovine sera. The sensitivity of the test was studied mainly in parallel testings of sera from animals in two infected herds and of sera taken from a bullock during one month following nasal infection. In agreement with conclusions in previous papers, the test appeared to be inadequately sensitive, and the test regularly gave titers that were about 4 in logs higher than the titers, which means that the test is about 16 times as sensitive as the conventional test. From examinations of 4902 sera from four different groups of cattle it was concluded that with the technique described and with the use of undiluted serum, the specificity of the test would be as good as 0.999. The observed high sensitivity and specificity of the test give high diagnostic values of both negative and positive reactions.     

Evaluation of a blocking ELISA for the detection of bovine viral diarrhoea virus (BVDV) antibodies in serum and milk

The performance characteristics of a blocking ELISA test applied to serum and individual milk for the detection of antibodies to bovine viral diarrhoea virus (BVDV) were assessed using 1189 matched milk/serum samples collected from cows of 42 dairy herds located in Brittany (west of France). This test was based on a monoclonal antibody directed against non-structural protein NS2-3 of pestiviruses. All tests were performed blind. For each type of sample, negative/positive cut-off values were determined using receiver operating characteristic (ROC) analysis. Sensitivity and specificity were estimated using the virus neutralisation test as a reference. For sera, the ROC analysis provided a negative/positive inhibition percentage cut-off value of 50% giving a sensitivity and a specificity of 96.9 and 97.8%. For individual milk samples, the cut-off was fixed at 30%, leading to a sensitivity and a specificity of 96.9 and 97.3%. Using this test, a good overall agreement was found between results obtained on matched milk/serum samples (Kappavalue=0.95). The present results indicate that this blocking ELISA test is reliable enough for use in a mass screening and control scheme on BVDV.     

基孔肯亚病毒和辛德毕斯病毒检测基因芯片的建立

根据GenBank中收录的基孔肯亚病毒和辛德毕斯病毒基因的保守序列,合成2种病毒E基因序列及引物,设计针对2种病毒的寡核苷酸探针,制备基孔肯亚病毒与辛德毕斯病毒特异性检测基因芯片,并对该芯片的灵敏性、特异性和重复性进行了验证。结果显示,所建立基因芯片检测方法的灵敏度是普通PCR方法的100倍。利用所制备的基因芯片,能检测到基孔肯亚病毒和辛德毕斯病毒特异性杂交信号,阴性对照病毒（基因Ⅰ型流行性乙型脑炎病毒,基因Ⅲ型流行性乙型脑炎病毒,猪繁殖与呼吸综合征病毒及流感病毒）均无杂交信号。本试验初步建立了基孔肯亚病毒与辛德毕斯病毒特异性基因芯片检测方法,该方法灵敏度高、特异性强,适用于基孔肯亚病毒与辛德毕斯病毒的流行病学调查和种特异性鉴定。