Comparison of three serotests for the detection of pseudorabies antibodies in pigs

The serum-neutralization test (SN), enzyme-linked immunosorbent assay (ELISA) and the radial immunodiffusion enzyme assay (RIDEA) were compared for the detection of pseudorabies (PRV) antibodies in swine sera. A total of 1285 serum samples were tested. All three tests were considered useful in determining the PRV antibody status of swine on a herd basis, but available evidence supports the continued use of SN as the definitive test because of possible false positive reactions associated with ELISA and RIDEA.     

两株抗猪伪狂犬病毒的单克隆抗体制备与鉴定

采用猪伪狂犬病毒(PRV)ZJ01株纯化病毒免疫BALB/c小鼠,利用融合细胞技术和间接ELISA抗体筛选技术,制备并获得2株能稳定分泌抗PRV单克隆抗体的杂交瘤细胞株2B3和5C10,其中,2B3单抗为Ig G2a亚类,5C10为Ig G1亚类,轻链均为κ链。间接免疫荧光检测结果表明,2株单克隆抗体均能与PRV发生特异性反应。Western-blot结果表明,2B3单抗针对PRV g C蛋白,5C10单抗针对PRV g E蛋白。本研究为建立快速检测伪狂犬病毒感染的免疫学方法奠定了基础。     

Identification of the cross-reactive antigens between Marek's disease virus and pseudorabies virus by monoclonal antibodies

Among the 33 monoclonal antibodies (MAbs) against pseudorabies virus (PRV) examined, three MAbs (24-17, 74-26, and 8) were found to react with cells infected with Marek's disease virus (MDV)-related viruses by immunofluorescence test. Two of the MAbs (24-17 and 74-26) reacted with the nuclei of cells infected with MDV serotype 1 (MDV1), MDV serotype 2 (MDV2), and herpesvirus of turkeys (HVT), whereas MAb 8 reacted with the cytoplasm of MDV2- and HVT-infected cells. However, none of the MAbs against MDV1, MDV2, and HVT that were examined reacted with PRV-infected cells. None of these three MAbs against PRV reactive with MDV-related viruses cross-reacted with the cells infected with other herpesviruses, such as herpes simplex virus type 1, herpes simplex virus type 2, varicella zoster virus, Epstein-Barr virus, or human herpesvirus 6. Southern-blot hybridization under stringent or less-stringent conditions showed that no significant DNA homology was detected between PRV DNA and MDV DNA.     

猪伪狂犬病血清抗体两种检测方法的比较

应用酶联免疫吸附试验和免疫金标检测试纸两种方法，同时对64头份猪血清样品进行猪伪狂犬病血清抗体检测，试验结果显示：两种方法检测的64头份猪血清中伪狂犬病抗体阳性率均为62．5％(40／64)，符合率达到100％。两种方法都具有微量、特异、准确的优点。前者需要用酶标仪等仪器，试验时间相对较长，成本高，但能用准确的数字表达抗体水平；后者不需要任何仪器设备，试验时间短，成本低，适用于基层兽医站、养殖场使用，以及大面积开展猪伪狂犬病抗体检测的使用。     

Comparison between results of virus neutralization test and those of two ELISAs when screening for antibodies to pseudorabies virus in Thailand

The virus neutralization (VN) test and two enzyme-linked immunosorbent assays (blocking and indirect ELISAs) were used to detect antibodies to pseudorabies virus on serum samples of 1,000 pigs from the central part of Thailand. The results of these tests were compared to those of VN test. Using the VN test as standard, the blocking and indirect ELISAs showed respectively 95.12% and 99.37% relative sensitivity and 92.0% and 93.5% relative specificity. The two ELISAs were considered both as practical alternatives to the VN test. However, the indirect ELISA was the more suitable test for the routine screening for antibodies to pseudorabies virus in Thailand.     

多重PCR检测猪细小病毒和猪伪狂犬病病毒的研究

根据GenBank上已发表的猪细小病毒（Porcine parvovirus，PPV）的VP2基因序列和猪伪狂犬病病毒（Porcine pseudorabies virus，PRV）的gH基因序列，设计合成了两对特异引物，分别建立了PPV和PRV的单项PCR诊断方法，通过对扩增条件的筛选，最终成功地建立了PPV和PRV的复合PCR诊断方法，即利用一次PCR反应，可同时扩增PPV的751bp和PRV355bp的特异性片段，而扩增猪圆环病毒Ⅱ型（PCV．2）及相应的培养细胞（PK-15）核酸结果均为阴性，对PPV和PRV的最低检出量分别为100Pg和10Pg的DNA。该方法适合对PPV和PRV的联合检测和鉴别诊断。     

分泌抗猪伪狂犬病病毒(北京株)单克隆抗体杂交瘤细胞株的建立

用纯化的猪伪狂犬病病毒免疫Ｂａｌｂ／ｃ小鼠,取脾细胞与ＳＰ２／０骨髓瘤细胞融合 ,经间接ＥＬＩＳＡ筛选,３次有限稀释法克隆,获得了２株能稳定分泌抗伪狂犬病病毒单克隆抗体杂交瘤细胞株１Ｈ７和３Ｂ５,经鉴定两株单抗均为ＩｇＧ１亚类、Ｋａｐｐａ型轻链,杂交瘤细胞的平均染色体数为９７,细胞培养液上清及腹水效价分别为１：１０２４、１：１０２４和１：１０＾８、１：１０＾７;１Ｈ７、３Ｂ５单克隆抗体不与猪繁殖－呼吸综合征病毒、猪温病毒、猪细小病毒发生交叉反应,显示良好的特异性,为进一步应用奠定了基础。     

Evaluation of the sensitivity and specificity of two diagnostic tests for antibodies to pseudorabies virus glycoprotein X.

The diagnostic performance of 2 enzyme-linked immunosorbent assays (gX-T, gX-H) for antibodies to pseudorabies virus (PRV) glycoprotein X (gX) were evaluated using 311 serum samples from a nonvaccinated quarantined herd. When the standardized virus neutralization (VN) test, which uses the Shope strain (VN Shope), was used as the comparative diagnostic standard, the gX-T test had a 7% false-negative rate and a 52% false-positive rate, and the gX-H test had a 19% false-negative rate and a 19% false-positive rate. When the VN test with a Bartha recombinant strain (VN Bartha gIIIKa) was used as the diagnostic standard, the gX-T test had a 9% false-negative rate and a 26% false-positive rate, and the gX-H test had a 24% false-negative rate and a 11% false-positive rate. Thus, the gX-T test was more sensitive and the gX-H test was more specific. Additional diagnostic tests on 79 serum samples from a noninfected herd did not produce false positives for the gX-H test, but there was an 8% false-positive rate for the gX-T test. Previous studies from our laboratory have demonstrated that VN Bartha gIIIKa has higher sensitivity than VN Shope, without losing specificity, and thus is a better comparative diagnostic standard. When adding a suspect range to the gX-T test, using the same criteria as the suspect range for the gX-H test, the false-positive rate of the gX-T test was reduced to 5% when evaluated versus VN Bartha gIIIKa in the infected herd and to 1% for the PRV-negative herd.(ABSTRACT TRUNCATED AT 250 WORDS)     

猪伪狂犬病病毒变异毒株gE和gB蛋白单克隆抗体的制备与鉴定

为建立猪伪狂犬病病毒(PRV)疫苗免疫抗体与病毒感染抗体鉴别诊断方法,分别以PRV变异毒株的gE或gB重组蛋白免疫BALB/c小鼠,将免疫小鼠脾细胞与SP2/0骨髓瘤细胞进行融合,经免疫过氧化物酶单层细胞试验(IPMA)和酶联免疫吸附试验(ELISA)筛选所需单克隆抗体,获得6株抗PRV gE蛋白的单抗(3E6、7E1、7C5、10C3、15C1、14C1)和6株抗PRV gB蛋白的单抗(1C1、5D2、2F11、5G8、10C7、8H5)。IPMA检测结果显示,gE蛋白单抗能识别PRV变异毒株,但不识别疫苗株;而gB蛋白单抗可同时识别PRV变异毒株和疫苗株。gE单抗10C3和gB单抗10C7的ELISA效价和IPMA效价均分别达1∶5×10~(5.0)和1∶8×10~(3.0)以上。所筛选出的单抗均不与猪圆环病毒2型(PCV2)、猪繁殖与呼吸综合征病毒(PRRSV)、猪瘟病毒(CSFV)、猪流行性腹泻病毒(PEDV)、流行性乙型脑炎病毒(JEV)等其他常见病毒发生交叉反应。gE蛋白和gB蛋白的表达及单抗的制备为PRV感染抗体与疫苗免疫抗体鉴别诊断(DIVA)试纸的研发奠定基础。     

The detection of antibodies to the glycoprotein X antigen of pseudorabies virus

The persistence of antibodies to glycoprotein X (gpX) in the serum of pigs experimentally infected with pseudorabies virus (PRV) was determined using an anti-gpX enzyme-linked immunosorbent assay (ELISA). Antibodies to gpX were detected for at least 365 days postchallenge in nonvaccinated pigs. Previous sensitization of pigs by vaccination with S/PRV had no apparent effect on the antibody response of pigs to gpX postchallenge. In determining previous exposure of pigs to PRV strains containing the gpX gene, the anti-gpX ELISA was highly specific, but its sensitivity was lower than the standard serological procedures currently used for detecting PRV antibodies.     

猪伪狂犬病毒gE蛋白单克隆抗体制备与鉴定

为获得猪伪狂犬病毒(PRV)gE蛋白单克隆抗体,选择原核表达的重组gE蛋白免疫6周龄BALB/c雌性小鼠,将其脾细胞与SP2/0进行融合,经间接ELISA筛选阳性杂交瘤细胞,结果获得了2株能稳定分泌抗PRV gE蛋白的杂交瘤细胞,命名为E3B8和E5C11。间接ELISA检测2株杂交瘤细胞的培养上清液抗体效价为1∶6.4×10~3,腹水的抗体效价分别达到1∶3.28×10~6和1∶6.55×10~6。2株杂交瘤细胞的染色体数分别为105和108。E3B8亚类鉴定重链为IgG1,轻链为κ链;E5C11亚类鉴定重链为IgG2b,轻链为κ链。Western blot检测显示2株单克隆抗体腹水均能与PRV重组gE蛋白发生特异性反应,间接免疫荧光试验(IFA)检测显示2株单克隆抗体均能与PRV分离毒株感染的BHK-21细胞发生特异性反应,交叉反应性检测显示2株单克隆抗体与常见病毒不发生交叉反应。表明制备的2株gE蛋白单克隆抗体效价高、特异性强,为gE蛋白结构与功能分析以及PRV免疫诊断试剂盒的开发奠定了基础。     

Detection of latent pseudorabies virus in swine using in situ hybridization

We have examined methods for detection of pseudorabies virus (PRV) latency in three groups of swine; naturally infected animals obtained from a field case; animals which have been experimentally infected with Becker or Iowa strains of PRV; and single reactors (single seropositive animals within PRV-free herds). In situ hybridization was shown to be more sensitive than explanation/co-cultivation for the detection of latent virus. Nervous tissues, in particular the trigeminal ganglia, were found to be the most reliable source for detecting latent PRV. The presence of latent PRV was not detected in lymphoid tissues examined.     

猪流行伪狂犬病毒gE基因检测及生物信息学分析

为了解当前伪狂犬病毒(PrV)地方流行毒株gE基因的变异特点,根据Guizhou DY株设计引物序列扩增gE基因,并对扩增产物进行序列分析和蛋白特性预测。成功获得1 654bp目的片段,提交GenBank获得登录号KM079613。核酸序列分析在142~144和1 480~1 482位发现2处特征性CGA插入,并导致氨基酸序列在48和494位天冬氨酸插入。相对Ea株编码蛋白O-GlcNAc糖基化位点在563和571位出现偏移,抗原倾向性在420~460位区域明显下降。进化分析显示与2012年以来国内流行毒株WY、ZM、HuXT2012、HBBD、HBLF、XiangA和ZJNB2012同源性较高,达99.8%,而与欧洲、美洲毒株及2012年以前国内毒株同源性较低。表明所感染的伪狂犬病病毒已发生一定的变异,并且gE基因编码蛋白与国内早期分离的Ea株相比在O-GlcNAc糖基化位点和抗原倾向性上也发生了改变。     

荧光定量RT-PCR检测虾Taura综合征病毒方法建立及应用

由Taura综合征病毒（TaurasyndromevirusTSV）引起的虾Taura综合征（Taurasylldrome）是世界动物卫生组织（OIE）规定的必需上报的水生动物二类疫病。本研究采用Taqman探针技术建立了快速检测TSV的荧光定量RT—PCR方法，通过常规RT—PCR方法对比，证实其检测灵敏度和阳性检出率明显提高。     

Detection of African swine fever virus antibodies by immunoblotting assay.

An immunoblotting assay has been adapted to detect antibodies against African swine fever virus. The electrophoretic transfer of proteins and the immunoreaction conditions were optimized, using 4 mA/cm2 of current intensity and 10 micrograms of soluble cytoplasmic antigen of infected cells per strip. Filters of polyvinylidene difluoride showed the highest capacity for protein absorption, but nitrocellulose filters showed lower backgrounds. The specificity and the pattern of the proteins induced by African swine fever virus that react with the antisera were determined in immunoblotting assay, IP30 being the most reactive protein.     

Induction of antibodies in mice by a recombinant baculovirus expressing pseudorabies virus glycoprotein B in mammalian cells.

The glycoprotein gB of pseudorabies virus (PrV) was expressed in various mammalian cells by a recombinant baculovirus carrying the PrV gB gene under the control of the CAG promoter. When the recombinant baculovirus was inoculated into the stable porcine kidney cell line CPK, expression of PrV gB was detected by immunofluorescent antibody analysis and a 155 kDa of protein, which has the same molecular mass as the native PrV gB, was detected by Western blotting. High levels of expression of PrV gB were observed in BHK-21, HmLu-1 and SK-H cell lines. Furthermore, anti-PrV gB-specific antibodies against PrV gB protein were detected by the enzyme-linked immunosorbent assay in mice inoculated the recombinant baculovirus. The recombinant baculovirus containing the PrV glycoprotein gB gene under the CAG promoter could be a candidate for a pseudorabies vaccine.     

Protection of mice and swine from pseudorabies virus-induced mortality by administration of pseudorabies virus-specific mouse monoclonal antibodies

Hybridomas were selected for secretion of monoclonal antibodies directed against pseudorabies virus (PRV) glycoproteins. Each monoclonal antibody was capable of neutralizing PRV in vitro in the presence of complement. This panel of antibodies was used in passive immunization studies to protect mice and swine from PRV-induced mortality. The most protective antibody in mice was 3A4, specific for PRV glycoprotein gp50, which afforded as high as 100% protection. Although antibody 3A4 was partially protective in swine, antibody 3D11, which is specific for PRV glycoprotein glll, afforded greater protection--83% protection when ascitic fluid was used and 100% protection when immunoglobulin concentrated from cell cultures was used at a dose of 150 mg/pig. These studies demonstrated that monoclonal antibodies may be useful for short-term prophylaxis against PRV-induced disease and that antibody directed against either PRV glycoprotein glll or gp50 is sufficient to protect animals from PRV-induced mortality.     

Radial immunodiffusion enzyme assay for detection of antibodies to pseudorabies virus in swine serum

A radial immunodiffusion enzyme assay for the detection and quantitation of antibodies to pseudorabies virus in swine sera was developed and the methods were standardized. The assay combined the principle of radial immunodiffusion with enzyme-linked immunosorbent assay. Quantitation of pseudorabies virus antibody titers was determined by measuring the diameter of a colored circular zone after overnight incubation of antibody with antigen. The specificity and sensitivity of the radial immunodiffusion enzyme assay were compared with that of the standard virus-neutralization test, and the results were determined to be correlated highly (r = 0.694, P less than 0.0001). The assay also appeared to be highly reproducible and simple to perform.