Balasuriya U B, Patton J F, Rossitto P V, Timoney P J, McCollum W H, MacLachlan N J
Department of Pathology, Microbiology and Immunology, School of Veterinary Medicine, University of California, Davis 95616, USA.
Virology. 1997 May 26;232(1):114-28. doi: 10.1006/viro.1997.8551.
The N-terminal hydrophilic ectodomain of the G(L) envelope glycoprotein of equine arteritis virus (EAV) contains neutralization determinants of the virus. We developed a panel of 17 neutralizing murine monoclonal antibodies (MAbs) to further characterize the neutralization determinants of EAV. Included were 6 MAbs previously raised against a laboratory strain (EAVUCD) of the original Bucyrus strain of EAV, as well as 11 additional MAbs that were raised against a neutralization-resistant variant [escape mutant (EM)] virus (EM6D10) that was derived from EAVUCD. All MAbs raised against EAVUCD and 4 of the MAbs raised against EM6D10 (2B3, 5F8, 8D4, and 10B4) reacted with the corresponding G(L) envelope glycoprotein in a Western immunoblotting assay, whereas the remaining 7 MAbs raised against EM6D10 did not react with any viral protein in the immunoblotting assay but competitively inhibited the binding of MAbs 2B3, 5F8, 8D4, and 10B4, indicating that they also recognize epitopes on the G(L) protein. A panel of 18 EM viruses raised to the MAb panel, 19 field isolates of EAV from North America and Europe, the modified-live virus vaccine (ARVAC), and 3 other laboratory strains of EAV were characterized by microneutralization assay with the panel of neutralizing MAbs and polyclonal rabbit and horse antisera. Comparative analysis of the nucleotide sequences of ORF5 and the deduced amino acid sequences of the G(L) protein of individual EM viruses and field isolates of EAV identified four distinct neutralization sites. These sites include amino acids 49 (site A), 61 (site B), 67 through 90 (site C), and 99 through 106 (site D). With the notable exception of site A, the sites were all located in the V1 variable region (amino acids 61-121) within the second half of the N-terminal hydrophilic ectodomain of the G(L) protein. Site D includes several overlapping linear epitopes which appear to interact with amino acids in the other three sites to form conformationally dependent epitopes. Amino acid substitutions within any of these four sites can alter the neutralization phenotype of individual strains of EAV.
马动脉炎病毒（EAV）G(L)包膜糖蛋白的N端亲水性胞外结构域包含该病毒的中和决定簇。我们制备了一组17种中和性鼠单克隆抗体（MAb），以进一步表征EAV的中和决定簇。其中包括6种先前针对EAV原始比塞洛斯毒株的实验室毒株（EAVUCD）产生的MAb，以及另外11种针对源自EAVUCD的中和抗性变异株[逃逸突变体（EM）]病毒（EM6D10）产生的MAb。所有针对EAVUCD产生的MAb以及4种针对EM6D10产生的MAb（2B3、5F8、8D4和10B4）在Western免疫印迹分析中与相应的G(L)包膜糖蛋白发生反应，而其余7种针对EM6D10产生的MAb在免疫印迹分析中不与任何病毒蛋白发生反应，但竞争性抑制MAb 2B3、5F8、8D4和10B4的结合，表明它们也识别G(L)蛋白上的表位。通过用中和性MAb以及兔和马多克隆抗血清进行微量中和试验，对针对该MAb组产生的一组18种EM病毒、来自北美和欧洲的19种EAV野外分离株、改良活病毒疫苗（ARVAC）以及其他3种EAV实验室毒株进行了表征。对单个EM病毒和EAV野外分离株的ORF5核苷酸序列以及G(L)蛋白推导的氨基酸序列进行比较分析，确定了四个不同的中和位点。这些位点包括氨基酸49（位点A）、61（位点B）、67至90（位点C）和99至106（位点D）。除位点A外，这些位点均位于G(L)蛋白N端亲水性胞外结构域后半部分的V1可变区（氨基酸61 - 121）内。位点D包括几个重叠的线性表位，这些表位似乎与其他三个位点的氨基酸相互作用，形成构象依赖性表位。这四个位点中任何一个位点的氨基酸替换都可改变EAV单个毒株的中和表型。
