Bhatt Mukesh, Mohapatra Jajati K, Pandey Laxmi K, Mohanty Nihar N, Das Biswajit, Prusty Bikash R, Pattnaik Bramhadev
ICAR-Directorate of Foot and Mouth Disease, Mukteswar 263 138, Uttarakhand, India.
ICAR-Directorate of Foot and Mouth Disease, Mukteswar 263 138, Uttarakhand, India.
Virus Res. 2018 Jan 2;243:36-43. doi: 10.1016/j.virusres.2017.10.010. Epub 2017 Oct 14.
Inactivated purified whole virus vaccines are used for control of foot and mouth disease (FMD). ELISAs detecting antibodies to the nonstructural proteins (NSP), a marker of infection, are primarily used to differentiate FMD virus (FMDV) infected from vaccinated animals (DIVA). However, such DIVA assays have a limitation to their specificity since residual NSPs present in the relatively impure vaccines are suspected to induce an NSP-antibody response in the repeatedly vaccinated animals. Epitope-deleted negative marker vaccine strategy seems to have an advantage over the conventional vaccines in identifying the infected animals with accuracy. NSP 3AB contains an abundance of immunodominant B-cell epitopes of diagnostic importance. This study addresses the feasibility of producing 3AB-truncated FMDV mutant as a potential negative marker vaccine candidate. An infectious cDNA clone of FMDV serotype Asia 1 strain was used to engineer an array of deletion mutations in the established antigenic domain of 3AB. The maximum length of deletion tolerated by the virus was found to be restricted to amino acid residues 87-144 in the C-terminal half of 3A protein along with deletion of the first two copies of 3B peptide. The 3AB-truncated marker virus (Asia 1 IND 491/1997Δ3A3B+FLAG) demonstrated infectivity titres comparable to that of the parental virus in BHK-21 (log 7.42 TCID/ml) and LFBK-αβ (log 8.30 TCID/ml) cell monolayer culture. The protein fragment corresponding to the viable deletion in the 3AB region was expressed in a prokaryotic system to standardize a companion assay (3A3B I-ELISA) for the negative marker virus which showed reasonably high diagnostic sensitivity (96.9%) and specificity (100% for naïve and 97.1% for uninfected vaccinated samples). The marker virus and its companion ELISA designed in this study provide a basis to devise a marker vaccine strategy for FMD control.
灭活纯化全病毒疫苗用于控制口蹄疫（FMD）。检测针对非结构蛋白（NSP）抗体的ELISA方法主要用于区分感染口蹄疫病毒（FMDV）的动物和接种疫苗的动物（DIVA），NSP是感染的一个标志物。然而，此类DIVA检测方法的特异性存在局限性，因为相对不纯的疫苗中存在的残留NSP被怀疑会在多次接种疫苗的动物中诱导NSP抗体反应。在准确识别感染动物方面，表位缺失阴性标记疫苗策略似乎比传统疫苗具有优势。NSP 3AB包含大量具有诊断重要性的免疫显性B细胞表位。本研究探讨了生产3AB截短的FMDV突变体作为潜在阴性标记疫苗候选物的可行性。利用口蹄疫病毒亚洲1型毒株的感染性cDNA克隆，在已确定的3AB抗原结构域中设计了一系列缺失突变。发现病毒所能耐受的最大缺失长度限制在3A蛋白C端一半的第87 - 144个氨基酸残基，同时缺失3B肽的前两个拷贝。3AB截短的标记病毒（亚洲1型IND 491/1997Δ3A3B + FLAG）在BHK - 21（log 7.42 TCID/ml）和LFBK - αβ（log 8.30 TCID/ml）细胞单层培养中表现出与亲本病毒相当的感染滴度。在原核系统中表达了与3AB区域中可行缺失相对应的蛋白质片段，以标准化针对阴性标记病毒的配套检测方法（3A3B I - ELISA），该方法显示出相当高的诊断敏感性（96.9%）和特异性（未接触过疫苗的样本为100%，未感染的接种疫苗样本为97.1%）。本研究设计的标记病毒及其配套ELISA为制定口蹄疫控制的标记疫苗策略提供了基础。
