Department of Diagnostic Medicine and Pathobiology, College of Veterinary Medicine, Kansas State University, 1800 Denison Avenue, Manhattan, KS 66506, USA.
Department of Biochemistry and Molecular Biophysics, Kansas State University, 1711 Claflin Road, Manhattan, KS 66506, USA.
Virus Res. 2019 Aug;269:197632. doi: 10.1016/j.virusres.2019.05.010. Epub 2019 May 23.
Among the structural proteins that compose the virion of African swine fever virus (ASFV), p30 is one of the most immunogenic proteins and is produced during early stage of ASFV infection. These two characteristics make p30 a good target for diagnostic assays to detect ASFV infection. In this study, we describe a panel of newly generated p30-specific monoclonal antibodies (mAbs). The reactivity of these mAbs was confirmed by immunoprecipitation and Western blot analysis in Vero cells infected with alphavirus replicon particles that express p30 (RP-p30). Furthermore, this panel of mAbs recognized ASFV strains BA71 V (Genotype I) and Georgia/2007 (Genotype II) in immunofluorescence assays on virus-infected Vero cells and swine macrophages, respectively. These mAbs also detected p30 expression by immunohistochemistry in tissue samples from ASFV-infected pigs. Epitope mapping revealed that a selected mAb from the panel recognized a linear epitope within the 32-amino acid region, 61-93. In contrast, two of the mAbs recognize the C-terminal region of the protein, which is highly hydrophilic, enriched in glutamic acid residues, and predicted to contain an intrinsically disordered protein region (IDPR). This panel of mAbs and mAb-based diagnostic assays potentially represent valuable tools for ASFV detection, surveillance and disease control.
在构成非洲猪瘟病毒(ASFV)病毒粒子的结构蛋白中,p30 是最具免疫原性的蛋白之一,并且在 ASFV 感染的早期产生。这两个特征使 p30 成为用于检测 ASFV 感染的诊断测定的良好靶标。在本研究中,我们描述了一组新生成的 p30 特异性单克隆抗体(mAb)。通过用表达 p30 的阿尔发病毒复制子颗粒(RP-p30)感染 Vero 细胞进行免疫沉淀和 Western blot 分析,证实了这些 mAb 的反应性。此外,该 mAb 组在感染病毒的 Vero 细胞和猪巨噬细胞上的免疫荧光测定中分别识别 ASFV 株 BA71V(基因型 I)和格鲁吉亚/2007(基因型 II)。这些 mAb 还通过免疫组织化学在感染 ASFV 的猪的组织样本中检测到 p30 的表达。表位作图表明,该组中的一种选定的 mAb 识别 32 个氨基酸区域 61-93 内的线性表位。相比之下,两种 mAb 识别该蛋白的 C 末端区域,该区域高度亲水,富含谷氨酸残基,并且预测含有无规卷曲蛋白质区域(IDPR)。该 mAb 组和基于 mAb 的诊断测定法可能代表用于 ASFV 检测、监测和疾病控制的有价值的工具。