Institute of Life Sciences, Nalco Square, Bhubaneswar 751023, India.
Institute of Life Sciences, Nalco Square, Bhubaneswar 751023, India.
J Virol Methods. 2014 Apr;199:86-94. doi: 10.1016/j.jviromet.2014.01.008. Epub 2014 Jan 21.
The recent epidemics of Chikungunya viruses (CHIKV) with unprecedented magnitude and unusual clinical severity have raised a great public health concern worldwide, especially due to unavailability of vaccine or specific therapy. This emphasizes the need to understand the biological processes of this virus in details. Although CHIKV associated research has been initiated, the availability of CHIKV specific reagents for in-depth investigation of viral infection and replication are scanty. For Alphavirus replication, non-structural protein 2 (nsP2) is known to play a key regulatory role among all other non-structural proteins. The current study describes the development and characterization of nsP2 specific monoclonal antibody (mAb) against a synthetic peptide of CHIKV. Reactivity and efficacy of this mAb have been demonstrated by ELISA, Western blot, Flow cytometry and Immunofluorescence assay. Time kinetic study confirms that this mAb is highly sensitive to CHIKV-nsP2 as this protein has been detected very early during viral replication in infected cells. Homology analysis of the selected epitope sequence reveals that it is conserved among all the CHIKV strains of different genotypes, while analysis with other Alphavirus sequences shows that none of them are 100% identical to the epitope sequence. Moreover, using the mAb, three isoforms of CHIKV-nsP2 have been detected in 2D blot analysis during infection in mammalian cells. Accordingly, it can be suggested that the mAb reported in this study can be a sensitive and specific tool for experimental investigations of CHIKV replication and infection.
近期，基孔肯雅热病毒（CHIKV）的爆发规模空前，临床症状异常，引起了全球公共卫生的极大关注，尤其是因为目前尚无针对该病毒的疫苗或特效疗法。这凸显了深入了解该病毒生物学过程的必要性。虽然已经开展了针对基孔肯雅热病毒的相关研究，但可用于深入研究病毒感染和复制的基孔肯雅热病毒特异性试剂却十分匮乏。对于甲病毒复制而言，非结构蛋白 2（nsP2）在所有其他非结构蛋白中起着关键的调节作用。本研究描述了针对 CHIKV 合成肽的 nsP2 特异性单克隆抗体（mAb）的开发和特性鉴定。通过 ELISA、Western blot、流式细胞术和免疫荧光检测，证实了该 mAb 的反应性和功效。时程动力学研究证实，该 mAb 对 CHIKV-nsP2 高度敏感，因为在感染细胞中病毒复制的早期就可以检测到这种蛋白。对所选表位序列的同源性分析表明，它在不同基因型的所有 CHIKV 株中都保守，而与其他甲病毒序列的分析表明，没有一个序列与该表位序列完全一致。此外，在哺乳动物细胞感染过程中，使用该 mAb 在 2D 印迹分析中检测到了三种 CHIKV-nsP2 同工型。因此，可以认为本研究中报告的 mAb 可作为研究 CHIKV 复制和感染的敏感和特异性工具。
