Encephalitis Group, ICMR-National Institute of Virology, 130/1 Sus Road, Pashan, Pune, 411021, India.
Appl Microbiol Biotechnol. 2022 Mar;106(5-6):1945-1955. doi: 10.1007/s00253-022-11825-1. Epub 2022 Feb 17.
Japanese encephalitis virus (JEV) is one of the leading causes of epidemic encephalitis in South Asian countries. Due to the short-term viremia, detecting IgM antibodies by ELISA is treated as the front-line diagnostic assay. Co-circulation and multiple exposures to antigenically cross-reactive flaviviruses in India pose a challenge in serodiagnosis. Replacing the whole virus antigen currently used in the JE IgM detection kits (ELISA) may improve the specificity and sensitivity of the existing JE MAC ELISA kits. For this purpose, we developed a stably transfected cell clone, BHK-IE6, which expresses a high amount of VLPs up to 37 µg/ml and is consistent in expression up to 40 passages. For the expression of VLPs in the secretory form, we cloned the JEV G-I prM-E coding gene along with the C-terminal signal sequence of capsid protein in the BHK-21 cells using the pcDNA3.1 + mammalian expression vector. The immune assays performed demonstrated its immune reactivity equivalent to the parental JEV strain. Simultaneously performed ELISAs using the whole virus antigen and newly developed antigen gave comparable results for JE positive and negative samples, which established the utility of developed JEV E-VLP as an antigen. Reduced cross-reactivity and increased specificity were observed when tested with dual positive sera for anti-JEV and DENV antibodies. These findings confirm the efficiency and reliability of newly developed recombinant E-VLP antigen expressed by the BHK-IE6 cell clone as an antigen in serodiagnostic assays. The implementation and progress in developing cross-reactivity-reduced antigens would improve serodiagnosis and disease burden estimates of flavivirus infection. KEY POINTS: • pcDNA3.1/JE-Sig-prM-E plasmid transfected BHK-21 cells stably express VLPs. • Sodium butyrate induction enhanced the extracellular expression of VLPs. • Application of JEV-E VLPs increases the specificity of JE IgM ELISA.
日本脑炎病毒（JEV）是南亚国家流行脑炎的主要原因之一。由于短期病毒血症，通过 ELISA 检测 IgM 抗体被视为一线诊断检测。在印度，抗原交叉反应的黄病毒共同循环和多次暴露给血清学诊断带来了挑战。用目前用于 JEV IgM 检测试剂盒（ELISA）的整个病毒抗原替代物可能会提高现有的 JEV MAC ELISA 试剂盒的特异性和敏感性。为此，我们开发了一个稳定转染的细胞克隆 BHK-IE6，该细胞能够表达高达 37µg/ml 的大量 VLPs，并且在 40 个传代中表达一致。为了以分泌形式表达 VLPs，我们使用 pcDNA3.1 + 哺乳动物表达载体将 JEV G-I prM-E 编码基因与衣壳蛋白的 C 末端信号序列一起克隆到 BHK-21 细胞中。进行的免疫测定表明，其免疫反应性与亲本 JEV 株相当。同时使用整个病毒抗原和新开发的抗原进行的 ELISA 对 JE 阳性和阴性样本的结果相当，这证明了新开发的 JEV E-VLP 作为抗原的实用性。当用抗 JEV 和 DENV 抗体的双重阳性血清进行测试时，观察到交叉反应性降低和特异性增加。这些发现证实了 BHK-IE6 细胞克隆表达的新开发的重组 E-VLP 抗原作为血清学诊断检测中的抗原的效率和可靠性。开发交叉反应性降低抗原的实施和进展将改善黄病毒感染的血清学诊断和疾病负担估计。关键点： • pcDNA3.1/JE-Sig-prM-E 质粒转染 BHK-21 细胞稳定表达 VLPs。 • 丁酸钠诱导增强了 VLPs 的细胞外表达。 • JEV-E VLPs 的应用提高了 JE IgM ELISA 的特异性。
