An attenuated EMCV-HB10 strain acts as a live viral vector delivering a foreign gene.
An attenuated EMCV-HB10 strain acts as a live viral vector delivering a foreign gene.
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DOI：
10.1099/jgv.0.000541
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发表时间：
2016-09
期刊：
影响因子：
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通讯作者：
Huibin Yu;Li Huang;Yuanfeng Zhang;Liang Hu;Shengnan Wang;Jiangnan Li;Xuehui Cai;S. Cui;C. Weng
中科院分区：
文献类型：
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作者：
Huibin Yu;Li Huang;Yuanfeng Zhang;Liang Hu;Shengnan Wang;Jiangnan Li;Xuehui Cai;S. Cui;C. Weng
我们成功构建了脑心肌炎病毒（EMCV）HB10株的全长cDNA感染性克隆，并获得了具有较短poly（C）束的部分减毒的rEMCV-C9病毒。我们的结果表明，EMCV-HB10 Poly(C) 束的长度与 EMCV-HB10 菌株在体内的致病性相关。以pEMCV-C9为骨架，我们通过在2A蛋白中插入含有127个氨基酸缺失的cDNA片段、初级切割盒、FLAG标签和VP1和Δ2A连接处的多克隆位点（MCS），构建了新型病毒载体pC9-MCS-Δ2A。此外，增强型绿色荧光蛋白（egfp）基因被克隆到pC9-MCS-Δ2A的MCS中，以测试其表达外源蛋白的能力。插入egfp基因并不影响病毒复制，并且在连续5次传代内观察到EGFP表达减少。此外，我们发现 rC9-EGFP-Δ2A 在体内无毒，可诱导中和抗体产生，并赋予小鼠针对 EMCV 致命攻击的保护性免疫反应。总而言之，我们的结果表明，我们已经构建了一种基于 EMCV-HB10 毒株的减毒活载体，该毒株具有两种经过修饰的关键毒力因子（聚（C）束和 2A 蛋白），可用作候选活疫苗和用于外源抗原递送的潜在活病毒载体。
We successfully constructed a full-length cDNA infectious clone of the encephalomyocarditis virus (EMCV) HB10 strain and obtained a partially attenuated rEMCV-C9 virus with a shorter poly(C) tract. Our results showed that the length of the EMCV-HB10 poly(C) tract was related to the pathogenicity of the EMCV-HB10 strain in vivo. Using pEMCV-C9 as the backbone, we constructed the novel viral vector pC9-MCS-∆2A by inserting a cDNA fragment containing a 127 amino acid deletion in the 2A protein, a primary cleavage cassette, a FLAG tag and a multiple cloning site (MCS) at the junction of VP1 and ∆2A. Additionally, the enhanced green fluorescent protein (egfp) gene was cloned into the MCS of pC9-MCS-∆2A to test its capacity to express foreign proteins. Insertion of the egfp gene did not affect viral replication, and a decrease in EGFP expression was observed within five serial passages. Furthermore, we found that rC9-EGFP-∆2A was avirulent in vivo, induced neutralizing antibody production and conferred protective immune responses against lethal challenge with EMCV in mice. Taken together, our results demonstrated that we had constructed an attenuated live vector based on an EMCV-HB10 strain with two modified critical virulence factors (the poly(C) tract and 2A protein) that could be used as a candidate live vaccine and a potential live viral vector for foreign antigen delivery.