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手足口病是引起手、足和口等部位出现疱疹等症状的法定丙类传染病，主要感染5岁以下的婴幼儿，部分也可感染成人。而柯萨奇病毒A组16型（Coxsackievirus A16, CVA16）是引起手足口病主要的病原体之一。本研究利用反向遗传学技术，成功构建和表达外源荧光素酶（Nano luciferase, Nluc）的CVA16报告病毒（CVA16-Nluc）。该策略是将Nluc基因插入到CVA16的VP1和2A蛋白序列中间。在蛋白水平，2A蛋白酶识别Nluc基因两侧人为添加的2A蛋白酶切位点并实现精准剪切，从而不影响病毒蛋白的表达和颗粒装配。CVA16-Nluc报告病毒可连续传代12代，并稳定高效表达外源蛋白。另外病毒基因组中Nluc基因的插入也并未影响病毒的感染性和遗传稳定性，病毒结构蛋白的表达和病毒颗粒组装。本研究建立基于CVA16-Nluc报告病毒的快速微量中和试验，可缩短实验周期，提高检测结果的准确性，可用于人源、鼠源和兔源中和单克隆抗体的筛选。CVA16-Nluc报告病毒的成功构建也将成为研究CVA16病毒复制、致病机理、和抗病毒药物等方面有用的示踪工具。
Abstract：Hand, foot and mouth disease(HFMD) is a notifiable Class C infectious disease characterized by vesicular eruptions on the hands, feet and oral mucosa. It primarily affects children under five years of age, though adults may also be susceptible. Coxsackievirus A16(CVA16) is one of the principal etiological agents of HFMD. In this study, we constructed a recombinant CVA16 reporter virus(CVA16-Nluc) expressing NanoLuc luciferase(Nluc) using reverse genetics. The Nluc gene was inserted between the VP1 and 2A coding sequences of viral genome. At the protein level, the 2A protease c precisely cleaved at the engineered 2A protease recognition sites flanking the Nluc gene, thereby preserving viral protein expression and particle assembly. The CVA16-Nluc reporter virus was stably passaged for up to 12 generations with efficient and consistent expression of Nluc. Moreover, insertion of the Nluc gene did not compromise viral infectivity, genetic stability, structural protein expression, or virion assembly. Based on CVA16-Nluc, we established a rapid microneutralization assay, which can markedly shorten the experimental timeline and improved assay accuracy. This platform is suitable for screening neutralizing monoclonal antibodies derived from human, murine and rabbit sources. The successful construction of the CVA16-Nluc reporter virus also provides a valuable tool for investigating CVA16 replication, pathogenic mechanisms, and evaluating antiviral candidates.
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基本信息:
DOI：10.13242/j.cnki.bingduxuebao.250183
中图分类号:R373.23
引用信息:
[1]靳卫平,王文辉,郭靖,等.柯萨奇A组16型重组报告病毒的构建及快速中和实验的建立[J].病毒学报,2025,41(06):1731-1739.DOI:10.13242/j.cnki.bingduxuebao.250183.
基金信息:
浙江省疾病预防控制中心自主课题（项目号：2024-B-04）,题目：柯萨奇病毒A组16型重组报告基因病毒构建及快速中和实验的建立~~