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痘苗病毒C3L编码的补体控制蛋白(Vaccinia virus complement control protein,VCP)具有抑制补体系统激活等生物学功能，但其对病毒复制扩散及致病性的影响及具体机制尚不明确。本研究利用CRISPR/Cas9技术将痘苗病毒天坛株(Vaccinia virus Tiantan strain,VTT)C3L缺失构建了重组病毒rVTT-mCherryΔC3L，并通过细胞和小鼠模型系统评估其生物学特性。结果显示：在HeLa、Vero和BHK-21细胞中，rVTT-mCherryΔC3L(MOI=0.01)的复制效率与VTT无显著差异，但感染48h后形成的免疫噬斑面积显著增大(分别为VTT的1.3倍、2.1倍和1.6倍，P <0.01)；滴鼻感染BALB/c小鼠后，5×104 PFU rVTT-mCherryΔC3L感染的小鼠，体重下降幅度减轻且恢复时间提前，5×105 PFU感染时存活率显著高于VTT组(100%vs 16.7%)。研究结果表明，C3L缺失虽未显著影响病毒复制能力，但细胞间扩散能力增强，而小鼠体内毒力降低。该研究为痘苗病毒减毒疫苗设计及抗正痘病毒策略提供了重要理论依据。
Abstract：The complement control protein encoded by the C3L gene of Vaccinia virus(Vaccinia virus Complement Control Protein, VCP) exhibits biological functions such as inhibiting complement system activation. However, its impact on viral replication, spread, and pathogenicity, as well as the underlying mechanisms, remain unclear. In this study, CRISPR/Cas9 technology and homologous recombination were utilized to construct a recombinant virus, rVTT-mCherryΔC3L, by deleting the C3L gene from the Vaccinia virus Tiantan strain(VTT). The biological properties of this recombinant virus were systematically evaluated using cellular and murine models. Results showed that in HeLa, Vero, and BHK-21 cells, the replication efficiency of rVTT-mCherryΔC3L(MOI = 0.01) was not significantly different from that of wild-type VTT.However, the immune plaque areas formed at 48 hours post-infection were markedly enlarged(1.3-fold, 2.1-fold, and 1.6-fold larger than VTT, respectively; P < 0.01). In BALB/c mice intranasally infected with 5 ×104 PFU of rVTT-mCherryΔC3L, the severity of weight loss was reduced, and recovery time was accelerated.At a higher infection dose(5 × 105 PFU), the survival rate of the rVTT-mCherryΔC3L group was significantly higher than that of the VTT group(100% vs. 16.7%). These findings indicate that deletion of the C3L gene does not significantly impair viral replication but enhances intercellular spread while attenuating virulence in mice. This study provides critical theoretical insights for the design of attenuated Vaccinia virus vaccines and strategies against orthopoxviruses.
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基本信息:
DOI：10.13242/j.cnki.bingduxuebao.250084
中图分类号:R373
引用信息:
[1]韩一泽,翟玉倩,吴长城等.C3L敲除改变痘苗病毒天坛株生物学特性并降低小鼠体内致病性[J].病毒学报,2025,41(02):344-353.DOI:10.13242/j.cnki.bingduxuebao.250084.
基金信息:
北京市自然科学基金青年项目（项目号：7254390）,项目名称：重症猴痘病例中分离的大片段缺失毒株的生物学功能改变及其机制研究; 中国疾病预防控制中心青年科学基金（项目号：2024A103）,项目名称：基因组大片段缺失猴痘病毒新变异株的生物学功能改变及其机制研究~~