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目的 痘苗病毒N1L基因在抑制宿主凋亡与天然免疫中发挥关键作用，但其在痘苗病毒天坛株(Vaccinia virus Tiantan strain, VTT)中的具体功能尚未明确。本研究旨在阐明N1L缺失对VTT复制、扩散及体内致病性的影响。方法 利用CRISPR/Cas9基因编辑技术构建N1L缺失的重组病毒VTT-ΔN1L。通过细胞模型(HeLa、Vero、BHK-21)分析病毒复制动力学与噬斑形成能力；采用BALB/c小鼠滴鼻感染模型，以不同剂量(106 PFU、107 PFU)评价病毒毒力，监测体重变化与存活率。结果 在HeLa、Vero和BHK-21细胞中，VTT-ΔN1L的复制效率与野生型VTT无显著差异(P > 0.05)，但其形成的噬斑面积在感染后期(48 h)显著减小(P<0.0001)，表明病毒细胞间扩散能力受限。动物实验显示，VTT-ΔN1L感染组小鼠体重下降幅度显著低于VTT组(107 PFU组峰值下降16%vs 25%)，存活率提高(100%vs 83%)，但体重最低点出现时间较VTT组提前2-3d。结论 N1L缺失虽不影响VTT在细胞中的复制，但通过限制病毒扩散和解除其免疫抑制功能，显著降低病毒在小鼠体内的毒力，同时可能引起宿主早期免疫反应增强。该研究揭示了N1L在VTT致病过程中的关键调控作用，为基于VTT的减毒疫苗设计与抗正痘病毒策略提供了理论依据。
Abstract：Objective The vaccinia virus N1L gene plays a key role in suppressing host apoptosis and innate immune responses; however, its specific function in the vaccinia virus Tiantan strain(VTT) remains unclear. This study aimed to determine the effects of N1L deletion on viral replication, cell-to-cell spread, and in vivo pathogenicity of VTT. Methods A recombinant N1L-deleted virus(VTT-ΔN1L) was generated using CRISPR/Cas9 gene editing. Viral replication kinetics and plaque formation were evaluated in HeLa, Vero, and BHK-21 cells. Viral pathogenicity was assessed using an intranasal infection model in BALB/c mice with two inoculation doses(106 PFU and 107 PFU). Body weight changes and survival rates were monitored to evaluate disease severity. Results VTT-ΔN1L showed replication kinetics comparable to those of wild-type VTT in HeLa, Vero, and BHK-21 cells(P > 0.05). However, plaque areas produced by VTT-ΔN1L were significantly smaller at the late stage of infection(48 h; P < 0.0001), indicating reduced cell-to-cell spread. In vivo experiments demonstrated that mice infected with VTT-ΔN1L exhibited significantly less body weight loss than those infected with wild-type VTT(maximum loss: 16% vs. 25% in the 107 PFU group) and a higher survival rate(100% vs. 83%). Notably, the nadir of body weight occurred 2–3 days earlier in the VTT-ΔN1L group. Conclusion Deletion of the N1L gene does not affect VTT replication in vitro but significantly attenuates viral virulence in mice. This attenuation is associated with reduced viral dissemination and likely reflects diminished viral immune-evasion activity, potentially allowing enhanced early host immune responses. These findings highlight a critical regulatory role of N1L in VTT pathogenesis and provide insights for the rational design of attenuated VTT-based vaccines and antiviral strategies against orthopoxviruses.
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基本信息:
DOI：10.13242/j.cnki.bingduxuebao.250423
中图分类号:R373
引用信息:
[1]陈思研,翟玉倩,韩一泽,等.痘苗病毒天坛株N1L基因在病毒扩散与致病中的作用研究[J].病毒学报,2026,42(02):444-453.DOI:10.13242/j.cnki.bingduxuebao.250423.
基金信息:
国家重点研发计划课题（项目号：2025YFC2311803）,题目：2025mRNA疫苗原辅料国产化与免疫原迭代研究~~
2025-12-31
2025
2026-03-11
2026
2
2026-03-12
2026-03-12
2026-03-12