新型冠状病毒VSV假病毒的构建及其感染能力的初步研究  

Preparation and infectivity evaluation of SARS-CoV-2 VSV-Based pseudovirus

作　　者：李星霖 陈丹瑛 刘永梅 邱雅若 李国力[1] 宋川[1] 孔雅娴[1] 赵红心[1] 赵学森 LI Xing-lin;CHEN Dan-ying;LIU Yong-mei;QIU Ya-ruo;LI Guo-li;SONG chuan;KONG Ya-xian;ZHAO Hong-xin;ZHAO Xue-sen(Institute of Infectious Diseases,Beijing Ditan Hospital,Capital Medical University,100015,China)

机构地区：[1]首都医科大学附属北京地坛医院传染病研究所,100015 [2]北京大学地坛医院教学医院传染病研究所,100015

出　　处：《传染病信息》2022年第2期119-125,140,共8页Infectious Disease Information

基　　金：国家自然科学基金(81971916)。

摘　　要：目的 在二级生物安全实验室条件下,通过构建新型冠状病毒(新冠病毒)VSV假病毒,研究新冠病毒及其流行变异株的侵入特性。方法 通过购买的新冠病毒(Wuhan-Hu-1株)VSV-luc假病毒构建VSV-G/VSV-ΔG-luc假病毒;利用VSV-G/VSV-ΔG-luc假病毒构建新冠病毒VSV假病毒。通过分子克隆对新冠病毒S基因相应位点进行突变,构建新冠病毒不同变异株的刺突蛋白表达质粒,由此包装出新冠病毒各种变异株假病毒。比较新冠病毒Wuhan-Hu-1株与D614G株、Delta株在不同细胞系中的感染能力。结果 新冠病毒VSV假病毒构建成功。评价新型冠状病毒Wuhan-Hu-1株、D614G株、Delta株在4种不同细胞系中侵入情况,与Wuhan-Hu-1株相比,D614G株和Delta株的感染水平均明显增加(P均<0.05),同时在Calu-3和Caco-2细胞系中,Delta株的感染水平明显强于D614G株(P均<0.05)。利用模拟细胞-细胞间传播的感染试验发现,Delta株感染HEK-293T细胞与Vero细胞的能力明显高于D614G株(P均<0.05)。结论 新冠病毒Delta株的感染能力明显强于Wuhan-Hu-1株和D614G株。VSV假病毒系统能够模拟、替代新冠病毒活病毒在侵入细胞的过程中的生理功能,并且安全可靠、易于量化、应用广泛,在新冠病毒研究领域中能够发挥重要作用.Objective In BSL-2 laboratory, the SARS-CoV-2 VSV-based pseudovirus is developed to study the entry process of the SARS-CoV-2. Methods VSV-G/VSV-ΔG-lucpseudoviruswas established from the commercial SARS-CoV-2(WuhanHu-1) VSV-based pseudovirus;VSV-G/VSV-ΔG-lucpseudoviruswas used to construct more SARS-CoV-2 VSV-based pseudovirus;The plasmid containing spike(S) gene of SARS-CoV-2 variants of concern(VOC) was constructed by using PCR-directed mutagenesis,and thepseudoviruses of SARS-CoV-2 VOC were packaged;The infectivity of Wuhan-Hu-1 strain with D614G variant and Delta variant in different cell lines was compared. Results The VSV-based SARS-CoV-2 pseudovirus was successfully constructed. We compared the entry efficacy among SARS-CoV-2 Wuhan-Hu-1 strain, D614G variant and Delta variant in 4 different cell lines. Compared with WuhanHu-1 strain, the infection of D614G variant and Delta variant in the four cell lines were significantly increased(P < 0.05). Meanwhile,in Calu-3 and Caco-2 cell lines, the infection of Delta variant was significantly enhanced compared to the D614G variant(P < 0.05) The ability of Delta variant to infect HEK-293T cells and Vero cells was significantly higher than that of D614G variant(P < 0.05) in Spikemediated cell.-to-cell infection. Conclusions The infectivity of SARS-CoV-2 Delta variant is significantly stronger than Wuhan-Hu-1strain and D614G variant. The VSV-based pseudovirus is a secured, reliable, and practicable system that can mimic the physiological functions of live SARS-CoV-2 virus in the process of entry, which may play an important role in the research of SARS-CoV-2..

关 键 词：假病毒 血管紧张素转化酶Ⅱ 新型冠状病毒 Wuhan-Hu-1株 D614G株 Delta株 S蛋白 细胞-细胞间感染 跨膜丝氨酸蛋白酶2 

分 类 号：R512[医药卫生—内科学]