摘要
目的研究蝙蝠来源的重症急性呼吸综合征(SARS)样冠状病毒WIV1刺突蛋白利用浣熊狗血管紧张素转换酶Ⅱ(ACE2)受体侵入细胞的能力,并探究SARS样冠状病毒WIV1潜在的跨宿主传播风险。方法构建来自浣熊狗、果子狸、中华菊头蝠和人等不同动物来源的ACE2表达质粒,转染至293T细胞并利用免疫印迹方法检测其在293T细胞中的表达水平;建立SARS冠状病毒(Tor2株系)及蝙蝠SARS样冠状病毒(WIV1株系)假病毒感染系统,并进行假病毒感染实验;利用假病毒感染系统及荧光素酶报告基因检测WIV1刺突蛋白对浣熊狗等不同动物来源ACE2受体利用能力;构建跨膜丝氨酸蛋白酶2(TMPRSS2)质粒,并转染至T Rex 293细胞,借助假病毒感染系统检测TMPRSS2对WIV1侵入能力的影响。结果本研究所获赠及构建的不同动物来源的ACE2质粒可经瞬时转染至细胞进行表达;与pc DNA3.1载体相比,浣熊狗、中华菊头蝠、果子狸和人的ACE2均可使蝙蝠SARS样冠状病毒WIV1侵入细胞的能力增加上万倍,上述ACE2组与载体组的荧光素酶活性差异均具有统计学意义(t=27.744、P<0.001,t=18.740、P<0.001,t=32.297、P<0.001,t=15.902、P<0.001);与TMPRSS2阴性组相比,TMPRSS2在靶细胞的表达可使WIV1假病毒的感染能力增加10倍以上,两组荧光素酶活性差异具有统计学意义(t=29.460、P<0.001)。结论蝙蝠SARS样冠状病毒WIV1的刺突蛋白除了利用人类、果子狸及中华菊头蝠的ACE2受体感染细胞,还可利用浣熊狗的ACE2受体侵入细胞,且TMPRSS2可显著促进其侵入能力,提示WIV1可能存在多种跨宿主传播的风险。
Objective To investigate the ability of bat severe acute respiratory syndromes(SARS)-like coronavirus(WIV1 strain)to utilize angiotensin-converting enzymeⅡ(ACE2)from raccoon dog as a receptor for cellular entry,and to explore the potential cross-species transmissibility of WIV1.Methods Plasmids encoding ACE2 molecules from different animals including human(Homo sapiens),civet cat(Paguma larvata),Chinese horseshoe bat(Rhinolophus sinicus)and raccoon dog(Nyctereutes procyonoides)were constructed,and then transfected into 293 T cells.The expressions of ACE2 proteins in 293 T were detected by Western blot with anti-C9 antibody.The pseudoviral infection systems of SARS coronavirus(Tor2 strain)and bat SARS-like coronavirus(WIV1 strain)were established to detect receptor activity of different animals’ACE2 for WIV1 entry.The plasmid expressing transmembrane serine protease 2(TMPRSS2)was constructed,and then transfected into T Rex 293 cells.The effect of TMPRSS2 on WIV1 pp infectivity was examined by luciferase assay.Results ACE2 from raccoon dog,Chinese horseshoe bat,civet cat and human expressed well in 293 T cells and supported cellular entry mediated by WIV1 S protein.Compared with pcDNA3.1 vector,ACE2 molecules from raccoon dog,Chinese horseshoe bat,civet cat and human increased the WIV1 pp infection by thousands of times,all with significant differences in luciferase activity(t=27.744,P<0.001;t=18.740,P<0.001;t=32.297,P<0.001;t=15.902,P<0.001).Compared with the group without TMPRSS2,the expression of TMPRSS2 in target cells increased the infection mediated by WIV1-S protein by more than 10 folds,with significant difference in luciferase activity(t=29.460,P<0.001).Conclusions The ACE2 molecules from raccoon dog served as a functional receptor for cellular entry mediated by WIV1 spike protein,which was activated by the TMPRSS2 protease.Our findings herein suggest that there may exist a risk of multiple cross-species transmission of WIV1 among human,bat and raccoon dog.
作者
郑梅
郑双丽
陈丹瑛
蒋栋
曾辉
赵学森
Zheng Mei;Zheng Shuangli;Chen Danying;Jiang Dong;Zeng Hui;Zhao Xuesen(Institute of Infectious Diseases,Beijing Ditan Hospital,Capital Medical University,Beijing 100015,China)
出处
《中华实验和临床感染病杂志（电子版）》
CAS
2019年第5期370-376,共7页
Chinese Journal of Experimental and Clinical Infectious Diseases(Electronic Edition)
基金
国家自然科学基金（No.81571976）
