Severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)is the causative virus of the coronavirus disease 2019(COVID-19)pandemic.To establish a safe and convenient assay system for studying entry inhibitors and neu...Severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)is the causative virus of the coronavirus disease 2019(COVID-19)pandemic.To establish a safe and convenient assay system for studying entry inhibitors and neutralizing antibodies against SARS-CoV-2,we constructed a codon-optimized,full-length C-terminal mutant spike(S)gene of SARS-CoV-2.We generated a luciferase(Luc)-expressing pseudovirus containing the wild-type or mutant S protein of SARS-CoV-2 in the envelope-defective HIV-1 backbone.The key parameters for this pseudovirus-based assay,including the S mutants and virus incubation time,were optimized.This pseudovirus contains a Luc reporter gene that enabled us to easily quantify virus entry into angiotensin-converting enzyme 2(ACE2)-expressing 293T cells.Cathepsin(Cat)B/L inhibitor E64d could significantly block SARS-CoV-2 pseudovirus infection in 293T-ACE2 cells.Furthermore,the SARS-CoV-2 spike pseudotyped virus could be neutralized by sera from convalescent COVID-19 patients or recombinant ACE2 with the fused Fc region of human IgG1.Thus,we developed a pseudovirus-based assay for SARS-CoV-2,which will be valuable for evaluating viral entry inhibitors and neutralizing antibodies against this highly pathogenic virus.展开更多
Filoviruses cause severe and fatal viral hemorrhagic fever in humans. Filovirus research has been extensive since the 2014 Ebola outbreak. Due to their high pathogenicity and mortality, live filoviruses require Biosaf...Filoviruses cause severe and fatal viral hemorrhagic fever in humans. Filovirus research has been extensive since the 2014 Ebola outbreak. Due to their high pathogenicity and mortality, live filoviruses require Biosafety Level-4(BSL-4) facilities, which have restricted the development of anti-filovirus vaccines and drugs.An HIV-based pseudovirus cell infection assay is widely used for viral entry studies in BSL-2 conditions. Here,we successfully constructed nine in vitro pseudo-filovirus models covering all filovirus genera and three in vivo pseudo-filovirus-infection mouse models using Ebola virus, Marburg virus, and Lloviu virus as representative viruses. The pseudo-filovirus-infected mice showed visualizing bioluminescence in a dose-dependent manner. A bioluminescence peak in mice was reached on day 5 post-infection for Ebola virus and Marburg virus and on day4 post-infection for Lloviu virus. Two known filovirus entry inhibitors, clomiphene and toremiphene, were used to validate the model. Collectively, our study shows that all genera of filoviruses can be well-pseudotyped and are infectious in vitro. The pseudo-filovirus-infection mouse models can be used for in vivo activity evaluation of anti-filovirus drugs. This sequential in vitro and in vivo evaluation system of filovirus entry inhibitors provides a secure and efficient platform for screening and assessing anti-filovirus agents in BSL-2 facilities.展开更多
目的建立西尼罗病毒(West Nile virus,WNV)假病毒的体内感染模型,并对抗体WNV-XH1进行体内中和活性评价。方法利用前期建立的可包装WNV假病毒的稳定细胞株制备假病毒上清,将上清浓缩后感染BHK21细胞检测假病毒滴度。假病毒经腹腔注入C57...目的建立西尼罗病毒(West Nile virus,WNV)假病毒的体内感染模型,并对抗体WNV-XH1进行体内中和活性评价。方法利用前期建立的可包装WNV假病毒的稳定细胞株制备假病毒上清,将上清浓缩后感染BHK21细胞检测假病毒滴度。假病毒经腹腔注入C57BL/J小鼠体内后进行生物发光成像,观测小鼠体内假病毒感染情况。同时感染后取血,ELISA检测小鼠血清中NS1水平。利用建立的小鼠感染模型,评价抗体WNV-XH1的体内功能活性。结果C57BL/J小鼠感染WNV假病毒后,体内可检测到荧光,且感染假病毒的小鼠与未感染的小鼠相比外周血血清中WNV非结构蛋白NS1水平明显升高(1.453±0.09vs0.305±0.018)。攻毒前静脉给予抗体WNV-XH1后,小鼠体内荧光信号减弱,血清中NS1水平下降(0.384±0.015)。结论成功建立WNV假病毒体内感染模型,并证实抗体WNV-XH1在体内对WNV假病毒的感染具有保护作用。展开更多
With the development of the COVID-19 epidemic,there is an urgent need to establish a system for determining the effectiveness and neutralizing activity of vaccine candidates in biosafety level 2(BSL-2)facilities.Previ...With the development of the COVID-19 epidemic,there is an urgent need to establish a system for determining the effectiveness and neutralizing activity of vaccine candidates in biosafety level 2(BSL-2)facilities.Previously,researchers had developed a pseudotyped virus systemfor SARS-CoV andMERS-CoV,based onHIV-1 core,bearing virus spike protein.During the development of a pseudotyped SARS-CoV-2 system,a eukaryotic expression plasmid expressing SARSCoV-2 spike(S)protein was constructed and then co-transfectedwith HIV-1 based plasmid which containing the firefly luciferase reporter gene,into HEK293T cells to prepare the pseudotyped SARS-CoV-2 virus(ppSARS-2).We have successfully established the pseudotyped SARS-CoV-2 system for neutralization and entry inhibition assays.Huh7.5 cell line was found to be the most susceptible to our pseudotyped virus model.Different levels of neutralizing antibodies were detected in convalescent serum samples of COVID-19 patients using ppSARS-2.The recombinant,soluble,angiotensin-converting enzyme 2 protein was found to inhibit the entry of ppSARS-2 in Huh7.5 cells effectively.Furthermore,the neutralization results for ppSARS-2 were consistent with those of live SARS-CoV-2 and determined using the serum samples fromconvalescent patients.In conclusion,we have developed an easily accessible and reliable tool for studying the neutralizing efficiency of antibodies against SARS-CoV-2 and the entry process of the virus in a BSL-2 laboratory.展开更多
基金This work was supported by the Emergency Project from the Science&Technology Commission of Chongqing(cstc2020jscx-fyzx0053)a Major National Science&Technology Program grant(2017ZX10202203)from the Science&Technology Commission of China,the Leading Talent Program of CQ CSTC(CSTCCXLJRC201719)+1 种基金the Scientific Research Innovation Project for Postgraduate in Chongqing(CYB19168)the Emergency Project for Novel Coronavirus Pneumonia from the Chongqing Medical University(CQMUNCP0302).
文摘Severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)is the causative virus of the coronavirus disease 2019(COVID-19)pandemic.To establish a safe and convenient assay system for studying entry inhibitors and neutralizing antibodies against SARS-CoV-2,we constructed a codon-optimized,full-length C-terminal mutant spike(S)gene of SARS-CoV-2.We generated a luciferase(Luc)-expressing pseudovirus containing the wild-type or mutant S protein of SARS-CoV-2 in the envelope-defective HIV-1 backbone.The key parameters for this pseudovirus-based assay,including the S mutants and virus incubation time,were optimized.This pseudovirus contains a Luc reporter gene that enabled us to easily quantify virus entry into angiotensin-converting enzyme 2(ACE2)-expressing 293T cells.Cathepsin(Cat)B/L inhibitor E64d could significantly block SARS-CoV-2 pseudovirus infection in 293T-ACE2 cells.Furthermore,the SARS-CoV-2 spike pseudotyped virus could be neutralized by sera from convalescent COVID-19 patients or recombinant ACE2 with the fused Fc region of human IgG1.Thus,we developed a pseudovirus-based assay for SARS-CoV-2,which will be valuable for evaluating viral entry inhibitors and neutralizing antibodies against this highly pathogenic virus.
基金supported by grants from the National Natural Science Foundation of China (81202568, 81473256, and 81273561)the National Science and Technology Major Project (2015ZX09102-023-004)+1 种基金the CAMS Innovation Fund for Medical Sciences (2016-I2M-1–014)the Beijing Key Laboratory of New Drug Mechanisms and Pharmacological Evaluation Study (BZ0150)
文摘Filoviruses cause severe and fatal viral hemorrhagic fever in humans. Filovirus research has been extensive since the 2014 Ebola outbreak. Due to their high pathogenicity and mortality, live filoviruses require Biosafety Level-4(BSL-4) facilities, which have restricted the development of anti-filovirus vaccines and drugs.An HIV-based pseudovirus cell infection assay is widely used for viral entry studies in BSL-2 conditions. Here,we successfully constructed nine in vitro pseudo-filovirus models covering all filovirus genera and three in vivo pseudo-filovirus-infection mouse models using Ebola virus, Marburg virus, and Lloviu virus as representative viruses. The pseudo-filovirus-infected mice showed visualizing bioluminescence in a dose-dependent manner. A bioluminescence peak in mice was reached on day 5 post-infection for Ebola virus and Marburg virus and on day4 post-infection for Lloviu virus. Two known filovirus entry inhibitors, clomiphene and toremiphene, were used to validate the model. Collectively, our study shows that all genera of filoviruses can be well-pseudotyped and are infectious in vitro. The pseudo-filovirus-infection mouse models can be used for in vivo activity evaluation of anti-filovirus drugs. This sequential in vitro and in vivo evaluation system of filovirus entry inhibitors provides a secure and efficient platform for screening and assessing anti-filovirus agents in BSL-2 facilities.
文摘目的建立西尼罗病毒(West Nile virus,WNV)假病毒的体内感染模型,并对抗体WNV-XH1进行体内中和活性评价。方法利用前期建立的可包装WNV假病毒的稳定细胞株制备假病毒上清,将上清浓缩后感染BHK21细胞检测假病毒滴度。假病毒经腹腔注入C57BL/J小鼠体内后进行生物发光成像,观测小鼠体内假病毒感染情况。同时感染后取血,ELISA检测小鼠血清中NS1水平。利用建立的小鼠感染模型,评价抗体WNV-XH1的体内功能活性。结果C57BL/J小鼠感染WNV假病毒后,体内可检测到荧光,且感染假病毒的小鼠与未感染的小鼠相比外周血血清中WNV非结构蛋白NS1水平明显升高(1.453±0.09vs0.305±0.018)。攻毒前静脉给予抗体WNV-XH1后,小鼠体内荧光信号减弱,血清中NS1水平下降(0.384±0.015)。结论成功建立WNV假病毒体内感染模型,并证实抗体WNV-XH1在体内对WNV假病毒的感染具有保护作用。
基金support this work:The National Key Research and Development Program of China(No.2016YFD0500301,No.2020YFC0842100)the National Major Project for Control and Pre-vention of Infectious Disease in China(No.2018ZX10101002).
文摘With the development of the COVID-19 epidemic,there is an urgent need to establish a system for determining the effectiveness and neutralizing activity of vaccine candidates in biosafety level 2(BSL-2)facilities.Previously,researchers had developed a pseudotyped virus systemfor SARS-CoV andMERS-CoV,based onHIV-1 core,bearing virus spike protein.During the development of a pseudotyped SARS-CoV-2 system,a eukaryotic expression plasmid expressing SARSCoV-2 spike(S)protein was constructed and then co-transfectedwith HIV-1 based plasmid which containing the firefly luciferase reporter gene,into HEK293T cells to prepare the pseudotyped SARS-CoV-2 virus(ppSARS-2).We have successfully established the pseudotyped SARS-CoV-2 system for neutralization and entry inhibition assays.Huh7.5 cell line was found to be the most susceptible to our pseudotyped virus model.Different levels of neutralizing antibodies were detected in convalescent serum samples of COVID-19 patients using ppSARS-2.The recombinant,soluble,angiotensin-converting enzyme 2 protein was found to inhibit the entry of ppSARS-2 in Huh7.5 cells effectively.Furthermore,the neutralization results for ppSARS-2 were consistent with those of live SARS-CoV-2 and determined using the serum samples fromconvalescent patients.In conclusion,we have developed an easily accessible and reliable tool for studying the neutralizing efficiency of antibodies against SARS-CoV-2 and the entry process of the virus in a BSL-2 laboratory.
