Current serum neutralization assays based on the inhibition of the cytopathic effect（Nt-CPE） need to ma nipulate live viruses, which are time-consuming, labor-intensive, and have the potential exposure to infectious...Current serum neutralization assays based on the inhibition of the cytopathic effect（Nt-CPE） need to ma nipulate live viruses, which are time-consuming, labor-intensive, and have the potential exposure to infectious agents, so a safe and objective assay via pseudovirus for the fast and efficient detection of enterovirus 71（EV71） neutralizing antibodies was developed. First, we generated EV71 pseudovirus containing firefly luciferase gene in place of the capsid gene P1 in EV71 genome. Vero cells infected with 200 CCID50（50% cell culture infective dose） of EV71 pseudovirus for 24 h were found to have the best performance. Seval sera were measured by EV71 pseudoparticle neutralization assay（Nt-PPN） and the conventional serological method Nt-CPE. Neutralizing antibody titers measured by Nt-PPN and those obtained by Nt-CPE demonstrate a high correlation between the two methods. Overall, the PPN assay represents a valid alternative to conventional serological methods for the evaluation of EV71 neutralizing anti bodies. This method can be used for detecting neutralizing antibodies of other picornaviruses, such as hepatitis A vi rus（HAV） and coxsackievirus 16（CVA16）, and make it possible to determine whether there is cross-reactivity be tween EV71 and CVA16.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
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
The use of antiviral drugs such as influenza neuraminidase (NA) inhibitors is a critical strategy to prevent and control flu pandemic, but this strategy faces the challenge of emerging drug-resistant strains. For a hi...The use of antiviral drugs such as influenza neuraminidase (NA) inhibitors is a critical strategy to prevent and control flu pandemic, but this strategy faces the challenge of emerging drug-resistant strains. For a highly pathogenic avian influenza (HPAI) H5N1 virus, biosafety restrictions have significantly limited the efforts to monitor its drug responses and mechanisms involved. In this study, a rapid and biosafe assay based on NA pseudovirus was developed to study the resistance of HPAI H5N1 virus to NA inhibitor drugs. The H5N1 NA pseudovirus was comprehensively tested using oseltamivir-sensitive strains and their resistant mutants. Results were consistent with those in previous studies, in which live H5N1 viruses were used. Several oseltamivir-resistant mutations reported in human H1N1 were also identified to cause decreased oseltamivir sensitivity in H5N1 NA by using the H5N1 NA pseudovirus. Thus, H5N1 NA pseudoviruses could be used to monitor HPAI H5N1 drug resistance rapidly and safely.展开更多
The spread of coronavirus disease 2019(COVID-19)throughout the world has resulted in stressful healthcare burdens and global health crises.Developing an effective measure to protect people from infection is an urgent ...The spread of coronavirus disease 2019(COVID-19)throughout the world has resulted in stressful healthcare burdens and global health crises.Developing an effective measure to protect people from infection is an urgent need.The blockage of interaction between angiotensin-converting enzyme 2(ACE2)and S protein is considered an essential target for anti-severe acute respiratory syndrome coronavirus 2(SARS-Co V-2)drugs.A full-length ACE2 protein could be a potential drug to block early entry of SARS-Co V-2 into host cells.In this study,a therapeutic strategy was developed by using extracellular vesicles(EVs)with decoy receptor ACE2 for neutralization of SARS-Co V-2.The EVs embedded with engineered ACE2(EVs-ACE2)were prepared;the EVs-ACE2 were derived from an engineered cell line with stable ACE2 expression.The potential effect of the EVs-ACE2 on anti-SARS-Co V-2 was demonstrated by both in vitro and in vivo neutralization experiments using the pseudovirus with the S protein(S-pseudovirus).EVs-ACE2 can inhibit the infection of S-pseudovirus in various cells,and importantly,the mice treated with intranasal administration of EVs-ACE2 can suppress the entry of S-pseudovirus into the mucosal epithelium.Therefore,the intranasal EVs-ACE2 could be a preventive medicine to protect from SARS-Co V-2 infection.This EVs-based strategy offers a potential route to COVID-19 drug development.展开更多
Even with implementation of current influenza vaccines,influenza still claims up to 500,000 lives worldwide annually,indicating a need for a better vaccine strategy.We have developed a technology to generate unique S_...Even with implementation of current influenza vaccines,influenza still claims up to 500,000 lives worldwide annually,indicating a need for a better vaccine strategy.We have developed a technology to generate unique S_(60)-HA1 pseudovirus nanoparticles(PVNPs)that display the receptor-binding HA1 domains of influenza viruses.Each self-assembled S_(60)-HA1 PVNP consists of a T=1 icosahedral S_(60) nanoparticle that resembles the inner shell of norovirus capsid and 60 surface-displayed HA1 antigens that are excellent vaccine targets.Soluble S_(60)-HA1 PVNPs presenting HA1 antigens of H7N9 influenza virus subtypes have been produced efficiently in large amount.Their three-dimensional(3D)structures have been solved by cryogenic electron microscopy.The PVNP-displayed HA1 antigens react with HA-specific antibody,and retain authentic sialic acid binding specificity and hemagglutinate human erythrocytes.The PVNPs are highly immunogenic,eliciting high titers of HA1-specific antibodies in mice and the mouse sera strongly inhibited hemagglutinations of homologous and heterologous influenza virus HA proteins.Therefore,the S_(60)-HA1 PVNPs may provide useful reagents to study influenza viruses and offer a potential new vaccine tactic to fight the deadly influenza disease.展开更多
The coronavirus disease 2019(COVID‐19)is still causing a wide range of infections and deaths due to the high variability of the severe acute respiratory syndrome coronavirus 2(SARS‐CoV‐2).Therefore,it is necessary ...The coronavirus disease 2019(COVID‐19)is still causing a wide range of infections and deaths due to the high variability of the severe acute respiratory syndrome coronavirus 2(SARS‐CoV‐2).Therefore,it is necessary to establish a reliable and convenient pseudovirus‐based neutralization assay to develop drug targeted variants of SARS‐CoV‐2.Based on the HIV‐1 backbone,we generated a high titer luciferase(Luc)‐expressing pseudovirus packaging system.Three dominant S mutant substitution pseudovirus were also established and identified compared to wide type in hACE2‐overexpressingHEK‐293T cells(293T‐ACE2 cells).Compared to serine protease inhibitor camostat mesylate,the cysteine protease inhibitor E‐64d could significantly block all SARS‐CoV‐2 mutant S pseudovirus infection in 293T‐ACE2 cells.Furthermore,the neutralization ability of two antibodies targeted receptor‐binding domain(RBD)of SARS‐CoV‐2 spike protein(S)was evaluated,which showeddifferent inhibition dose–effect curves among four types of S pseudovirus.Overall,we developed a pseudovirus‐based neutralization assay for SARS‐CoV‐2,which would be readily adapted to SARS‐CoV‐2 variants for evaluating antibodies.展开更多
The serum samples and corresponding cervical swabs were collected from 50 women with genital warts from Tianjin city, China. The neutralizing antibodies against HPV-16, -18, -58, -45, -6 and -11 in serum samples were ...The serum samples and corresponding cervical swabs were collected from 50 women with genital warts from Tianjin city, China. The neutralizing antibodies against HPV-16, -18, -58, -45, -6 and -11 in serum samples were tested by using pseudovirus-based neutralization assays and HPV DNAs in cervical swabs were also tested by using a typing kit that can detect 21 types of HPV. The results revealed that 36% （18/50） of sera were positive for type-specific neutralizing antibodies with a titer range of 160-2560, of which 22%（11/50）, 12%（6/50）, 10%（5/50）, 4%（2/50）, 4%（2/50） and 2%（1/50） were against HPVs -6, -16, -18, -58, -45 and -1 l, respectively. Additionally, 60% （30/50） of samples were HPV DNA-positive, in which the most common types detected were HPV-68（18%）, HPV-16（14%）, HPV-58（12%）, HPV-33（8%） and HPV-6, HPV-11, HPV-18 and HPV-52 （6% each）. The concordance between HPV DNA and corresponding neutralizing antibodies was 56% （28/50） with a significant difference （P〈0.05）. The full-length sequences of five HPV types （HPV -42, -52, -53, -58 and -68） were determined and exhibited 98%-100% identities with their reported genomes. The present data may have utility for investigating the natural history of HPV infection and promote the development of HPV vaccines.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
The pandemic caused by severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)has led to unprecedented social and economic disruption.Many nucleic acid testing(NAT)laboratories in China have been established to co...The pandemic caused by severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)has led to unprecedented social and economic disruption.Many nucleic acid testing(NAT)laboratories in China have been established to control the epidemic better.This proficiency testing(PT)aims to evaluate the participants’performance in qualitative and quantitative SARS-CoV-2 NAT and to explore the factors that contribute to differences in detection capabilities.Two different concentrations of RNA samples(A,B)were used for quantitative PT.Pseudovirus samples D,E(different concentrations)and negative sample(F)were used for qualitative PT.50 data sets were reported for qualitative PT,of which 74.00%were entirely correct for all samples.Fortytwo laboratories participated in the quantitative PT.37 submitted all gene results,of which only 56.76%were satisfactory.For qualitative detection,it is suggested that laboratories should strengthen personnel training,select qualified detection kits,and reduce cross-contamination to improve detection accuracy.For quantitative detection,the results of the reverse transcription digital PCR(RT-dPCR)method were more comparable and reliable than those of reverse transcription quantitative PCR(RT-qPCR).The copy number concentration of ORF1ab and N in samples A and B scattered in 85,223,50,and 106 folds,respectively.The differences in the quantitative result of RT-qPCR was mainly caused by the non-standard use of reference materials and the lack of personnel operating skills.Comparing the satisfaction of participants participating in both quantitative and qualitative proficiency testing,95.65%of the laboratories with satisfactory quantitative results also judged the qualitative results correctly,while 85.71%of the laboratories with unsatisfactory quantitative results were also unsatisfied with their qualitative judgments.Therefore,the quantitative ability is the basis of qualitative judgment.Overall,participants from hospitals reported more satisfactory results than those from enterprises and universities.Therefore,surveillance,daily qualitiy control and standardized operating procedures should be strengthened to improve the capability of SARS-CoV-2 NAT.展开更多
基金Supported by the National Natural Science Foundation of China(No.20872048)
文摘Current serum neutralization assays based on the inhibition of the cytopathic effect（Nt-CPE） need to ma nipulate live viruses, which are time-consuming, labor-intensive, and have the potential exposure to infectious agents, so a safe and objective assay via pseudovirus for the fast and efficient detection of enterovirus 71（EV71） neutralizing antibodies was developed. First, we generated EV71 pseudovirus containing firefly luciferase gene in place of the capsid gene P1 in EV71 genome. Vero cells infected with 200 CCID50（50% cell culture infective dose） of EV71 pseudovirus for 24 h were found to have the best performance. Seval sera were measured by EV71 pseudoparticle neutralization assay（Nt-PPN） and the conventional serological method Nt-CPE. Neutralizing antibody titers measured by Nt-PPN and those obtained by Nt-CPE demonstrate a high correlation between the two methods. Overall, the PPN assay represents a valid alternative to conventional serological methods for the evaluation of EV71 neutralizing anti bodies. This method can be used for detecting neutralizing antibodies of other picornaviruses, such as hepatitis A vi rus（HAV） and coxsackievirus 16（CVA16）, and make it possible to determine whether there is cross-reactivity be tween EV71 and CVA16.
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.
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 the Chinese Ministry of Science and Technology(http://www.most.gov.cn/eng/)under Major National earmark Project for Infectious Diseases(2013ZX10004611-002)the National Basic Research Program(973 program)(No.2009CB918503)the National Foundation of Talent Youth(31125016 to T.J.).
文摘The use of antiviral drugs such as influenza neuraminidase (NA) inhibitors is a critical strategy to prevent and control flu pandemic, but this strategy faces the challenge of emerging drug-resistant strains. For a highly pathogenic avian influenza (HPAI) H5N1 virus, biosafety restrictions have significantly limited the efforts to monitor its drug responses and mechanisms involved. In this study, a rapid and biosafe assay based on NA pseudovirus was developed to study the resistance of HPAI H5N1 virus to NA inhibitor drugs. The H5N1 NA pseudovirus was comprehensively tested using oseltamivir-sensitive strains and their resistant mutants. Results were consistent with those in previous studies, in which live H5N1 viruses were used. Several oseltamivir-resistant mutations reported in human H1N1 were also identified to cause decreased oseltamivir sensitivity in H5N1 NA by using the H5N1 NA pseudovirus. Thus, H5N1 NA pseudoviruses could be used to monitor HPAI H5N1 drug resistance rapidly and safely.
基金support of National Special Project for Significant Drugs Development(2018ZX09711002-010-002,China)National Natural Science Foundation of China(81925035 and 81521005,China)+3 种基金Shanghai Sci-Tech Innovation Initiative(19431903100,18430740800,China)the Shanghai Collaborative Innovation Group of Early Diagnosis and Precise Treatment of Hemangiomas and Vascular Malformations(SSMUZDCX20180701,China)the Sanofi-SIBS Yong Faculty Award,and The Youth Innovation Promotion Association。
文摘The spread of coronavirus disease 2019(COVID-19)throughout the world has resulted in stressful healthcare burdens and global health crises.Developing an effective measure to protect people from infection is an urgent need.The blockage of interaction between angiotensin-converting enzyme 2(ACE2)and S protein is considered an essential target for anti-severe acute respiratory syndrome coronavirus 2(SARS-Co V-2)drugs.A full-length ACE2 protein could be a potential drug to block early entry of SARS-Co V-2 into host cells.In this study,a therapeutic strategy was developed by using extracellular vesicles(EVs)with decoy receptor ACE2 for neutralization of SARS-Co V-2.The EVs embedded with engineered ACE2(EVs-ACE2)were prepared;the EVs-ACE2 were derived from an engineered cell line with stable ACE2 expression.The potential effect of the EVs-ACE2 on anti-SARS-Co V-2 was demonstrated by both in vitro and in vivo neutralization experiments using the pseudovirus with the S protein(S-pseudovirus).EVs-ACE2 can inhibit the infection of S-pseudovirus in various cells,and importantly,the mice treated with intranasal administration of EVs-ACE2 can suppress the entry of S-pseudovirus into the mucosal epithelium.Therefore,the intranasal EVs-ACE2 could be a preventive medicine to protect from SARS-Co V-2 infection.This EVs-based strategy offers a potential route to COVID-19 drug development.
基金The research described in this study was supported by the National Institute of Allergy and Infectious Diseases(NIAID,No.R56 AI148426-01A1 to M.T.)Cincinnati Children Hospital Medical Center(CCHMC,Innovation Funds 2018-2020,GAP Fund 2020-2021,and Research Innovation and Pilot Grant 2020-2021 to M.T.)+1 种基金the Center for Clinical and Translational Science and Training(CCTST)of the University of Cincinnati College of Medicine(Pilot Collaborative Studies Grant 2018-2019 to M.T.)that was supported by the National Center for Advancing Translational Sciences of the National Institutes of Health(No.UL1TR001425).
文摘Even with implementation of current influenza vaccines,influenza still claims up to 500,000 lives worldwide annually,indicating a need for a better vaccine strategy.We have developed a technology to generate unique S_(60)-HA1 pseudovirus nanoparticles(PVNPs)that display the receptor-binding HA1 domains of influenza viruses.Each self-assembled S_(60)-HA1 PVNP consists of a T=1 icosahedral S_(60) nanoparticle that resembles the inner shell of norovirus capsid and 60 surface-displayed HA1 antigens that are excellent vaccine targets.Soluble S_(60)-HA1 PVNPs presenting HA1 antigens of H7N9 influenza virus subtypes have been produced efficiently in large amount.Their three-dimensional(3D)structures have been solved by cryogenic electron microscopy.The PVNP-displayed HA1 antigens react with HA-specific antibody,and retain authentic sialic acid binding specificity and hemagglutinate human erythrocytes.The PVNPs are highly immunogenic,eliciting high titers of HA1-specific antibodies in mice and the mouse sera strongly inhibited hemagglutinations of homologous and heterologous influenza virus HA proteins.Therefore,the S_(60)-HA1 PVNPs may provide useful reagents to study influenza viruses and offer a potential new vaccine tactic to fight the deadly influenza disease.
基金This work was supported by grants 2020‐SKT‐14 and 2021‐YKT‐01 from the Shanghai Institute for Food and Drug ControlLX‐2021‐06 from the Shanghai Drug Administration19DZ2294600 from the Shanghai Science and Technology Committee.
文摘The coronavirus disease 2019(COVID‐19)is still causing a wide range of infections and deaths due to the high variability of the severe acute respiratory syndrome coronavirus 2(SARS‐CoV‐2).Therefore,it is necessary to establish a reliable and convenient pseudovirus‐based neutralization assay to develop drug targeted variants of SARS‐CoV‐2.Based on the HIV‐1 backbone,we generated a high titer luciferase(Luc)‐expressing pseudovirus packaging system.Three dominant S mutant substitution pseudovirus were also established and identified compared to wide type in hACE2‐overexpressingHEK‐293T cells(293T‐ACE2 cells).Compared to serine protease inhibitor camostat mesylate,the cysteine protease inhibitor E‐64d could significantly block all SARS‐CoV‐2 mutant S pseudovirus infection in 293T‐ACE2 cells.Furthermore,the neutralization ability of two antibodies targeted receptor‐binding domain(RBD)of SARS‐CoV‐2 spike protein(S)was evaluated,which showeddifferent inhibition dose–effect curves among four types of S pseudovirus.Overall,we developed a pseudovirus‐based neutralization assay for SARS‐CoV‐2,which would be readily adapted to SARS‐CoV‐2 variants for evaluating antibodies.
文摘The serum samples and corresponding cervical swabs were collected from 50 women with genital warts from Tianjin city, China. The neutralizing antibodies against HPV-16, -18, -58, -45, -6 and -11 in serum samples were tested by using pseudovirus-based neutralization assays and HPV DNAs in cervical swabs were also tested by using a typing kit that can detect 21 types of HPV. The results revealed that 36% （18/50） of sera were positive for type-specific neutralizing antibodies with a titer range of 160-2560, of which 22%（11/50）, 12%（6/50）, 10%（5/50）, 4%（2/50）, 4%（2/50） and 2%（1/50） were against HPVs -6, -16, -18, -58, -45 and -1 l, respectively. Additionally, 60% （30/50） of samples were HPV DNA-positive, in which the most common types detected were HPV-68（18%）, HPV-16（14%）, HPV-58（12%）, HPV-33（8%） and HPV-6, HPV-11, HPV-18 and HPV-52 （6% each）. The concordance between HPV DNA and corresponding neutralizing antibodies was 56% （28/50） with a significant difference （P〈0.05）. The full-length sequences of five HPV types （HPV -42, -52, -53, -58 and -68） were determined and exhibited 98%-100% identities with their reported genomes. The present data may have utility for investigating the natural history of HPV infection and promote the development of HPV vaccines.
基金supported by the National Key R&D Program of China(2022YFE0210300,2022YFC2303401,2016YFD0500300,2021YFC0863300,and 2021YFC2300101)the National Natural Science Foundation of China(32070407)the special fund for Science and Technology Innovation Teams of Shanxi Province(202204051001022)。
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.
基金NIM(National Institute of Metrology,China)(AKYZZ2126/AKYYJ2009).
文摘The pandemic caused by severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)has led to unprecedented social and economic disruption.Many nucleic acid testing(NAT)laboratories in China have been established to control the epidemic better.This proficiency testing(PT)aims to evaluate the participants’performance in qualitative and quantitative SARS-CoV-2 NAT and to explore the factors that contribute to differences in detection capabilities.Two different concentrations of RNA samples(A,B)were used for quantitative PT.Pseudovirus samples D,E(different concentrations)and negative sample(F)were used for qualitative PT.50 data sets were reported for qualitative PT,of which 74.00%were entirely correct for all samples.Fortytwo laboratories participated in the quantitative PT.37 submitted all gene results,of which only 56.76%were satisfactory.For qualitative detection,it is suggested that laboratories should strengthen personnel training,select qualified detection kits,and reduce cross-contamination to improve detection accuracy.For quantitative detection,the results of the reverse transcription digital PCR(RT-dPCR)method were more comparable and reliable than those of reverse transcription quantitative PCR(RT-qPCR).The copy number concentration of ORF1ab and N in samples A and B scattered in 85,223,50,and 106 folds,respectively.The differences in the quantitative result of RT-qPCR was mainly caused by the non-standard use of reference materials and the lack of personnel operating skills.Comparing the satisfaction of participants participating in both quantitative and qualitative proficiency testing,95.65%of the laboratories with satisfactory quantitative results also judged the qualitative results correctly,while 85.71%of the laboratories with unsatisfactory quantitative results were also unsatisfied with their qualitative judgments.Therefore,the quantitative ability is the basis of qualitative judgment.Overall,participants from hospitals reported more satisfactory results than those from enterprises and universities.Therefore,surveillance,daily qualitiy control and standardized operating procedures should be strengthened to improve the capability of SARS-CoV-2 NAT.
