The recent emergence of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)caused serious harm to human health and struck a blow to global economic development.Research on SARS-CoV-2 has greatly benefited from...The recent emergence of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)caused serious harm to human health and struck a blow to global economic development.Research on SARS-CoV-2 has greatly benefited from the use of reverse genetics systems,which have been established to artificially manipulate the viral genome,generating recombinant and reporter infectious viruses or biosafety level 2(BSL-2)-adapted non-infectious replicons with desired modifications.These tools have been instrumental in studying the molecular biological characteristics of the virus,investigating antiviral therapeutics,and facilitating the development of attenuated vaccine candidates.Here,we review the construction strategies,development,and applications of reverse genetics systems for SARS-CoV-2,which may be applied to other CoVs as well.展开更多
Dear Editor,Newcastle disease virus(NDV),also known as avian paramyxovirus serotype 1(APMV-1),is a member of the genus Avulavirus within the family Paramyxoviridae,order Mononegavirales(Miller et al.,2010).Although al...Dear Editor,Newcastle disease virus(NDV),also known as avian paramyxovirus serotype 1(APMV-1),is a member of the genus Avulavirus within the family Paramyxoviridae,order Mononegavirales(Miller et al.,2010).Although all isolated NDV strains belong to a single serotype,epidemiological studies have revealed that the genotype展开更多
Reverse genetics via targeted modification of gene sequences to obtain a phenotype and the inference of a gene's function or regulatory mechanism is widely used as a potent tool in viral biology and application.Ho...Reverse genetics via targeted modification of gene sequences to obtain a phenotype and the inference of a gene's function or regulatory mechanism is widely used as a potent tool in viral biology and application.However,while reverse genetics has contributed significantly to our understanding of molecular biology and the pathogenesis of viruses,its accessibility(operation)and openness(data)have raised many concerns regarding biosafety and biosecurity.In this review,we retrospectively examine the development of reverse genetics and its applications in virology,then emphasize global biosafety and biosecurity concerns regarding reverse genetics,and summarize global regulations,governance,and laws on reverse genetics.This review seeks to enhance our understanding and rational application of reverse genetics technology for the benefit of humankind.展开更多
Objective In China, 24 cases of human infection with highly pathogenic avian influenza(HPAI) H5 N6 virus have been confirmed since the first confirmed case in 2014. Therefore, we developed and assessed two H5 N6 candi...Objective In China, 24 cases of human infection with highly pathogenic avian influenza(HPAI) H5 N6 virus have been confirmed since the first confirmed case in 2014. Therefore, we developed and assessed two H5 N6 candidate vaccine viruses(CVVs).Methods In accordance with the World Health Organization(WHO) recommendations, we constructed two reassortant viruses using reverse genetics(RG) technology to match the two different epidemic H5 N6 viruses. We performed complete genome sequencing to determine the genetic stability. We assessed the growth ability of the studied viruses in MDCK cells and conducted a hemagglutination inhibition assay to analyze their antigenicity. Pathogenicity attenuation was also evaluated in vitro and in vivo.Results The results showed that no mutations occurred in hemagglutinin or neuraminidase, and both CVVs retained their original antigenicity. The replication capacity of the two CVVs reached a level similar to that of A/Puerto Rico/8/34 in MDCK cells. The two CVVs showed low pathogenicity in vitro and in vivo, which are in line with the WHO requirements for CVVs.Conclusion We obtained two genetically stable CVVs of HPAI H5N6 with high growth characteristics,which may aid in our preparedness for a potential H5N6 pandemic.展开更多
Recombinant Newcastle disease virus(rNDV)has shown an anti-cancer effect in preclinical studies,but has never been tested for lung cancer models.This study explored the anti-cancer activity of genetically modified NDV...Recombinant Newcastle disease virus(rNDV)has shown an anti-cancer effect in preclinical studies,but has never been tested for lung cancer models.This study explored the anti-cancer activity of genetically modified NDV expressing IL-2(rNDV–IL-2)in lung cancer models.This study used Lewis lung carcinoma cell line(LLC)to create tumor models in C57 female mice,the tumor-bearing mice were treated with rNDV-IL-2,rNDV and phosphate-buffered saline(PBS),respectively.In vitro results revealed that rNDV effectively infected malignant cells and expressed IL-2,in vivo results revealed that rNDV expressing IL-2 was highly efficient in inhibiting lung cancer tumors,with an average tumor size of 291.255 mm^3 in rNDV-IL-2 group compared to 763.068 mm^3 in rNDV group and 1101.68 mm^3 in PBS group.For the survival studies,treatment with rNDV-IL-2 enhanced the survival rates of tumor-bearing mice by 36%compared to those of rNDV treated mice and by 80%compared to those of vehicle-treated mice(survival rate:12 out of 15 for rNDV-IL-2 group;seven out of 15 for rNDV group and zero out of 15 for vehicle group).These results demonstrated that rNDV-expressed IL-2 enhanced the intrinsic anti-tumor ability of Newcastle disease virus in lung cancer models by further restrain of lung tumor growth and improvement of the survival rates of the tumor-bearing mice.The genetically modified rNDV-IL-2 was a good candidate for lung cancer therapy.展开更多
Background Mumps is a common type of respiratory infectious disease caused by mumps virus(MuV),and can be effectively prevented by vaccination.In this study,a reverse genetic system of MuV that can facilitate the rati...Background Mumps is a common type of respiratory infectious disease caused by mumps virus(MuV),and can be effectively prevented by vaccination.In this study,a reverse genetic system of MuV that can facilitate the rational design of safer,more efficient mumps vaccine candidates is established.Methods MuV-S79 cDNA clone was assembled into a full-length plasmid by means of the GeneArtTM High-Order Genetic Assembly System,and was rescued via reverse genetic technology.RT-PCR,sequencing,and immunofluorescence assays were used for rMuV-S79 authentication.Viral replication kinetics and in vivo experimental models were used to evaluate the replication,safety,and immunogenicity of rMuV-S79.Results A full-length cDNA clone of MuV-S79 in the assembly process was generated by a novel plasmid assemble strategy,and a robust reverse genetic system of MuV-S79 was successfully established.The established rMuV-S79 strain could reach a high virus titer in vitro.The average viral titer of rMuV-S79 in the lung tissues was 2.68±0.14 log10PFU/g lung tissue,and rMuV-S79 group did not induce inflammation in the lung tissues in cotton rats.Neutralizing antibody titers induced by rMuV-S79 were high,long-lasting and could provide complete protection against MuV wild strain challenge.Conclusion We have established a robust reverse genetic system of MuV-S79 which can facilitate the optimization of mumps vaccines.rMuV-S79 rescued could reach a high virus titer and the safety was proven in vivo.It could also provide complete protection against MuV wild strain challenge.展开更多
Severe fever with thrombocytopenia syndrome virus(SFTSV)is an emerging tick-borne bunyavirus that causes hemorrhagic fever-like disease(SFTS)in humans with a case fatality rate up to 30%.To date,the molecular biology ...Severe fever with thrombocytopenia syndrome virus(SFTSV)is an emerging tick-borne bunyavirus that causes hemorrhagic fever-like disease(SFTS)in humans with a case fatality rate up to 30%.To date,the molecular biology involved in SFTSV infection remains obscure.There are seven major genotypes of SFTSV(C1-C4 and J1-J3)and previously a reverse genetic system was established on a C3 strain of SFTSV.Here,we reported successfully establishment of a reverse genetics system based on a SFTSV C4 strain.First,we obtained the 5’-and 3’-terminal untranslated region(UTR)sequences of the Large(L),Medium(M)and Small(S)segments of a laboratory-adapted SFTSV C4 strain through rapid amplification of cDNA ends analysis,and developed functional T7 polymerase-based L-,M-and S-segment minigenome assays.Then,fulllength cDNA clones were constructed and infectious SFTSV were recovered from co-transfected cells.Viral infectivity,growth kinetics,and viral protein expression profile of the rescued virus were compared with the laboratory-adapted virus.Focus formation assay showed that the size and morphology of the foci formed by the rescued SFTSV were indistinguishable with the laboratory-adapted virus.However,one-step growth curve and nucleoprotein expression analyses revealed the rescued virus replicated less efficiently than the laboratory-adapted virus.Sequence analysis indicated that the difference may be due to the mutations in the laboratory-adapted strain which are more prone to cell culture.The results help us to understand the molecular biology of SFTSV,and provide a useful tool for developing vaccines and antivirals against SFTS.展开更多
Severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)has caused a devastating pandemic worldwide.Vaccines and antiviral drugs are the most promising candidates for combating this global epidemic,and scientists a...Severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)has caused a devastating pandemic worldwide.Vaccines and antiviral drugs are the most promising candidates for combating this global epidemic,and scientists all over the world have made great efforts to this end.However,manipulation of the SARS-CoV-2 should be performed in the biosafety level3 laboratory.This makes experiments complicated and time-consuming.Therefore,a safer system for working with this virus is urgently needed.Here,we report the construction of plasmid-based,non-infectious SARS-CoV-2 replicons with turbo-green fluorescent protein and/or firefly luciferase reporters by reverse genetics using transformation-associated recombination cloning in Saccharomyces cerevisiae.Replication of these replicons was achieved simply by direct transfection of cells with the replicon plasmids as evident by the expression of reporter genes.Using SARS-CoV-2 replicons,the inhibitory effects of E64-D and remdesivir on SARS-CoV-2 replication were confirmed,and the halfmaximal effective concentration(EC50)value of remdesivir and E64-D was estimated by different quantification methods respectively,indicating that these SARS-CoV-2 replicons are useful tools for antiviral drug evaluation.展开更多
SARS-CoV-2 causes the pandemic of COVID-19 and no effective drugs for this disease are available thus far.Due to the high infectivity and pathogenicity of this virus,all studies on the live virus are strictly confined...SARS-CoV-2 causes the pandemic of COVID-19 and no effective drugs for this disease are available thus far.Due to the high infectivity and pathogenicity of this virus,all studies on the live virus are strictly confined in the biosafety level 3(BSL3)laboratory but this would hinder the basic research and antiviral drug development of SARS-CoV-2 because the BSL3 facility is not commonly available and the work in the containment is costly and laborious.In this study,we constructed a reverse genetics system of SARS-CoV-2 by assembling the viral cDNA in a bacterial artificial chromosome(BAC)vector with deletion of the spike(S)gene.Transfection of the cDNA into cells results in the production of an RNA replicon that keeps the capability of genome or subgenome replication but is deficient in virion assembly and infection due to the absence of S protein.Therefore,such a replicon system is not infectious and can be used in ordinary biological laboratories.We confirmed the efficient replication of the replicon by demonstrating the expression of the subgenomic RNAs which have similar profiles to the wild-type virus.By mutational analysis of nsp12 and nsp14,we showed that the RNA polymerase,exonuclease,and cap N7 methyltransferase play essential roles in genome replication and sgRNA production.We also created a SARS-CoV-2 replicon carrying a luciferase reporter gene and this system was validated by the inhibition assays with known anti-SARS-CoV-2 inhibitors.Thus,such a one-plasmid system is biosafe and convenient to use,which will benefit both fundamental research and development of antiviral drugs.展开更多
Coxsackievirus A10(CVA10)is one of the major etiological agents of hand,foot,and mouth disease.There are no vaccine and antiviral drugs for controlling CVA10 infection.Reverse genetic tools for CVA10 will benefit its ...Coxsackievirus A10(CVA10)is one of the major etiological agents of hand,foot,and mouth disease.There are no vaccine and antiviral drugs for controlling CVA10 infection.Reverse genetic tools for CVA10 will benefit its mechanistic study and development of vaccines and antivirals.Here,two infectious clones for the prototype and a Myc-tagged CVA10 were constructed.Viable CVA10 viruses were harvested by transfecting the viral m RNA into human rhabdomyosarcoma(RD)cells.Rescued CVA10 was further confirmed by next generation sequencing and characterized experimentally.We also constructed the vectors for CVA10 subgenomic replicon with luciferase reporter and viral capsid with EGFP reporter,respectively.Co-transfection of the viral replicon RNA and capsid expresser in human embryonic kidney 293 T(HEK293 T)cells led to the production of single round infectious particles(SRIPs).Based on CVA10 replicon RNA,SRIPs with either the enterovirus A71(EVA71)capsid or the CVA10 capsid were generated.Infection by EVA71 SRIPs required SCARB2,while CVA10 SRIPs did not.Finally,we showed great improvement of the replicon activity and SRIPs production by insertion of a cis-active hammerhead ribozyme(HHRib)before the 50-untranslated region(UTR).In summary,reverse genetic tools for prototype strain of CVA10,including both the infectious clone and the SRIPs system,were successfully established.These tools will facilitate the basic and translational study of CVA10.展开更多
Species in the microalgal genus Nannochloropsis are increasingly used as models for theoretical and applied studies. Herewe attempt to generate InDei variations in the genome of Nannochloropsis oceanica, and then deci...Species in the microalgal genus Nannochloropsis are increasingly used as models for theoretical and applied studies. Herewe attempt to generate InDei variations in the genome of Nannochloropsis oceanica, and then decipher the genetic basisof its economic and biological traits with bulked mutant analysis modified from bulked segregant analysis. In addition, wedescribe our efforts to construct site-tagged and gene-traceable mutant libraries to clone its genes through reverse geneticapproaches. Currently, more than a half of N. oceanica protein-encoding genes are annotated against databanks. However, nofunctional gene has been de novo cloned from N. oceanica and no new function has been assigned to any of its annotatablegenes. Here, we discuss the possible methods and potential benefits of de novo cloning of N. oceanica genes.展开更多
Getah virus(GETV)is a mosquito-borne virus of the genus Alphavirus in the family Togaviridae and,in recent years,it has caused several outbreaks in animals.The molecular basis for GETV pathogenicity is not well unders...Getah virus(GETV)is a mosquito-borne virus of the genus Alphavirus in the family Togaviridae and,in recent years,it has caused several outbreaks in animals.The molecular basis for GETV pathogenicity is not well understood.Therefore,a reverse genetic system of GETV is needed to produce genetically modified viruses for the study of the viral replication and its pathogenic mechanism.Here,we generated a CMV-driven infectious cDNA clone based on a previously isolated GETV strain,GX201808(pGETV-GX).Transfection of pGETV-GX into BHK-21 cells resulted in the recovery of a recombinant virus(rGETV-GX)which showed similar growth characteristics to its parental virus.Then three-day-old mice were experimentally infected with either the parental or recombinant virus.The recombinant virus showed milder pathogenicity than the parental virus in the mice.Based on the established CMV-driven cDNA clone,subgenomic promoter and two restriction enzyme sites(BamHI and EcoRI)were introduced into the region between E1 protein and 3’UTR.Then the green fluorescent protein(GFP),red fluorescent protein(RFP)and improved light-oxygen-voltage(iLOV)genes were inserted into the restriction enzyme sites.Transfection of the constructs carrying the reporter genes into BHK-21 cells proved the rescue of the recombinant reporter viruses.Taken together,the establishment of a reverse genetic system for GETV provides a valuable tool for the study of the virus life cycle,and to aid the development of genetically engineered GETVs as vectors for foreign gene expression.展开更多
Background To describe mumps virus(MuV)used as a vector to express enhanced green fluorescent protein(EGFP)or red fluorescent protein(RFP)genes.Methods Molecular cloning technique was applied to establish the cDNA clo...Background To describe mumps virus(MuV)used as a vector to express enhanced green fluorescent protein(EGFP)or red fluorescent protein(RFP)genes.Methods Molecular cloning technique was applied to establish the cDNA clones of recombinant mumps viruses(rMuVs).rMuVs were recovered based on our reverse genetic system of MuV-S79.The properties of rMuVs were determined by growth curve,plaque assay,fluorescent microscopy and determination of fluorescent intensity.Results Three recombinant viruses replicated well in Vero cells and similarly as parental rMuV-S79,expressed heterologous genes in high levels,and were genetically stable in at least 15 passages.Conclusion rMuV-S79 is a promising platform to accommodate foreign genes like marker genes,other antigens and immunomodulators for addressing various diseases.展开更多
基金the Major Research Plan of the National Natural Science Foundation of China(92269105)the Zhejiang Provincial Natural Science Foundation(LZ22C180002).
文摘The recent emergence of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)caused serious harm to human health and struck a blow to global economic development.Research on SARS-CoV-2 has greatly benefited from the use of reverse genetics systems,which have been established to artificially manipulate the viral genome,generating recombinant and reporter infectious viruses or biosafety level 2(BSL-2)-adapted non-infectious replicons with desired modifications.These tools have been instrumental in studying the molecular biological characteristics of the virus,investigating antiviral therapeutics,and facilitating the development of attenuated vaccine candidates.Here,we review the construction strategies,development,and applications of reverse genetics systems for SARS-CoV-2,which may be applied to other CoVs as well.
基金supported by the Innovation Foundation Project of China Animal Health and Epidemiology Center,Ministry of Agriculture,P.R.China(project no:CAHEC-2015-Y105)supported by the National Natural Scientific Fund(31272561)
文摘Dear Editor,Newcastle disease virus(NDV),also known as avian paramyxovirus serotype 1(APMV-1),is a member of the genus Avulavirus within the family Paramyxoviridae,order Mononegavirales(Miller et al.,2010).Although all isolated NDV strains belong to a single serotype,epidemiological studies have revealed that the genotype
文摘Reverse genetics via targeted modification of gene sequences to obtain a phenotype and the inference of a gene's function or regulatory mechanism is widely used as a potent tool in viral biology and application.However,while reverse genetics has contributed significantly to our understanding of molecular biology and the pathogenesis of viruses,its accessibility(operation)and openness(data)have raised many concerns regarding biosafety and biosecurity.In this review,we retrospectively examine the development of reverse genetics and its applications in virology,then emphasize global biosafety and biosecurity concerns regarding reverse genetics,and summarize global regulations,governance,and laws on reverse genetics.This review seeks to enhance our understanding and rational application of reverse genetics technology for the benefit of humankind.
基金This study was supported by the National Major Science and Technology Project for Control and Prevention of Major Infectious Diseases in China[No.2018ZX10711001,2018ZX10305409-004-002]Emergency Prevention and Control Project of Ministry of Science and Technology of China[No.10600100000015001206].
文摘Objective In China, 24 cases of human infection with highly pathogenic avian influenza(HPAI) H5 N6 virus have been confirmed since the first confirmed case in 2014. Therefore, we developed and assessed two H5 N6 candidate vaccine viruses(CVVs).Methods In accordance with the World Health Organization(WHO) recommendations, we constructed two reassortant viruses using reverse genetics(RG) technology to match the two different epidemic H5 N6 viruses. We performed complete genome sequencing to determine the genetic stability. We assessed the growth ability of the studied viruses in MDCK cells and conducted a hemagglutination inhibition assay to analyze their antigenicity. Pathogenicity attenuation was also evaluated in vitro and in vivo.Results The results showed that no mutations occurred in hemagglutinin or neuraminidase, and both CVVs retained their original antigenicity. The replication capacity of the two CVVs reached a level similar to that of A/Puerto Rico/8/34 in MDCK cells. The two CVVs showed low pathogenicity in vitro and in vivo, which are in line with the WHO requirements for CVVs.Conclusion We obtained two genetically stable CVVs of HPAI H5N6 with high growth characteristics,which may aid in our preparedness for a potential H5N6 pandemic.
基金Supported by the National Key R&D Program of China(2017YFD0501102)the National Natural Science Foundation of China(31200121)。
文摘Recombinant Newcastle disease virus(rNDV)has shown an anti-cancer effect in preclinical studies,but has never been tested for lung cancer models.This study explored the anti-cancer activity of genetically modified NDV expressing IL-2(rNDV–IL-2)in lung cancer models.This study used Lewis lung carcinoma cell line(LLC)to create tumor models in C57 female mice,the tumor-bearing mice were treated with rNDV-IL-2,rNDV and phosphate-buffered saline(PBS),respectively.In vitro results revealed that rNDV effectively infected malignant cells and expressed IL-2,in vivo results revealed that rNDV expressing IL-2 was highly efficient in inhibiting lung cancer tumors,with an average tumor size of 291.255 mm^3 in rNDV-IL-2 group compared to 763.068 mm^3 in rNDV group and 1101.68 mm^3 in PBS group.For the survival studies,treatment with rNDV-IL-2 enhanced the survival rates of tumor-bearing mice by 36%compared to those of rNDV treated mice and by 80%compared to those of vehicle-treated mice(survival rate:12 out of 15 for rNDV-IL-2 group;seven out of 15 for rNDV group and zero out of 15 for vehicle group).These results demonstrated that rNDV-expressed IL-2 enhanced the intrinsic anti-tumor ability of Newcastle disease virus in lung cancer models by further restrain of lung tumor growth and improvement of the survival rates of the tumor-bearing mice.The genetically modified rNDV-IL-2 was a good candidate for lung cancer therapy.
基金supported partly by the Natural Science Foundation for Young Scholars of Zhejiang Province(LQ19H100005).
文摘Background Mumps is a common type of respiratory infectious disease caused by mumps virus(MuV),and can be effectively prevented by vaccination.In this study,a reverse genetic system of MuV that can facilitate the rational design of safer,more efficient mumps vaccine candidates is established.Methods MuV-S79 cDNA clone was assembled into a full-length plasmid by means of the GeneArtTM High-Order Genetic Assembly System,and was rescued via reverse genetic technology.RT-PCR,sequencing,and immunofluorescence assays were used for rMuV-S79 authentication.Viral replication kinetics and in vivo experimental models were used to evaluate the replication,safety,and immunogenicity of rMuV-S79.Results A full-length cDNA clone of MuV-S79 in the assembly process was generated by a novel plasmid assemble strategy,and a robust reverse genetic system of MuV-S79 was successfully established.The established rMuV-S79 strain could reach a high virus titer in vitro.The average viral titer of rMuV-S79 in the lung tissues was 2.68±0.14 log10PFU/g lung tissue,and rMuV-S79 group did not induce inflammation in the lung tissues in cotton rats.Neutralizing antibody titers induced by rMuV-S79 were high,long-lasting and could provide complete protection against MuV wild strain challenge.Conclusion We have established a robust reverse genetic system of MuV-S79 which can facilitate the optimization of mumps vaccines.rMuV-S79 rescued could reach a high virus titer and the safety was proven in vivo.It could also provide complete protection against MuV wild strain challenge.
基金supported by grants from the National Natural Science Foundation of China(No.31900146Open Research Fund Program of the State Key Laboratory of Virology of China(No.2020IOV003)Team project of Health Commission of Hubei Province(WJ2019C003)。
文摘Severe fever with thrombocytopenia syndrome virus(SFTSV)is an emerging tick-borne bunyavirus that causes hemorrhagic fever-like disease(SFTS)in humans with a case fatality rate up to 30%.To date,the molecular biology involved in SFTSV infection remains obscure.There are seven major genotypes of SFTSV(C1-C4 and J1-J3)and previously a reverse genetic system was established on a C3 strain of SFTSV.Here,we reported successfully establishment of a reverse genetics system based on a SFTSV C4 strain.First,we obtained the 5’-and 3’-terminal untranslated region(UTR)sequences of the Large(L),Medium(M)and Small(S)segments of a laboratory-adapted SFTSV C4 strain through rapid amplification of cDNA ends analysis,and developed functional T7 polymerase-based L-,M-and S-segment minigenome assays.Then,fulllength cDNA clones were constructed and infectious SFTSV were recovered from co-transfected cells.Viral infectivity,growth kinetics,and viral protein expression profile of the rescued virus were compared with the laboratory-adapted virus.Focus formation assay showed that the size and morphology of the foci formed by the rescued SFTSV were indistinguishable with the laboratory-adapted virus.However,one-step growth curve and nucleoprotein expression analyses revealed the rescued virus replicated less efficiently than the laboratory-adapted virus.Sequence analysis indicated that the difference may be due to the mutations in the laboratory-adapted strain which are more prone to cell culture.The results help us to understand the molecular biology of SFTSV,and provide a useful tool for developing vaccines and antivirals against SFTS.
基金supported by the CAMS Innovation Fund for Medical Science(CIFMS 2016-I2M-1-014)the National Key Plan for Scientific Research and Development of China(2016YFD0500300)+1 种基金National Key R&D Program of China(2020YFA0707600)Beijing Municipal Science and Technology Project(Z201100001020005)。
文摘Severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)has caused a devastating pandemic worldwide.Vaccines and antiviral drugs are the most promising candidates for combating this global epidemic,and scientists all over the world have made great efforts to this end.However,manipulation of the SARS-CoV-2 should be performed in the biosafety level3 laboratory.This makes experiments complicated and time-consuming.Therefore,a safer system for working with this virus is urgently needed.Here,we report the construction of plasmid-based,non-infectious SARS-CoV-2 replicons with turbo-green fluorescent protein and/or firefly luciferase reporters by reverse genetics using transformation-associated recombination cloning in Saccharomyces cerevisiae.Replication of these replicons was achieved simply by direct transfection of cells with the replicon plasmids as evident by the expression of reporter genes.Using SARS-CoV-2 replicons,the inhibitory effects of E64-D and remdesivir on SARS-CoV-2 replication were confirmed,and the halfmaximal effective concentration(EC50)value of remdesivir and E64-D was estimated by different quantification methods respectively,indicating that these SARS-CoV-2 replicons are useful tools for antiviral drug evaluation.
基金supported by Grants(the National Natural Science Foundation of China#32041002,#31971161,#31900546 and#81620108020)the Guangdong Science and Technology Department(#2019A1515011332)+1 种基金the Shenzhen Science and Technology Innovation Program(JSGG20200225150431472,JCYJ20190807160615255,JCYJ20190807153203560,and KQTD20180411143323605)supported by the Guangdong Zhujiang Leading Talents Programme and the National Tenthousand Talents Program。
文摘SARS-CoV-2 causes the pandemic of COVID-19 and no effective drugs for this disease are available thus far.Due to the high infectivity and pathogenicity of this virus,all studies on the live virus are strictly confined in the biosafety level 3(BSL3)laboratory but this would hinder the basic research and antiviral drug development of SARS-CoV-2 because the BSL3 facility is not commonly available and the work in the containment is costly and laborious.In this study,we constructed a reverse genetics system of SARS-CoV-2 by assembling the viral cDNA in a bacterial artificial chromosome(BAC)vector with deletion of the spike(S)gene.Transfection of the cDNA into cells results in the production of an RNA replicon that keeps the capability of genome or subgenome replication but is deficient in virion assembly and infection due to the absence of S protein.Therefore,such a replicon system is not infectious and can be used in ordinary biological laboratories.We confirmed the efficient replication of the replicon by demonstrating the expression of the subgenomic RNAs which have similar profiles to the wild-type virus.By mutational analysis of nsp12 and nsp14,we showed that the RNA polymerase,exonuclease,and cap N7 methyltransferase play essential roles in genome replication and sgRNA production.We also created a SARS-CoV-2 replicon carrying a luciferase reporter gene and this system was validated by the inhibition assays with known anti-SARS-CoV-2 inhibitors.Thus,such a one-plasmid system is biosafe and convenient to use,which will benefit both fundamental research and development of antiviral drugs.
基金supported by the Shanghai Public Health Clinical Center(KY-GW2018-17)Shanghai Municipal Commission of Health and Family Planning(20174Y0099)。
文摘Coxsackievirus A10(CVA10)is one of the major etiological agents of hand,foot,and mouth disease.There are no vaccine and antiviral drugs for controlling CVA10 infection.Reverse genetic tools for CVA10 will benefit its mechanistic study and development of vaccines and antivirals.Here,two infectious clones for the prototype and a Myc-tagged CVA10 were constructed.Viable CVA10 viruses were harvested by transfecting the viral m RNA into human rhabdomyosarcoma(RD)cells.Rescued CVA10 was further confirmed by next generation sequencing and characterized experimentally.We also constructed the vectors for CVA10 subgenomic replicon with luciferase reporter and viral capsid with EGFP reporter,respectively.Co-transfection of the viral replicon RNA and capsid expresser in human embryonic kidney 293 T(HEK293 T)cells led to the production of single round infectious particles(SRIPs).Based on CVA10 replicon RNA,SRIPs with either the enterovirus A71(EVA71)capsid or the CVA10 capsid were generated.Infection by EVA71 SRIPs required SCARB2,while CVA10 SRIPs did not.Finally,we showed great improvement of the replicon activity and SRIPs production by insertion of a cis-active hammerhead ribozyme(HHRib)before the 50-untranslated region(UTR).In summary,reverse genetic tools for prototype strain of CVA10,including both the infectious clone and the SRIPs system,were successfully established.These tools will facilitate the basic and translational study of CVA10.
文摘Species in the microalgal genus Nannochloropsis are increasingly used as models for theoretical and applied studies. Herewe attempt to generate InDei variations in the genome of Nannochloropsis oceanica, and then decipher the genetic basisof its economic and biological traits with bulked mutant analysis modified from bulked segregant analysis. In addition, wedescribe our efforts to construct site-tagged and gene-traceable mutant libraries to clone its genes through reverse geneticapproaches. Currently, more than a half of N. oceanica protein-encoding genes are annotated against databanks. However, nofunctional gene has been de novo cloned from N. oceanica and no new function has been assigned to any of its annotatablegenes. Here, we discuss the possible methods and potential benefits of de novo cloning of N. oceanica genes.
基金funded by the Natural Science Foundation of Guangxi Province(No.2018GXNSFDA281021)the Foundation of Guangxi University(No.XGZ130959)。
文摘Getah virus(GETV)is a mosquito-borne virus of the genus Alphavirus in the family Togaviridae and,in recent years,it has caused several outbreaks in animals.The molecular basis for GETV pathogenicity is not well understood.Therefore,a reverse genetic system of GETV is needed to produce genetically modified viruses for the study of the viral replication and its pathogenic mechanism.Here,we generated a CMV-driven infectious cDNA clone based on a previously isolated GETV strain,GX201808(pGETV-GX).Transfection of pGETV-GX into BHK-21 cells resulted in the recovery of a recombinant virus(rGETV-GX)which showed similar growth characteristics to its parental virus.Then three-day-old mice were experimentally infected with either the parental or recombinant virus.The recombinant virus showed milder pathogenicity than the parental virus in the mice.Based on the established CMV-driven cDNA clone,subgenomic promoter and two restriction enzyme sites(BamHI and EcoRI)were introduced into the region between E1 protein and 3’UTR.Then the green fluorescent protein(GFP),red fluorescent protein(RFP)and improved light-oxygen-voltage(iLOV)genes were inserted into the restriction enzyme sites.Transfection of the constructs carrying the reporter genes into BHK-21 cells proved the rescue of the recombinant reporter viruses.Taken together,the establishment of a reverse genetic system for GETV provides a valuable tool for the study of the virus life cycle,and to aid the development of genetically engineered GETVs as vectors for foreign gene expression.
基金supported partly by the Natural Science Foundation for Young Scholars of Zhejiang Province(LQ19H100005).
文摘Background To describe mumps virus(MuV)used as a vector to express enhanced green fluorescent protein(EGFP)or red fluorescent protein(RFP)genes.Methods Molecular cloning technique was applied to establish the cDNA clones of recombinant mumps viruses(rMuVs).rMuVs were recovered based on our reverse genetic system of MuV-S79.The properties of rMuVs were determined by growth curve,plaque assay,fluorescent microscopy and determination of fluorescent intensity.Results Three recombinant viruses replicated well in Vero cells and similarly as parental rMuV-S79,expressed heterologous genes in high levels,and were genetically stable in at least 15 passages.Conclusion rMuV-S79 is a promising platform to accommodate foreign genes like marker genes,other antigens and immunomodulators for addressing various diseases.
