Avian metapneumovirus(aMPV),a paramyxovirus,causes acute respiratory diseases in turkeys and swollen head syndrome in chickens.This study established a reverse genetics system for aMPV subtype B LN16-A strain based on...Avian metapneumovirus(aMPV),a paramyxovirus,causes acute respiratory diseases in turkeys and swollen head syndrome in chickens.This study established a reverse genetics system for aMPV subtype B LN16-A strain based on T7 RNA polymerase.Full-length cDNA of the LN16-A strain was constructed by assembling 5 cDNA fragments between the T7 promoter and hepatitis delta virus ribozyme.Transfection of this plasmid,along with the supporting plasmids encoding the N,P,M2-1,and L proteins of LN16-A into BSR-T7/5 cells,resulted in the recovery of aMPV subtype B.To identify an effective insertion site,the enhanced green fluorescent protein(EGFP)gene was inserted into different sites of the LN16-A genome to generate recombinant LN16-As.The results showed that the expression levels of EGFP at the site between the G and L genes of LN16-A were significantly higher than those at the other two sites(between the leader and N genes or replacing the SH gene).To verify the availability of the site between G and L for foreign gene expression,the VP2 gene of very virulent infectious bursal disease virus(vvIBDV)was inserted into this site,and recombinant LN16-A(rLN16A-vvVP2)was successfully rescued.Single immunization of specificpathogen-free chickens with rLN16A-vvVP2 induced high levels of neutralizing antibodies and provided 100%protection against the virulent aMPV subtype B and vvIBDV.Establishing a reverse genetics system here provides an important foundation for understanding aMPV pathogenesis and developing novel vector vaccines.展开更多
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, or- der Mononegavirales （Miller et al., 2...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, or- der Mononegavirales （Miller et al., 2010）. Although all isolated NDV strains belong to a single serotype, epi- demiological studies have revealed that the genotype VII strain is currently the most prevalent circulating geno- type worldwide and is associated with many of the most recent outbreaks in China since 1997 （Liu et al., 2007）.展开更多
The antigenic variation of influenza A virus hemagglutinin (HA) glycoproteins requires frequent changes in vaccine formulation. The new strategy of creating influenza seed strains for vaccine production is to generate...The antigenic variation of influenza A virus hemagglutinin (HA) glycoproteins requires frequent changes in vaccine formulation. The new strategy of creating influenza seed strains for vaccine production is to generate 7 + 1 reassortants that contain seven genes from a high-yield virus A/Puerto Rico/8/34[A/PR/8/34](H1N1) and the HA gene from the circulating strains. By using this DNA-based cotransfection technique, we generated 7 + 1 reassortants rgH3N1 which had the antigenic determinants of influenza virus A/Songbird/HongKong/102/00[SB/HK/01](H3N8) and 7 other genes from A/PR/ 8/34. The hemagglutinin of A/Songbird/HongKong/102/00 is 96.3% homologous to that of A/Equine/Jilin/98[Eq/Jl/89] (H3N8). The resulting virus rgH3N1 grows to high HA titers in chicken embryonated eggs, allowing vaccine preparation in unconcentrated allantoic fluid. The rgH3N1 is stable after multiple passages in embryonated eggs. The reassortant rgH3N1 virus could be used as vaccine candidate to reduce the reemergence of equine influenza outbreaks.展开更多
Unveiling the molecular mechanisms underlying rotavirus replication and pathogenesis has been hampered by the lack of a reverse genetics(RG)system in the past.Since 2017,multiple plasmid-based RG systems for simian,hu...Unveiling the molecular mechanisms underlying rotavirus replication and pathogenesis has been hampered by the lack of a reverse genetics(RG)system in the past.Since 2017,multiple plasmid-based RG systems for simian,human,and murine-like rotaviruses have been established.However,none of the described methods have supported the recovery of bovine rotaviruses(BRVs).Here,we established an optimized plasmid-based RG system for BRV culture-adapted strain(BRV G10P[15]BLR)and clinical isolates(BRV G6P[1]C73,G10P[11]HM26)based on a BHK-T7 cell clone stably expressing T7 polymerase.Furthermore,using this optimized RG system,we successfully rescued the reporter virus BRV rC73/Zs,rHM26/Zs and rBLR/Zs,harboring a genetically modified 1.8-kb segment 7 encoding full-length nonstructural protein 3(NSP3)fused to ZsGreen,a 232-amino acid green fluorescent protein.Analysis of the stability of genomic insertions showed that the rC73/Zs and rBLR/Zs replicated efficiently and were genetically stable in seven rounds of serial passaging,while rHM26/Zs can be stabilized only up to the third generation,indicating that the BRV segment composition may influence the viral fitness.In addition,we adopted the recombinant reporter viruses for high-throughput screening application and discovered 12 candidates out of 1440 compounds with potential antiviral activities against rotavirus.In summary,this improved RG system of BRVs represents an important tool with great potential for understanding the molecular biology of BRV and facilitates the development of novel therapeutics and vaccines for BRV.展开更多
Over the past decades,oncolytic viruses have emerged as prominent therapeutic agents with significant potential for anticancer treatment.Enteroviruses(EVs)have garnered particular attention due to their specific tropi...Over the past decades,oncolytic viruses have emerged as prominent therapeutic agents with significant potential for anticancer treatment.Enteroviruses(EVs)have garnered particular attention due to their specific tropism for various types of tumor cells.The rapid advancement of reverse genetics has enabled its application in the genetic modification of enteroviruses and the investigation of viral infection mechanisms.The utilization of reverse genetics has significantly enhanced our understanding of the infection mechanisms and pathogenesis of enteroviruses,while concurrently facilitating the development of translational therapies related to these viruses.In this review,we summarize the progress in the application of reverse genetics to oncolytic enteroviruses and their potential clinical applications.Specifically,we discuss the characteristics of EVs and the applications of reverse genetics in EV research.We review the utilization of reverse genetics in mechanistic investigations of EVs and in exploring the oncolytic potential of EVs.Further,we discuss the oncolytic roles of specific EVs including EV-A71,coxsackievirus B3(CV-B3),echovirus 7(Echo-7),CV-A21,and poliovirus.Our review highlights the advances in oncolytic therapy utilizing EVs with specific tumor tropism,which holds significant potential for immunotherapy.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
Homeobox transcription factors participate in the growth and development of plants by regulating cell differentiation, morphogenesis and environmental signal response. To reveal the functions of these transcription fa...Homeobox transcription factors participate in the growth and development of plants by regulating cell differentiation, morphogenesis and environmental signal response. To reveal the functions of these transcription factors in rice, we constructed the RNAi vectors of OsHox9, a member of homeobox family, and analyzed the function of OsHox9 using reverse genetics. The plant height and tillering number of RNAi transgenic plants decreased compared with those of wild-type plants. Reverse transcdption-polymerase chain reaction analysis showed that OsHox9 expression reduced in the transgenic plants with phenotypic variance, whereas that in the transgenic plants without phenotypic variance was similar to that in the wild-type plants. This result suggests that the phenotypes of the transgenic plants were caused by RNAi effects. The tissue-specificity of OsHox9 expression indicated that it was expressed in different organs, with high expression in stem apical medstem and young panicles. Subcellular location of OsHox9 demonstrated that it was localized on the cell membrane.展开更多
Zebrafish（Danio rerio） is a well-established vertebrate animal model.A comprehensive collection of reverse genetics tools has been developed for studying gene function in this useful organism.Morpholino is the most ...Zebrafish（Danio rerio） is a well-established vertebrate animal model.A comprehensive collection of reverse genetics tools has been developed for studying gene function in this useful organism.Morpholino is the most widely used reagent to knock down target gene expression post-transcriptionally.For a long time,targeted genome modification has been heavily relied on large-scale traditional forward genetic screens,such as ENU（N-ethyl-N-nitrosourea） mutagenesis derived TILLING（Targeting Induced Local Lesions IN Genomes） strategy and pseudo-typed retrovirus mediated insertional mutagenesis.Recently,engineered endonucleases,including ZFNs（zinc finger nucleases） and TALENs（transcription activator-like effector nucleases）,provide new and efficient strategies to directly generate site-specific indel mutations by inducing double strand breaks in target genes.Here we summarize the major reverse genetic approaches for loss-of-function studies used and emerging in zebrafish,including strategies based on genome-wide mutagenesis and methods for site-specific gene targeting.Future directions and expectations will also be discussed.展开更多
The live attenuated hepatitis A virus vaccine H2 strain was developed by passaging a wild-type H2w isolate in cell cultures.Currently,the mechanism underlying its attenuation phenotype remain largely unknown.In this s...The live attenuated hepatitis A virus vaccine H2 strain was developed by passaging a wild-type H2w isolate in cell cultures.Currently,the mechanism underlying its attenuation phenotype remain largely unknown.In this study,we generated a full-length infectious cDNA clone of the H2 strain using in-fusion techniques.The recovered H2 strain(H2ic)from the cDNA clone exhibited an efficient replication in both the hepatoma cell line Huh7.5.1 and the 2BS cell line used for vaccine production,similar to the parental H2 strain.Additionally,H2ic did not cause disease in Ifnar1-/- C57 mice,consistent with the H2 strain.To explore the cell-adaptive mutations of the H2 strain,chimeric viruses were generated by replacing its non-structural proteins with corresponding regions from H2w using the infectious cDNA clone as a genetic backbone.The chimeric viruses carrying the 3C or 3D proteins from H2w showed decreased replication in Huh7.5.1 and 2BS cell lines compared to H2ic.Other chimeric viruses containing the 2B,2C,or 3A proteins from H2w failed to be recovered.Furthermore,there were no significant differences in disease manifestation in mice between H2ic and the recovered chimeric viruses.These results demonstrate that adaptive mutations in the 2B,2C,and 3A proteins are essential for efficient replication of the H2 strain in cell cultures.Mutations in the 3C and 3D proteins contribute to enhanced replication in cell cultures but did not influence the attenuated phenotypes in mice.Together,this study presents the first reverse genetic system of the H2 strain and identifies viral proteins essential for adaptation to cell cultures.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
To obtain the helper plasmids for a reverse genetics system of rabies virus, the cDNAs of the complete open reading frames of the N, P, G, and L genes of rabies street virus stain HN10 were each cloned into expression...To obtain the helper plasmids for a reverse genetics system of rabies virus, the cDNAs of the complete open reading frames of the N, P, G, and L genes of rabies street virus stain HN10 were each cloned into expression vector pVAX1, These four plasmids were identified by restriction enzyme digestion and gene sequencing. The plasmid encoding the N protein was selected to determine the expression effect of these plasmids in NA cells. The results showed that the helper plasmids for a reverse genetics system of rabies street virus strain HN10 had been successfully constructed.展开更多
Immunomodulatory cancer therapy is witnessing the rise of viral immunotherapy.The oncolytic influenza A virus,although promising in preclinical investigations,remains to be implemented in clinical practice.Recent prog...Immunomodulatory cancer therapy is witnessing the rise of viral immunotherapy.The oncolytic influenza A virus,although promising in preclinical investigations,remains to be implemented in clinical practice.Recent progress in genetic engineering,coupled with experiential insights,offers opportunities to enhance the therapeutic efficacy of the influenza A virus.This review explores the use of the influenza virus,its attenuated forms,and associated vaccines in cancer immunotherapy,highlighting their respective advantages and challenges.We further elucidate methods for engineering influenza viruses and innovative approaches to augment them with cytokines or immune checkpoint inhibitors,aiming to maximize their clinical impact.Our goal is to provide insights essential for refining influenza A virus-based viral tumor immunotherapies.展开更多
Technology development has always been one of the forces driving breakthroughs in biomedical research. Since the time of Thomas Morgan, Drosophilists have, step by step, developed powerful genetic tools for manipulati...Technology development has always been one of the forces driving breakthroughs in biomedical research. Since the time of Thomas Morgan, Drosophilists have, step by step, developed powerful genetic tools for manipulating and functionally dissecting the Drosophila genome, but room for improving these technologies and developing new techniques is still large, especially today as biologists start to study systematically the functional genomics of different model organisms, including humans, in a high-throughput manner. Here, we report, for the first time in Drosophila, a rapid, easy, and highly specific method for modifying the Drosophila genome at a very high efficiency by means of an improved transcription activator-like effector nuclease （TALEN） strategy. We took advantage of the very recently developed ＂unit assembly＂ strategy to assemble two pairs of specific TALENs designed to modify the yellow gene （on the sex chromosome） and a novel autosomal gene. The mRNAs of TALENs were subsequently injected into Drosophila embryos. From 31.2% of the injected Fo fertile flies, we detected inheritable modification involving the yellow gene. The entire process from construction of specific TALENs to detection of inheritable modifications can be accomplished within one month. The potential applications of this TALEN-mediated genome modification method in Drosophila are discussed.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
One influenza H3N2 virus, A/swine/Shandong/3/2005 (Sw/SD/3/2005), was isolated from pigs with respiratory disease on a farm in eastern China. Genetic analysis revealed that Sw/SD/3/2005 was a triple-reassortant virus ...One influenza H3N2 virus, A/swine/Shandong/3/2005 (Sw/SD/3/2005), was isolated from pigs with respiratory disease on a farm in eastern China. Genetic analysis revealed that Sw/SD/3/2005 was a triple-reassortant virus with a PB2 gene from human-like H1N1, NS from classical swine H1N1, and the remaining genes from human-like H3N2 virus. These findings further support the concept that swine can serve as reservoir or mixing vessels of influenza virus strains and maintain genetic and antigenic stability of viruses. Furthermore, we have successfully established a reverse genetics system based on eight plasmids and rescued Sw/SD/3/2005 through cell transfection. HI tests and RT-PCR confirmed that the rescued virus maintained the biological properties of the wild type Sw/SD/3/2005. The successful establishment of the reverse genetics system of Sw/SD/3/2005 will enable us to conduct extensive studies of the molecular evolution of H3N2 influenza viruses in swine.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
The reverse genetics for classical swine fever virus （CSFV） is currently based on the transfection of in vitro transcribed RNA from a viral genomic cDNA clone, which is inefficient and time-consuming. This study was...The reverse genetics for classical swine fever virus （CSFV） is currently based on the transfection of in vitro transcribed RNA from a viral genomic cDNA clone, which is inefficient and time-consuming. This study was aimed to develop an improved method for rapid recovery of CSFV directly from cloned cDNA. Full-length genomic cDNA from the CSFV Shimen strain, which was flanked by a T7 promoter, the hepatitis delta virus ribozyme and T7 terminator sequences, was cloned into the low- copy vector pOK12, producing pOKShimen-RzTФ. Direct transfection of pOKShimen-RzTqb into PK/T7 cells, a PK-15- derived cell line stably expressing bacteriophage T7 RNA polymerase, allowed CSFV to be rescued rapidly and efficiently, i.e., at least 12 h faster and 31.6-fold greater viral titer when compared with the in vitro transcription-based rescue system. Furthermore, the progeny virus rescued from PK/T7 cells was indistinguishable, both in vitro and in vivo, from its parent virus and the virus rescued from classical reverse genetics. The reverse genetics based on intracellular transcription is efficient, convenient and cost-effective. The PK/T7 cell line can be used to rescue CSFV directly from cloned cDNA and it can also be used as an intracellular transcription and expression system for studying the structure and function of viral genes.展开更多
One of the major objectives of the Human Genome Project is to understand the biological function of the gene and genome as well as to develop clinical applications for human diseases. For this purpose, the experimenta...One of the major objectives of the Human Genome Project is to understand the biological function of the gene and genome as well as to develop clinical applications for human diseases. For this purpose, the experimental validations and preclinical trails by using animal models are indispensable. The mouse （Mus musculus） is one of the best animal models because genetics is well established in the mouse and embryonic manipulation technologies are also well developed. Large-scale mouse mutagenesis projects have been conducted to develop various mouse models since 1997. Originally, the phenotype-driven mutagenesis with N-ethyl-N-nitrosourea （ENU） has been the major efforts internationally then knockout/conditional mouse projects and gene-driven mutagenesis have been following. At the beginning, simple monogenic traits in the experimental condition have been elucidated. Then, more complex traits with variety of environmental interactions and gene-to-gene interactions （epistasis） have been challenged with mutant mice. In addition, chromosomal substitution strains and collaborative cross strains are also available to elucidate the complex traits in the mouse. Altogether, mouse models with mutagenesis and various laboratory strains will accelerate the studies of functional genomics in the mouse as well as in human.展开更多
基金supported by the grants from the National Key Research and Development Program of China(2022YFD1800604)the China Agriculture Research System(CARS-41)the Heilongjiang Touyan Innovation Team Program,China。
文摘Avian metapneumovirus(aMPV),a paramyxovirus,causes acute respiratory diseases in turkeys and swollen head syndrome in chickens.This study established a reverse genetics system for aMPV subtype B LN16-A strain based on T7 RNA polymerase.Full-length cDNA of the LN16-A strain was constructed by assembling 5 cDNA fragments between the T7 promoter and hepatitis delta virus ribozyme.Transfection of this plasmid,along with the supporting plasmids encoding the N,P,M2-1,and L proteins of LN16-A into BSR-T7/5 cells,resulted in the recovery of aMPV subtype B.To identify an effective insertion site,the enhanced green fluorescent protein(EGFP)gene was inserted into different sites of the LN16-A genome to generate recombinant LN16-As.The results showed that the expression levels of EGFP at the site between the G and L genes of LN16-A were significantly higher than those at the other two sites(between the leader and N genes or replacing the SH gene).To verify the availability of the site between G and L for foreign gene expression,the VP2 gene of very virulent infectious bursal disease virus(vvIBDV)was inserted into this site,and recombinant LN16-A(rLN16A-vvVP2)was successfully rescued.Single immunization of specificpathogen-free chickens with rLN16A-vvVP2 induced high levels of neutralizing antibodies and provided 100%protection against the virulent aMPV subtype B and vvIBDV.Establishing a reverse genetics system here provides an important foundation for understanding aMPV pathogenesis and developing novel vector vaccines.
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, or- der Mononegavirales （Miller et al., 2010）. Although all isolated NDV strains belong to a single serotype, epi- demiological studies have revealed that the genotype VII strain is currently the most prevalent circulating geno- type worldwide and is associated with many of the most recent outbreaks in China since 1997 （Liu et al., 2007）.
文摘The antigenic variation of influenza A virus hemagglutinin (HA) glycoproteins requires frequent changes in vaccine formulation. The new strategy of creating influenza seed strains for vaccine production is to generate 7 + 1 reassortants that contain seven genes from a high-yield virus A/Puerto Rico/8/34[A/PR/8/34](H1N1) and the HA gene from the circulating strains. By using this DNA-based cotransfection technique, we generated 7 + 1 reassortants rgH3N1 which had the antigenic determinants of influenza virus A/Songbird/HongKong/102/00[SB/HK/01](H3N8) and 7 other genes from A/PR/ 8/34. The hemagglutinin of A/Songbird/HongKong/102/00 is 96.3% homologous to that of A/Equine/Jilin/98[Eq/Jl/89] (H3N8). The resulting virus rgH3N1 grows to high HA titers in chicken embryonated eggs, allowing vaccine preparation in unconcentrated allantoic fluid. The rgH3N1 is stable after multiple passages in embryonated eggs. The reassortant rgH3N1 virus could be used as vaccine candidate to reduce the reemergence of equine influenza outbreaks.
基金supported by the Heilongjiang Provincial Natural Science Foundation of China(grant no.LH2033C107)the National Key Research and Development Program of China(2023YFD1801302)the Central Public-interest Scientific Institution Basal Research Fund(grant no.1610302022010).
文摘Unveiling the molecular mechanisms underlying rotavirus replication and pathogenesis has been hampered by the lack of a reverse genetics(RG)system in the past.Since 2017,multiple plasmid-based RG systems for simian,human,and murine-like rotaviruses have been established.However,none of the described methods have supported the recovery of bovine rotaviruses(BRVs).Here,we established an optimized plasmid-based RG system for BRV culture-adapted strain(BRV G10P[15]BLR)and clinical isolates(BRV G6P[1]C73,G10P[11]HM26)based on a BHK-T7 cell clone stably expressing T7 polymerase.Furthermore,using this optimized RG system,we successfully rescued the reporter virus BRV rC73/Zs,rHM26/Zs and rBLR/Zs,harboring a genetically modified 1.8-kb segment 7 encoding full-length nonstructural protein 3(NSP3)fused to ZsGreen,a 232-amino acid green fluorescent protein.Analysis of the stability of genomic insertions showed that the rC73/Zs and rBLR/Zs replicated efficiently and were genetically stable in seven rounds of serial passaging,while rHM26/Zs can be stabilized only up to the third generation,indicating that the BRV segment composition may influence the viral fitness.In addition,we adopted the recombinant reporter viruses for high-throughput screening application and discovered 12 candidates out of 1440 compounds with potential antiviral activities against rotavirus.In summary,this improved RG system of BRVs represents an important tool with great potential for understanding the molecular biology of BRV and facilitates the development of novel therapeutics and vaccines for BRV.
基金supported in part by funding from the National Natural Science Foundation of China(32222005,82372226,and 82172246)the Open Project of Key Laboratory of Organ Regeneration and Transplantation,Ministry of Education,Program for JLU Science and Technology Innovative Research Team(2017TD-08)the Fundamental Research Funds for Central Universities.
文摘Over the past decades,oncolytic viruses have emerged as prominent therapeutic agents with significant potential for anticancer treatment.Enteroviruses(EVs)have garnered particular attention due to their specific tropism for various types of tumor cells.The rapid advancement of reverse genetics has enabled its application in the genetic modification of enteroviruses and the investigation of viral infection mechanisms.The utilization of reverse genetics has significantly enhanced our understanding of the infection mechanisms and pathogenesis of enteroviruses,while concurrently facilitating the development of translational therapies related to these viruses.In this review,we summarize the progress in the application of reverse genetics to oncolytic enteroviruses and their potential clinical applications.Specifically,we discuss the characteristics of EVs and the applications of reverse genetics in EV research.We review the utilization of reverse genetics in mechanistic investigations of EVs and in exploring the oncolytic potential of EVs.Further,we discuss the oncolytic roles of specific EVs including EV-A71,coxsackievirus B3(CV-B3),echovirus 7(Echo-7),CV-A21,and poliovirus.Our review highlights the advances in oncolytic therapy utilizing EVs with specific tumor tropism,which holds significant potential for immunotherapy.
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supported by the National Natural Science Foundation of China (Grant NO. 31171515)the Tianjin Natural Science Foundation of China (Grant NO. 11JCZDJC17900)the Knowledge Innovation and Training Program of Tianjin, Tianjin Municipal Education Commission, China (Grant NO. 2013-1-2015 -12)
文摘Homeobox transcription factors participate in the growth and development of plants by regulating cell differentiation, morphogenesis and environmental signal response. To reveal the functions of these transcription factors in rice, we constructed the RNAi vectors of OsHox9, a member of homeobox family, and analyzed the function of OsHox9 using reverse genetics. The plant height and tillering number of RNAi transgenic plants decreased compared with those of wild-type plants. Reverse transcdption-polymerase chain reaction analysis showed that OsHox9 expression reduced in the transgenic plants with phenotypic variance, whereas that in the transgenic plants without phenotypic variance was similar to that in the wild-type plants. This result suggests that the phenotypes of the transgenic plants were caused by RNAi effects. The tissue-specificity of OsHox9 expression indicated that it was expressed in different organs, with high expression in stem apical medstem and young panicles. Subcellular location of OsHox9 demonstrated that it was localized on the cell membrane.
基金partially supported by the grants from National Program on Key Basic Research Project(973 program)(Nos.2012CB945101 and 201 ICBAO 1000)National Natural Science Foundation of China(NSFC)(Nos. 31110103904 and 30730056)
文摘Zebrafish（Danio rerio） is a well-established vertebrate animal model.A comprehensive collection of reverse genetics tools has been developed for studying gene function in this useful organism.Morpholino is the most widely used reagent to knock down target gene expression post-transcriptionally.For a long time,targeted genome modification has been heavily relied on large-scale traditional forward genetic screens,such as ENU（N-ethyl-N-nitrosourea） mutagenesis derived TILLING（Targeting Induced Local Lesions IN Genomes） strategy and pseudo-typed retrovirus mediated insertional mutagenesis.Recently,engineered endonucleases,including ZFNs（zinc finger nucleases） and TALENs（transcription activator-like effector nucleases）,provide new and efficient strategies to directly generate site-specific indel mutations by inducing double strand breaks in target genes.Here we summarize the major reverse genetic approaches for loss-of-function studies used and emerging in zebrafish,including strategies based on genome-wide mutagenesis and methods for site-specific gene targeting.Future directions and expectations will also be discussed.
基金funding from the National Key Research and Development Project of China(2021YFC2300202)the National Natural Science Foundation of China(82171820)+2 种基金C.-F.Q.received support from the National Science Fund for Distinguished Young Scholar(81925025)the Innovative Research Group(81621005)from the National Natural Science Foundation Chinathe Innovation Fund for Medical Sciences(No.2019-I2M-5-049)from the Chinese Academy of Medical Sciences.
文摘The live attenuated hepatitis A virus vaccine H2 strain was developed by passaging a wild-type H2w isolate in cell cultures.Currently,the mechanism underlying its attenuation phenotype remain largely unknown.In this study,we generated a full-length infectious cDNA clone of the H2 strain using in-fusion techniques.The recovered H2 strain(H2ic)from the cDNA clone exhibited an efficient replication in both the hepatoma cell line Huh7.5.1 and the 2BS cell line used for vaccine production,similar to the parental H2 strain.Additionally,H2ic did not cause disease in Ifnar1-/- C57 mice,consistent with the H2 strain.To explore the cell-adaptive mutations of the H2 strain,chimeric viruses were generated by replacing its non-structural proteins with corresponding regions from H2w using the infectious cDNA clone as a genetic backbone.The chimeric viruses carrying the 3C or 3D proteins from H2w showed decreased replication in Huh7.5.1 and 2BS cell lines compared to H2ic.Other chimeric viruses containing the 2B,2C,or 3A proteins from H2w failed to be recovered.Furthermore,there were no significant differences in disease manifestation in mice between H2ic and the recovered chimeric viruses.These results demonstrate that adaptive mutations in the 2B,2C,and 3A proteins are essential for efficient replication of the H2 strain in cell cultures.Mutations in the 3C and 3D proteins contribute to enhanced replication in cell cultures but did not influence the attenuated phenotypes in mice.Together,this study presents the first reverse genetic system of the H2 strain and identifies viral proteins essential for adaptation to cell cultures.
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National High Technology Research and Development Program of China (2006AA02Z110, 2007AA02Z402)Major Program of the National Natural Science Foundation of China (30630049)
文摘To obtain the helper plasmids for a reverse genetics system of rabies virus, the cDNAs of the complete open reading frames of the N, P, G, and L genes of rabies street virus stain HN10 were each cloned into expression vector pVAX1, These four plasmids were identified by restriction enzyme digestion and gene sequencing. The plasmid encoding the N protein was selected to determine the expression effect of these plasmids in NA cells. The results showed that the helper plasmids for a reverse genetics system of rabies street virus strain HN10 had been successfully constructed.
基金supported by the Leading Innovative and Entrepreneur Team Introduction Program of Zhejiang(No.2022R01002)the National Natural Science Foundation of China(Nos.82272300 and 82102893)the Fundamental Research Funds for the Central Universities(No.226-2024-00062),China.
文摘Immunomodulatory cancer therapy is witnessing the rise of viral immunotherapy.The oncolytic influenza A virus,although promising in preclinical investigations,remains to be implemented in clinical practice.Recent progress in genetic engineering,coupled with experiential insights,offers opportunities to enhance the therapeutic efficacy of the influenza A virus.This review explores the use of the influenza virus,its attenuated forms,and associated vaccines in cancer immunotherapy,highlighting their respective advantages and challenges.We further elucidate methods for engineering influenza viruses and innovative approaches to augment them with cytokines or immune checkpoint inhibitors,aiming to maximize their clinical impact.Our goal is to provide insights essential for refining influenza A virus-based viral tumor immunotherapies.
基金supported by the grants from the 973 Program(Nos.2009CB918702 and 2012CB945101)the NSFC(Nos.31071087 and 31100889)+1 种基金W.-M.D.is supported by NIH grant R01GM072562National Science Foundation of USA(IOS-1052333)
文摘Technology development has always been one of the forces driving breakthroughs in biomedical research. Since the time of Thomas Morgan, Drosophilists have, step by step, developed powerful genetic tools for manipulating and functionally dissecting the Drosophila genome, but room for improving these technologies and developing new techniques is still large, especially today as biologists start to study systematically the functional genomics of different model organisms, including humans, in a high-throughput manner. Here, we report, for the first time in Drosophila, a rapid, easy, and highly specific method for modifying the Drosophila genome at a very high efficiency by means of an improved transcription activator-like effector nuclease （TALEN） strategy. We took advantage of the very recently developed ＂unit assembly＂ strategy to assemble two pairs of specific TALENs designed to modify the yellow gene （on the sex chromosome） and a novel autosomal gene. The mRNAs of TALENs were subsequently injected into Drosophila embryos. From 31.2% of the injected Fo fertile flies, we detected inheritable modification involving the yellow gene. The entire process from construction of specific TALENs to detection of inheritable modifications can be accomplished within one month. The potential applications of this TALEN-mediated genome modification method in Drosophila are discussed.
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.
文摘One influenza H3N2 virus, A/swine/Shandong/3/2005 (Sw/SD/3/2005), was isolated from pigs with respiratory disease on a farm in eastern China. Genetic analysis revealed that Sw/SD/3/2005 was a triple-reassortant virus with a PB2 gene from human-like H1N1, NS from classical swine H1N1, and the remaining genes from human-like H3N2 virus. These findings further support the concept that swine can serve as reservoir or mixing vessels of influenza virus strains and maintain genetic and antigenic stability of viruses. Furthermore, we have successfully established a reverse genetics system based on eight plasmids and rescued Sw/SD/3/2005 through cell transfection. HI tests and RT-PCR confirmed that the rescued virus maintained the biological properties of the wild type Sw/SD/3/2005. The successful establishment of the reverse genetics system of Sw/SD/3/2005 will enable us to conduct extensive studies of the molecular evolution of H3N2 influenza viruses in swine.
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.
基金supported by the National Basic Research Program of China (2005CB523202)
文摘The reverse genetics for classical swine fever virus （CSFV） is currently based on the transfection of in vitro transcribed RNA from a viral genomic cDNA clone, which is inefficient and time-consuming. This study was aimed to develop an improved method for rapid recovery of CSFV directly from cloned cDNA. Full-length genomic cDNA from the CSFV Shimen strain, which was flanked by a T7 promoter, the hepatitis delta virus ribozyme and T7 terminator sequences, was cloned into the low- copy vector pOK12, producing pOKShimen-RzTФ. Direct transfection of pOKShimen-RzTqb into PK/T7 cells, a PK-15- derived cell line stably expressing bacteriophage T7 RNA polymerase, allowed CSFV to be rescued rapidly and efficiently, i.e., at least 12 h faster and 31.6-fold greater viral titer when compared with the in vitro transcription-based rescue system. Furthermore, the progeny virus rescued from PK/T7 cells was indistinguishable, both in vitro and in vivo, from its parent virus and the virus rescued from classical reverse genetics. The reverse genetics based on intracellular transcription is efficient, convenient and cost-effective. The PK/T7 cell line can be used to rescue CSFV directly from cloned cDNA and it can also be used as an intracellular transcription and expression system for studying the structure and function of viral genes.
基金supported by Grants-in-Aid for Scientific Research (A) (Nos. KAKENHI 15200032 and KAKENHI 21240043)
文摘One of the major objectives of the Human Genome Project is to understand the biological function of the gene and genome as well as to develop clinical applications for human diseases. For this purpose, the experimental validations and preclinical trails by using animal models are indispensable. The mouse （Mus musculus） is one of the best animal models because genetics is well established in the mouse and embryonic manipulation technologies are also well developed. Large-scale mouse mutagenesis projects have been conducted to develop various mouse models since 1997. Originally, the phenotype-driven mutagenesis with N-ethyl-N-nitrosourea （ENU） has been the major efforts internationally then knockout/conditional mouse projects and gene-driven mutagenesis have been following. At the beginning, simple monogenic traits in the experimental condition have been elucidated. Then, more complex traits with variety of environmental interactions and gene-to-gene interactions （epistasis） have been challenged with mutant mice. In addition, chromosomal substitution strains and collaborative cross strains are also available to elucidate the complex traits in the mouse. Altogether, mouse models with mutagenesis and various laboratory strains will accelerate the studies of functional genomics in the mouse as well as in human.
