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
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
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
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
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
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
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
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
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
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
There are only eight approved small molecule antiviral drugs for treating COVID-19.Among them,four are nucleotide analogues(remdesivir,JT001,molnupiravir,and azvudine),while the other four are protease inhibitors(nirm...There are only eight approved small molecule antiviral drugs for treating COVID-19.Among them,four are nucleotide analogues(remdesivir,JT001,molnupiravir,and azvudine),while the other four are protease inhibitors(nirmatrelvir,ensitrelvir,leritrelvir,and simnotrelvir-ritonavir).Antiviral resistance,unfavourable drug‒drug interaction,and toxicity have been reported in previous studies.Thus there is a dearth of new treatment options for SARS-CoV-2.In this work,a three-tier cell-based screening was employed to identify novel compounds with anti-SARS-CoV-2 activity.One compound,designated 172,demonstrated broad-spectrum antiviral activity against multiple human pathogenic coronaviruses and different SARS-CoV-2 variants of concern.Mechanistic studies validated by reverse genetics showed that compound 172 inhibits the 3-chymotrypsin-like protease(3CLpro)by binding to an allosteric site and reduces 3CLpro dimerization.A drug synergistic checkerboard assay demonstrated that compound 172 can achieve drug synergy with nirmatrelvir in vitro.In vivo studies confirmed the antiviral activity of compound 172 in both Golden Syrian Hamsters and K18 humanized ACE2 mice.Overall,this study identified an alternative druggable site on the SARS-CoV-23CLpro,proposed a potential combination therapy with nirmatrelvir to reduce the risk of antiviral resistance and shed light on the development of allosteric protease inhibitors for treating a range of coronavirus diseases.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
The G-H loop of the foot-and-mouth disease virus（FMDV） virion contains certain dominant immunogenic epitopes, as well as an arginine-glycine-aspartic acid（RGD） motif that is recognized by cell surface integrin rec...The G-H loop of the foot-and-mouth disease virus（FMDV） virion contains certain dominant immunogenic epitopes, as well as an arginine-glycine-aspartic acid（RGD） motif that is recognized by cell surface integrin receptors. Previous experiments indicate that it is critical to maintain virus structural integrity when inserting an exogenous epitope into the surface of an FMDV structural protein. However, it remains to be determined how factors such as different insertion positions affect interactions among the virus, cells and host immune system. In this study, one infectious c DNA clone of the swine FMDV Cathay topotype strain O/CHA/90 was constructed. Then, a FLAG marker（DYKDDDDK） was inserted upstream（–4） or downstream（＋10） of the RGD motif to generate tagged viruses vFLAG-O/CHA/90 or vO/CHA/90-FLAG, investigating the possibility of expressing foreign antigen and effect on its immunogenicity. Compared to the parental virus, both tagged viruses exhibited similar plaque phenotypes, suckling mouse pathogenicity and antigenicity. Additionally, the FLAGtag insertion position did not change the use of integrin-mediated cell entry by the tagged viruses. Interestingly, both tagged vaccines protected pigs against challenge with the parental virus O/CHA/90 and induced immune responses against FMDV in BALB/c mice and pigs, but only vaccination with vFLAG-O/CHA/90 generated anti-FLAG antibodies. Our findings demonstrated that two sites（RGD–4 and RGD＋10） tolerated the insertion of an exogenous gene in the swine FMDV O/CHA/90 strain. However, only RGD–4 was a novel and appropriate inserting site which could tolerate exogenous FLAG. The resultant tagged virus is a promising candidate for FMD vaccine which can be differentiating infected from vaccinated animals（DIVA）.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
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
Identification of all genes involved in the phytochrome （phy）-medieted responses of plants to their light environment is an important goal in providing an overall understanding of light-regulated growth end developm...Identification of all genes involved in the phytochrome （phy）-medieted responses of plants to their light environment is an important goal in providing an overall understanding of light-regulated growth end development. This article highlights end integrates the central findings of two recent comprehensive studies in Arabidopsis that have identified the genome-wide set of phy-reguleted genes that respond rapidly to red-light signals upon first exposure of dark-grown seedlings, and have tested the functional relevance to normal seedling photomorphogenesis of an Initial subset of these genes. The data： （a） reveal considerable complexity in the channeling of the light signals through the different phy-femily members （phyA to phyE） to responsive genes; （b） identify a diversity of transcription-factor-encoding genes as major early, if not primary, targets of phy signaling, end, therefore, as potentially important regulators in the transcriptional-network hierarchy; and （c） identify auxin-related genes as the dominant class among rapidly-regulated, hormone-related genes. However, reverse-genetic functional profiling of a selected subset of these genes reveals that only a limited fraction are necessary for optimal phy-induced seedling deetioletion.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
The present mini-review describes newer methods and strategies, including transposon and T-DNA insertions, TILLING, Deleteagene, and RNA interference, to functionally analyze genes of interest in the model plant Arabi...The present mini-review describes newer methods and strategies, including transposon and T-DNA insertions, TILLING, Deleteagene, and RNA interference, to functionally analyze genes of interest in the model plant Arabidopsis. The relative advantages and disadvantages of the systems are also discussed.展开更多
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.
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 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 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.
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National Natural Science Foundation of China(NSFC)/Research Grants Council(RGC)Joint Research Scheme(N_HKU767/22 and 82261160398)Health and Medical Research Fund(COVID190121)+13 种基金the Food and Health Bureau,The Government of the Hong Kong Special Administrative Regionthe National Natural Science Foundation of China(32322087,32300134,and 82272337)Guangdong Natural Science Foundation(2023A1515012907)Health@-InnoHK,Innovation and Technology Commission,the Government of the Hong Kong Special Administrative Regionthe Collaborative Research Fund(C7060-21G and C7002-23Y)and Theme-Based Research Scheme(T11-709/21-N)of the Research Grants Council,The Government of the Hong Kong Special Administrative RegionPartnership Programme of Enhancing Laboratory Surveillance and Investigation of Emerging Infectious Diseases and Antimicrobial Resistance for the Department of Health of the Hong Kong Special Administrative Region GovernmentSanming Project of Medicine in Shenzhen,China(SZSM201911014)the High Level-Hospital Program,Health Commission of Guangdong Province,Chinathe research project of Hainan Academician Innovation Platform(YSPTZX202004)Emergency Collaborative Project of Guangzhou Laboratory(EKPG22-01)and the National Key R&D Program of China(projects 2021YFC0866100 and 2023YFC3041600)The University of Hong Kong Seed Fund for Collaborative Research(2207101537)and Hunan University(521119400156)donations of Providence Foundation Limited(in memory of the late Lui Hac Minh).
文摘There are only eight approved small molecule antiviral drugs for treating COVID-19.Among them,four are nucleotide analogues(remdesivir,JT001,molnupiravir,and azvudine),while the other four are protease inhibitors(nirmatrelvir,ensitrelvir,leritrelvir,and simnotrelvir-ritonavir).Antiviral resistance,unfavourable drug‒drug interaction,and toxicity have been reported in previous studies.Thus there is a dearth of new treatment options for SARS-CoV-2.In this work,a three-tier cell-based screening was employed to identify novel compounds with anti-SARS-CoV-2 activity.One compound,designated 172,demonstrated broad-spectrum antiviral activity against multiple human pathogenic coronaviruses and different SARS-CoV-2 variants of concern.Mechanistic studies validated by reverse genetics showed that compound 172 inhibits the 3-chymotrypsin-like protease(3CLpro)by binding to an allosteric site and reduces 3CLpro dimerization.A drug synergistic checkerboard assay demonstrated that compound 172 can achieve drug synergy with nirmatrelvir in vitro.In vivo studies confirmed the antiviral activity of compound 172 in both Golden Syrian Hamsters and K18 humanized ACE2 mice.Overall,this study identified an alternative druggable site on the SARS-CoV-23CLpro,proposed a potential combination therapy with nirmatrelvir to reduce the risk of antiviral resistance and shed light on the development of allosteric protease inhibitors for treating a range of coronavirus diseases.
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 the National Key Research and Development Program of China(2016YFD0501500)the Special Fund for Agro-scientific Research in the Public Interest,China(201303046)
文摘The G-H loop of the foot-and-mouth disease virus（FMDV） virion contains certain dominant immunogenic epitopes, as well as an arginine-glycine-aspartic acid（RGD） motif that is recognized by cell surface integrin receptors. Previous experiments indicate that it is critical to maintain virus structural integrity when inserting an exogenous epitope into the surface of an FMDV structural protein. However, it remains to be determined how factors such as different insertion positions affect interactions among the virus, cells and host immune system. In this study, one infectious c DNA clone of the swine FMDV Cathay topotype strain O/CHA/90 was constructed. Then, a FLAG marker（DYKDDDDK） was inserted upstream（–4） or downstream（＋10） of the RGD motif to generate tagged viruses vFLAG-O/CHA/90 or vO/CHA/90-FLAG, investigating the possibility of expressing foreign antigen and effect on its immunogenicity. Compared to the parental virus, both tagged viruses exhibited similar plaque phenotypes, suckling mouse pathogenicity and antigenicity. Additionally, the FLAGtag insertion position did not change the use of integrin-mediated cell entry by the tagged viruses. Interestingly, both tagged vaccines protected pigs against challenge with the parental virus O/CHA/90 and induced immune responses against FMDV in BALB/c mice and pigs, but only vaccination with vFLAG-O/CHA/90 generated anti-FLAG antibodies. Our findings demonstrated that two sites（RGD–4 and RGD＋10） tolerated the insertion of an exogenous gene in the swine FMDV O/CHA/90 strain. However, only RGD–4 was a novel and appropriate inserting site which could tolerate exogenous FLAG. The resultant tagged virus is a promising candidate for FMD vaccine which can be differentiating infected from vaccinated animals（DIVA）.
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 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 National Institute of Health Grant GM47475, Department of Energy Grant DE-FG03-87ER13742, and U.S. Department of Agriculture Grant 5335-21000-010-00D. Publication of this paper is supported by the National Natural Science Foundation of China （30624808） and Science Publication Foundation of the Chinese Academy of Sciences.Acknowledgements Thank the coauthors of the original research publications used as the basis for this article, and Jim Tepperman for figure preparation and help with the manuscript.
文摘Identification of all genes involved in the phytochrome （phy）-medieted responses of plants to their light environment is an important goal in providing an overall understanding of light-regulated growth end development. This article highlights end integrates the central findings of two recent comprehensive studies in Arabidopsis that have identified the genome-wide set of phy-reguleted genes that respond rapidly to red-light signals upon first exposure of dark-grown seedlings, and have tested the functional relevance to normal seedling photomorphogenesis of an Initial subset of these genes. The data： （a） reveal considerable complexity in the channeling of the light signals through the different phy-femily members （phyA to phyE） to responsive genes; （b） identify a diversity of transcription-factor-encoding genes as major early, if not primary, targets of phy signaling, end, therefore, as potentially important regulators in the transcriptional-network hierarchy; and （c） identify auxin-related genes as the dominant class among rapidly-regulated, hormone-related genes. However, reverse-genetic functional profiling of a selected subset of these genes reveals that only a limited fraction are necessary for optimal phy-induced seedling deetioletion.
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The present mini-review describes newer methods and strategies, including transposon and T-DNA insertions, TILLING, Deleteagene, and RNA interference, to functionally analyze genes of interest in the model plant Arabidopsis. The relative advantages and disadvantages of the systems are also discussed.
