Dear Editor,Coxsackievirus A16(CA16)is one of the major viral pathogens associated with hand,foot,and mouth disease.CA16 belongs to the Enterovirus genus of the Picornaviridae family and possesses a single-stranded po...Dear Editor,Coxsackievirus A16(CA16)is one of the major viral pathogens associated with hand,foot,and mouth disease.CA16 belongs to the Enterovirus genus of the Picornaviridae family and possesses a single-stranded positivesense RNA genome(Mao et al.,2014).Reverse genetics is an important tool for CA16 research.Previously,a reverse genetics T7 polymerase-based system was de-展开更多
The biological features of most foamy viruses(FVs) are poorly understood, including bovine foamy virus(BFV). BFV strain 3026(BFV3026) was isolated from the peripheral blood mononuclear cells of an infected cow in Zhan...The biological features of most foamy viruses(FVs) are poorly understood, including bovine foamy virus(BFV). BFV strain 3026(BFV3026) was isolated from the peripheral blood mononuclear cells of an infected cow in Zhangjiakou, China. A full-length genomic clone of BFV3026 was obtained from BFV3026-infected cells, and it exhibited more than 99% amino acid(AA) homology to another BFV strain isolated in the USA. Upon transfection into fetal canine thymus cells, the full-length BFV3026 clone produced viral structural and auxiliary proteins, typical cytopathic effects, and virus particles. These results demonstrate that the full-length BFV3026 clone is fully infectious and can be used in further BFV3026 research.展开更多
Construction of infectious clones by full-length cDNA is basic and key for recovering RNA virus and is core of reverse genetics.In this article,basic consideration and key technology were viewed and factors affecting ...Construction of infectious clones by full-length cDNA is basic and key for recovering RNA virus and is core of reverse genetics.In this article,basic consideration and key technology were viewed and factors affecting infectivity of clones were also summarized.Some research advances were briefly introduced about positive-strand RNA viruses infectious clones.Finally,this article also reviewed the application of infectious clones.展开更多
The objective of this study was to construct the infectious clone of encephalomyocarditis virus (EMCV) BJC3 strain.The genomic cDNA of the virus was amplified by three overlapping segments using RT-PCR,and cloned into...The objective of this study was to construct the infectious clone of encephalomyocarditis virus (EMCV) BJC3 strain.The genomic cDNA of the virus was amplified by three overlapping segments using RT-PCR,and cloned into low-copy plasmid pWSK29 to construct the full-length cDNA clone pWSKBJC3/ w.The pWSKBJC3/w was in vitro transcribed and transfected into BHK-21 cells to rescue the virus.The results showed that the full-length cDNA clone was infectious and the virus could be rescued in BHK-21 cells.The rescued virus designated RvBJC3W was identified by RT-PCR and indirect immunofluorescence assay(IFA).The rescued virus had similar growth characteristics to its parental virus BJC3 and retained pathogenicity for mice.Our results indicate that the first infectious cDNA clone of EMCV in China has been successfully established and provides an essential tool for investigating the molecular basis of pathogenicity of EMCV.展开更多
Coronaviruses can infect humans,mammals,and birds,leading to respiratory,gastrointestinal,and neurological diseases.These viruses are significant zoonotic pathogens with nine known types capable of infecting humans.Th...Coronaviruses can infect humans,mammals,and birds,leading to respiratory,gastrointestinal,and neurological diseases.These viruses are significant zoonotic pathogens with nine known types capable of infecting humans.The coronavirus genome,approximately 30 kb in size,is the largest known ribonucleic acid(RNA)virus genome,and its complexity makes assembly and manipulation time-consuming and labor-intensive.Reverse genetic systems are widely used to engineer recombinant viruses that can be adapted at Biosafety Level 2(BSL-2)for studying viral gene function,replication,pathogenesis,vaccines,and therapeutics.The infectious clones,which enabled the recovery of various viruses after DNA recombinant technology,were indispensable tools for the reverse genetics of viruses.Various techniques for constructing infectious clones of human coronaviruses(HCoV)have been developed,encompassing methods such as vaccinia virus vectors method,in vitro ligation,bacterial artificial chromosome systems,yeast artificial chromosome systems,circular polymerase extension reaction,and the recently reported infectious sub-genomic amplicons technology.This review summarizes the status of various techniques for constructing infectious clones of human coronaviruses and related applications.展开更多
Viral infectious clones(ICs)serve as robust platforms for studying viral biology and screening antiviral agents using reverse genetics.However,the molecular profiles and complex limitations of human coronaviruses(HCoV...Viral infectious clones(ICs)serve as robust platforms for studying viral biology and screening antiviral agents using reverse genetics.However,the molecular profiles and complex limitations of human coronaviruses(HCoVs)pose a challenge to ICs development.In this study,we report a novel platform to develop the ICs for HCoV-OC43-VR1558 using a one-step assembly method in yeast by transformation-associated recombination(TAR)technology.Recombinant HCoV-OC43-VR1558,named as rOC43(1558)-WT,was rapidly generated by TAR.In addition,recombinant HCoV-OC43-VR1558-expressing reporter genes,named as rOC43(1558)-ns2FusionRluc,was also generated based on TAR by inserting the ns2 region of the IC with Renilla luciferase(Rluc).We further characterized their replication through virus titration using 50%tissue culture infective dose(TCID50)and indirect immunofluorescence assay(IFA),luciferase reporter assay,and western blotting(WB)assay.The genetic stability of the recombinant HCoV-OC43 was assessed through viral genome sequencing following passaging in BHK-21 cells.These reporter viruses were validated as screening tools for inhibitorsin vitro by evaluating the antiviral activities of remdesivir and chloroquine.The phenotypes of HCoV-OC43-VR1558 and HCoV-OC43-VR759 were comparedin vitro andin vivo.The TAR-based one-step assembly of IC was successfully applied,facilitating the rapid generation of recombinant HCoV-OC43 and providing a useful platform for the investigation of biological mechanisms,development of vaccines and diagnostic tests,and screening inhibitors of HCoVs.展开更多
Tomato leaf curl New Delhi virus(ToLCNDV)is a member of the genus Begomovirus,and causes devastating disease in the world.In recent years,ToLCNDV was rapidly spreading in China and induces severe economic losses in ag...Tomato leaf curl New Delhi virus(ToLCNDV)is a member of the genus Begomovirus,and causes devastating disease in the world.In recent years,ToLCNDV was rapidly spreading in China and induces severe economic losses in agriculture.In this study,we sequenced and characterized the complete genome of ToLCNDV isolates from melon plants showing leaf curling and stunting symptoms in Jiangsu Province of China.We constructed a full-length infectious cDNA clone of ToLCNDV,which could induce systemic infection with typical symptoms in Nicotiana benthamiana,Cit-rullus melo,and Citrullus lanatus plants through agrobacterium-mediated inoculation.Further experimental evidence demonstrated that the virions produced in plants infected with the infectious clone of ToLCNDV are biologically active and sap-transmissible.We also evaluated the resistance of commercial melon cultivars to ToLCNDV and found all testing melon cultivars were susceptible to ToLCNDV.Collectively,the reverse genetic system developed herein will facilitate further research on biological functions of proteins encoded by ToLCNDV and plant-ToLCNDV interactions,which might provide new insights into breeding resistance germplasm in crops.展开更多
Tick-borne encephalitis virus(TBEV) is a pathogenic virus known to cause central nervous system(CNS) diseases in humans, and has become an increasing public health threat nowadays. The rates of TBEV infection in the e...Tick-borne encephalitis virus(TBEV) is a pathogenic virus known to cause central nervous system(CNS) diseases in humans, and has become an increasing public health threat nowadays. The rates of TBEV infection in the endemic countries are increasing. However, there is no effective antiviral against the disease. This underscores the urgent need for tools to study the emergence and pathogenesis of TBEV and to accelerate the development of vaccines and antivirals. In this study, we reported an infectious c DNA clone of TBEV that was isolated in China(the WH2012 strain). A beta-globin intron was inserted in the coding region of nonstructural protein 1(NS1) gene to improve the stability of viral genome in bacteria. In mammalian cells, the inserted intron was excised and spliced precisely, which did not lead to the generation of inserted mutants. High titers of infectious progeny viruses were generated after the transfection of the infectious clone. The cDNA-derived TBEV replicated efficiently, and caused typical cytopathic effect(CPE) and plaques in BHK-21 cells. In addition, the CPE and growth curve of cDNA-derived virus were similar to that of its parental isolate in cells. Together, we have constructed the first infectious TBEV cDNA clone in China, and the clone can be used to investigate the genetic determinants of TBEV virulence and disease pathogenesis, and to develop countermeasures against the virus.展开更多
Dear Editor,Since April 2010,an outbreak of a new disease has elicited symptoms of high fever,loss of appetite,and reduction in egg production in layer ducks in eastern China;this phenomenon has now spread throughout ...Dear Editor,Since April 2010,an outbreak of a new disease has elicited symptoms of high fever,loss of appetite,and reduction in egg production in layer ducks in eastern China;this phenomenon has now spread throughout China(Cao et al.,2011;Su et al.,2011).The causative agent of the disease was identified as Tembusu virus(TMUV),which was classified into the genus Flavivirus,展开更多
Ligularia jaluensis is an important medicinal and ornamental plant in China.However,the viruses capable of infecting Ligularia jaluensis remains unknown.Here,we identified a novel carlavirus,tentatively named ligulari...Ligularia jaluensis is an important medicinal and ornamental plant in China.However,the viruses capable of infecting Ligularia jaluensis remains unknown.Here,we identified a novel carlavirus,tentatively named ligularia jaluensis carlavirus(LJCV),as well as a known iris severe mosaic virus(ISMV),in L.jaluensis plants displaying chlorosis and yellow ring spot symptoms,using RNA-seq analysis.The LJCV genome consists of an 8497 nt positive-sense,single-stranded RNA[excluding the poly(A)tail],and contains six open reading frames(ORFs).Phylogenetic analyses based on the fulllength genome and RNA-dependent RNA polymerase(RdRp)amino acid(aa)sequences revealed that LJCV clusters within an evolutionary branch alongside known viruses in the Carlavirus genus.The RdRp protein encoded by ORF1 of LJCV shared 45.38%–67.41%identity with the corresponding proteins of eight closely related carlaviruses.ORFs 2–4 constitute the triple gene block(TGB),with TGBp1 and TGBp3 localized in the endoplasmic reticulum(ER),while TGBp2 is localized at plasmodesmata(PD)and facilitates viral intercellular movement,as demonstrated by its ability to complement the potato virus X with movement-deficient mutant(PVX-Δp25-GFP).Additionally,ORF6 encodes a cysteine-rich protein(CRP)that is localized in the chloroplast and functions as a viral pathogenicity factor,inducing severe viral symptoms in the heterologous PVX expression system.Furthermore,we successfully constructed an infectious cDNA clone of LJCV,and found that it can infect Nicotiana benthamiana plants through mechanical inoculation or agrobacterium-mediated infiltration of the LJCV infectious clone.These findings enhance our understanding of the characteristics and host range of carlaviruses,as well as the viruses capable of infecting L.jaluensis.展开更多
Human bocavirus 1(HBoV1;family:Parvoviridae)causes a wide spectrum of respiratory diseases in children and gastroenteritis in adults.A lack of sensitive cell lines and efficient animal models hinders research on HBoV,...Human bocavirus 1(HBoV1;family:Parvoviridae)causes a wide spectrum of respiratory diseases in children and gastroenteritis in adults.A lack of sensitive cell lines and efficient animal models hinders research on HBoV,including the development of anti-HBoV drugs or vaccines.Although the construction of a wild-type HBoV1 infectious clone has been reported,generating HBoV1 infectious clone carrying foreign reporter genes with suitable insertion sites in its genome while retaining replicative ability remains challenging.Here,HBoV1 infectious clones harboring the 11-amino-acid HiBiT tag at five distinct insertion sites were constructed and evaluated.Only the recombinant HBoV1 carrying the HiBiT tag in the N-terminus of the NS1 protein(HBoV1-HiBiTNS1)displayed comparable characteristics to wild-type HBoV1 as determined via the analysis of viral DNA copy number,NanoLuc activity,viral protein expression,and the formation of replication intermediates.Notably,the replication kinetics of HBoV1-HiBiTNS1 could be examined by monitoring NanoLuc activity,which was noted to be correlated with the viral DNA level.Additionally,we successfully applied HiBiT-tagged HBoV1 for the evaluation of antiviral drug activity and identified ivermectin(EC50=2.27μM)as a potent anti-HBoV1 replication drug.Overall,our study demonstrated that the HBoV1-HiBiTNS1 reporter can serve as a convenient platform for screening candidate drugs targeting HBoV1 replication and may also be useful for investigating the life cycle of the virus.展开更多
The utilization of enteroviruses engineered with reporter genes serves as a valuable tool for advancing our understanding of enterovirus biology and its applications,enabling the development of effective therapeutic a...The utilization of enteroviruses engineered with reporter genes serves as a valuable tool for advancing our understanding of enterovirus biology and its applications,enabling the development of effective therapeutic and preventive strategies.In this study,our initial attempts to introduce a NanoLuc luciferase(NLuc)reporter gene into recombinant enteroviruses were unsuccessful in rescuing viable progenies.We hypothesized that the size of the inserted tag might be a determining factor in the rescue of the virus.Therefore,we inserted the 11-amino-acid HiBiT tag into the genomes of enterovirus A71(EV-A71),coxsackievirus A10(CVA10),coxsackievirus A7(CVA7),coxsackievirus A16(CVA16),namely EV-A71-HiBiT,CVA16-HiBiT,CVA10-HiBiT,CVA7-HiBiT,and observed that the HiBiT-tagged viruses exhibited remarkably high rescue efficiency.Notably,the HiBiT-tagged enteroviruses displayed comparable characteristics to the wild-type viruses.A direct comparison between CVA16-NLuc and CVA16-HiBiT recombinant viruses revealed that the tiny HiBiT insertion had minimal impact on virus infectivity and replication kinetics.Moreover,these HiBiT-tagged enteroviruses demonstrated high genetic stability in different cell lines over multiple passages.In addition,the HiBiT-tagged viruses were successfully tested in antiviral drug assays,and the sensitivity of the viruses to drugs was not affected by the HiBiT tag.Ultimately,our findings provide definitive evidence that the integration of HiBiT into enteroviruses presents a universal,convenient,and invaluable method for advancing research in the realm of enterovirus virology.Furthermore,HiBiT-tagged enteroviruses exhibit great potential for diverse applications,including the development of antivirals and the elucidation of viral infection mechanisms.展开更多
It has been reported that squash leaf curl China virus(SLCCNV)infects some Cucurbitaceae crops except for melon(Cucumis melo L.).A new disease of melon exhibiting severe leaf curl and dwarfing was observed in Hainan P...It has been reported that squash leaf curl China virus(SLCCNV)infects some Cucurbitaceae crops except for melon(Cucumis melo L.).A new disease of melon exhibiting severe leaf curl and dwarfing was observed in Hainan Province of China.In this study,the pathogen was identified as SLCCNV through biological and molecular characterization.The isolate(SLCCNV-HN)possess a bipartite genome,DNA-A(HM566112.1)with the highest nucleotide identity(99%)to SLCCNV-Hn(MF062251.1)pumpkin and SLCCNV-Hn61(AM260205.1)squash isolates from China,whereas DNA-B(HM566113.1)with the highest nucleotide identity(99%)to SLCCNV-Hn(MF062252.1).Phylogenetic analyses based on the full-length SLCCNV-HN DNA-A and-B sequences indicated that SLCCNV-HN melon isolate is clustered with SLCCNV-Hn pumpkin,SLCCNV-Hn61 and SLCCNV-SY squash isolates from southern China,forming an independent cluster.Infectious clone of SLCCNV-HN was constructed and the melon plants were inoculated and the infection rate is 100%,the systemic symptoms in melon showed identical to those of melon plants infected in fields.Additionally,melon plants transmission of this virus by Bemisia tabaci with a transmission rate of 95%(19/20)showed leaf curl and dwarf symptoms 15 days post transmission,thereby fulfilling Koch’s postulates.Analysis of genomic organization and phylogenetic trees indicated that SLCCNV-HN melon isolate belongs to the Begomovirus genus.To the best of our knowledge,this is the first characterization of meloninfecting SLCCNV through its genome,infectious clone and transmission.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
It was rarely reported about strawberry vein banding virus(SVBV)genome sequence in China and most countries worldwide.In this work,we determined the complete genome sequences of two SVBV isolates in China,designated S...It was rarely reported about strawberry vein banding virus(SVBV)genome sequence in China and most countries worldwide.In this work,we determined the complete genome sequences of two SVBV isolates in China,designated SVBV-AH and SVBV-BJ,that were obtained from naturally infected strawberry samples from Anhui province and Beijing city of China,respectively.The complete genomes of SVBV-AH and SVBV-BJ were 7,862 nucleotides(nts)and 7,863 nts long,respectively,and both constituted with seven genes typical of the caulimoviruses.Alignment of complete nucleotide sequences showed that SVBV-AH and SVBV-BJ shared a significant nucleotide sequence identity of 97.7%of each other and had 85.7%and 86.0%sequence identity related to SVBV from the United States(SVBV-US),respectively.Phylogenetic trees,based on the alignment of complete nucleotide sequences and amino acid sequences of Coat Protein(CP),both showed that SVBV-AH and SVBV-BJ clustered into one branch with all the other SVBV isolates,and other species of caulimoviruses clustered into another tree branch.It illustrated that all the SVBV isolates had an extremely high relationship but had a distant relationship with other species of caulimoviruses.We further confirmed that SVBV-AH infectious clone could cause similar symptoms to SVBVinfected in strawberry under natural conditions.Taken together,our study provided valuable information to elucidate the origin and dissemination of SVBV Chinese isolates,meanwhile providing the necessary vector for studying the gene functions of strawberry.展开更多
Cre/loxP,a site-specific recombination system,has been widely used for various purposes,including chromosomal translocations,generation of marker-free transgenic plants,tissue-specific activation of a reporter gene an...Cre/loxP,a site-specific recombination system,has been widely used for various purposes,including chromosomal translocations,generation of marker-free transgenic plants,tissue-specific activation of a reporter gene and efficient heterologous gene expression in plants.However,stable or transient expression of Cre recombinase in plants can cause chlorosis or necrosis.Here,we describe a modified Cre/loxP recombination system using a DNA fragment flanked with loxP sites in the same orientation in which necrosis induced by Cre recombinase in Nicotiana benthamiana leaves was alleviated.The modified system was successfully used to create functional GFP-tagged pepper mild mottle virus(PMMoV)and a chimeric virus with coat protein(CP)substitution assembled from separate pro-vector modules.Our results provide a new strategy and flexible technique to construct chimeric virus and infectious clones for plant viruses with large genomes.展开更多
Tick-borne encephalitis virus(TBEV)is a re-emerging pathogen in Kazakhstan,where the increasing risk of its spread underscores the need for improved healthcare preparedness,including the development of local vaccines....Tick-borne encephalitis virus(TBEV)is a re-emerging pathogen in Kazakhstan,where the increasing risk of its spread underscores the need for improved healthcare preparedness,including the development of local vaccines.However,the absence of reference TBEV strains in the country presented a major challenge.To address this,we generated a prototype strain(Vasilchenko)of the Siberian TBEV genotype,predominant in Kazakhstan,using synthetic genome and molecular infectious clone technology.A DNA-launched TBEV molecular clone was assembled from DNA fragments,enabling virus rescue upon plasmid transfection.During the propagation of the post-transfection virus in cell culture,a single amino acid substitution(E51K)in the envelope protein emerged,resulting in a 100-fold increase in the titer of the mutant variant.In vivo,this mutation significantly attenuated virulence:while wild-type TBEV caused 100%mortality in BALB/c mice,the E51K variant was non-lethal and exhibited reduced viremia,suggesting impaired neuroinvasiveness.To further exploit this attenuated,high-titer virus,we developed a GFP-expressing reporter TBEV variant.Using this reporter system,we demonstrated that favipiravir possesses antiviral activity against TBEV,with inhibitory concentrations within a pharmacologically relevant range.In conclusion,synthetic genomics enabled the generation of a reference TBEV strain to replenish Kazakhstan's collections.The E51K mutation enhances viral replication in vitro while attenuating pathogenicity in vivo,and the derived reporter virus is suitable for antiviral compound screening.展开更多
基金supported by grants from the Science and Technology Commission of Shanghai Municipality (13ZR1462900)the Shanghai Institutes for Biological Science (SIBS),Chinese Academy of Science (CAS) (2013KIP317)+1 种基金the support of the SA-SIBS scholarship programYouth Innovation Promotion Association of CAS (2016249)
文摘Dear Editor,Coxsackievirus A16(CA16)is one of the major viral pathogens associated with hand,foot,and mouth disease.CA16 belongs to the Enterovirus genus of the Picornaviridae family and possesses a single-stranded positivesense RNA genome(Mao et al.,2014).Reverse genetics is an important tool for CA16 research.Previously,a reverse genetics T7 polymerase-based system was de-
基金supported by grants from the National Natural Science Foundation of China (31070135, 31370182)the Tianjin Research Program of Application Foundation and Advanced Technology (12JCQNJC06100)New Century Excellent Talents in University (NCET-10-0508)
文摘The biological features of most foamy viruses(FVs) are poorly understood, including bovine foamy virus(BFV). BFV strain 3026(BFV3026) was isolated from the peripheral blood mononuclear cells of an infected cow in Zhangjiakou, China. A full-length genomic clone of BFV3026 was obtained from BFV3026-infected cells, and it exhibited more than 99% amino acid(AA) homology to another BFV strain isolated in the USA. Upon transfection into fetal canine thymus cells, the full-length BFV3026 clone produced viral structural and auxiliary proteins, typical cytopathic effects, and virus particles. These results demonstrate that the full-length BFV3026 clone is fully infectious and can be used in further BFV3026 research.
文摘Construction of infectious clones by full-length cDNA is basic and key for recovering RNA virus and is core of reverse genetics.In this article,basic consideration and key technology were viewed and factors affecting infectivity of clones were also summarized.Some research advances were briefly introduced about positive-strand RNA viruses infectious clones.Finally,this article also reviewed the application of infectious clones.
基金supported by the Key Project of National Natural Science Foundation of China(30530550)
文摘The objective of this study was to construct the infectious clone of encephalomyocarditis virus (EMCV) BJC3 strain.The genomic cDNA of the virus was amplified by three overlapping segments using RT-PCR,and cloned into low-copy plasmid pWSK29 to construct the full-length cDNA clone pWSKBJC3/ w.The pWSKBJC3/w was in vitro transcribed and transfected into BHK-21 cells to rescue the virus.The results showed that the full-length cDNA clone was infectious and the virus could be rescued in BHK-21 cells.The rescued virus designated RvBJC3W was identified by RT-PCR and indirect immunofluorescence assay(IFA).The rescued virus had similar growth characteristics to its parental virus BJC3 and retained pathogenicity for mice.Our results indicate that the first infectious cDNA clone of EMCV in China has been successfully established and provides an essential tool for investigating the molecular basis of pathogenicity of EMCV.
基金the National Key Research and Development Program of China(2022YFC2304101,2021YFA1201003).
文摘Coronaviruses can infect humans,mammals,and birds,leading to respiratory,gastrointestinal,and neurological diseases.These viruses are significant zoonotic pathogens with nine known types capable of infecting humans.The coronavirus genome,approximately 30 kb in size,is the largest known ribonucleic acid(RNA)virus genome,and its complexity makes assembly and manipulation time-consuming and labor-intensive.Reverse genetic systems are widely used to engineer recombinant viruses that can be adapted at Biosafety Level 2(BSL-2)for studying viral gene function,replication,pathogenesis,vaccines,and therapeutics.The infectious clones,which enabled the recovery of various viruses after DNA recombinant technology,were indispensable tools for the reverse genetics of viruses.Various techniques for constructing infectious clones of human coronaviruses(HCoV)have been developed,encompassing methods such as vaccinia virus vectors method,in vitro ligation,bacterial artificial chromosome systems,yeast artificial chromosome systems,circular polymerase extension reaction,and the recently reported infectious sub-genomic amplicons technology.This review summarizes the status of various techniques for constructing infectious clones of human coronaviruses and related applications.
基金supported by the National Key Research and Development Program of China(2022YFC2304100 and 2021YFA1201003).
文摘Viral infectious clones(ICs)serve as robust platforms for studying viral biology and screening antiviral agents using reverse genetics.However,the molecular profiles and complex limitations of human coronaviruses(HCoVs)pose a challenge to ICs development.In this study,we report a novel platform to develop the ICs for HCoV-OC43-VR1558 using a one-step assembly method in yeast by transformation-associated recombination(TAR)technology.Recombinant HCoV-OC43-VR1558,named as rOC43(1558)-WT,was rapidly generated by TAR.In addition,recombinant HCoV-OC43-VR1558-expressing reporter genes,named as rOC43(1558)-ns2FusionRluc,was also generated based on TAR by inserting the ns2 region of the IC with Renilla luciferase(Rluc).We further characterized their replication through virus titration using 50%tissue culture infective dose(TCID50)and indirect immunofluorescence assay(IFA),luciferase reporter assay,and western blotting(WB)assay.The genetic stability of the recombinant HCoV-OC43 was assessed through viral genome sequencing following passaging in BHK-21 cells.These reporter viruses were validated as screening tools for inhibitorsin vitro by evaluating the antiviral activities of remdesivir and chloroquine.The phenotypes of HCoV-OC43-VR1558 and HCoV-OC43-VR759 were comparedin vitro andin vivo.The TAR-based one-step assembly of IC was successfully applied,facilitating the rapid generation of recombinant HCoV-OC43 and providing a useful platform for the investigation of biological mechanisms,development of vaccines and diagnostic tests,and screening inhibitors of HCoVs.
基金supported by the National Key Research and Development Program of China(2021YFD1400400)the National Natural Science Foundation of China(31930089).
文摘Tomato leaf curl New Delhi virus(ToLCNDV)is a member of the genus Begomovirus,and causes devastating disease in the world.In recent years,ToLCNDV was rapidly spreading in China and induces severe economic losses in agriculture.In this study,we sequenced and characterized the complete genome of ToLCNDV isolates from melon plants showing leaf curling and stunting symptoms in Jiangsu Province of China.We constructed a full-length infectious cDNA clone of ToLCNDV,which could induce systemic infection with typical symptoms in Nicotiana benthamiana,Cit-rullus melo,and Citrullus lanatus plants through agrobacterium-mediated inoculation.Further experimental evidence demonstrated that the virions produced in plants infected with the infectious clone of ToLCNDV are biologically active and sap-transmissible.We also evaluated the resistance of commercial melon cultivars to ToLCNDV and found all testing melon cultivars were susceptible to ToLCNDV.Collectively,the reverse genetic system developed herein will facilitate further research on biological functions of proteins encoded by ToLCNDV and plant-ToLCNDV interactions,which might provide new insights into breeding resistance germplasm in crops.
基金This study was supported by grants from the National Key R&D Program of China(2018YFA0507201)。
文摘Tick-borne encephalitis virus(TBEV) is a pathogenic virus known to cause central nervous system(CNS) diseases in humans, and has become an increasing public health threat nowadays. The rates of TBEV infection in the endemic countries are increasing. However, there is no effective antiviral against the disease. This underscores the urgent need for tools to study the emergence and pathogenesis of TBEV and to accelerate the development of vaccines and antivirals. In this study, we reported an infectious c DNA clone of TBEV that was isolated in China(the WH2012 strain). A beta-globin intron was inserted in the coding region of nonstructural protein 1(NS1) gene to improve the stability of viral genome in bacteria. In mammalian cells, the inserted intron was excised and spliced precisely, which did not lead to the generation of inserted mutants. High titers of infectious progeny viruses were generated after the transfection of the infectious clone. The cDNA-derived TBEV replicated efficiently, and caused typical cytopathic effect(CPE) and plaques in BHK-21 cells. In addition, the CPE and growth curve of cDNA-derived virus were similar to that of its parental isolate in cells. Together, we have constructed the first infectious TBEV cDNA clone in China, and the clone can be used to investigate the genetic determinants of TBEV virulence and disease pathogenesis, and to develop countermeasures against the virus.
基金supported by the National Natural Science Foundation of China(no.31272583,31472199)China Agriculture Research System(CARS-43-10)
文摘Dear Editor,Since April 2010,an outbreak of a new disease has elicited symptoms of high fever,loss of appetite,and reduction in egg production in layer ducks in eastern China;this phenomenon has now spread throughout China(Cao et al.,2011;Su et al.,2011).The causative agent of the disease was identified as Tembusu virus(TMUV),which was classified into the genus Flavivirus,
基金financially supported by the Hainan Province Science and Technology Special Fund(No.ZDYF2022XDNY240)the National Key Research and Development Program of China(2022YFD1401200).
文摘Ligularia jaluensis is an important medicinal and ornamental plant in China.However,the viruses capable of infecting Ligularia jaluensis remains unknown.Here,we identified a novel carlavirus,tentatively named ligularia jaluensis carlavirus(LJCV),as well as a known iris severe mosaic virus(ISMV),in L.jaluensis plants displaying chlorosis and yellow ring spot symptoms,using RNA-seq analysis.The LJCV genome consists of an 8497 nt positive-sense,single-stranded RNA[excluding the poly(A)tail],and contains six open reading frames(ORFs).Phylogenetic analyses based on the fulllength genome and RNA-dependent RNA polymerase(RdRp)amino acid(aa)sequences revealed that LJCV clusters within an evolutionary branch alongside known viruses in the Carlavirus genus.The RdRp protein encoded by ORF1 of LJCV shared 45.38%–67.41%identity with the corresponding proteins of eight closely related carlaviruses.ORFs 2–4 constitute the triple gene block(TGB),with TGBp1 and TGBp3 localized in the endoplasmic reticulum(ER),while TGBp2 is localized at plasmodesmata(PD)and facilitates viral intercellular movement,as demonstrated by its ability to complement the potato virus X with movement-deficient mutant(PVX-Δp25-GFP).Additionally,ORF6 encodes a cysteine-rich protein(CRP)that is localized in the chloroplast and functions as a viral pathogenicity factor,inducing severe viral symptoms in the heterologous PVX expression system.Furthermore,we successfully constructed an infectious cDNA clone of LJCV,and found that it can infect Nicotiana benthamiana plants through mechanical inoculation or agrobacterium-mediated infiltration of the LJCV infectious clone.These findings enhance our understanding of the characteristics and host range of carlaviruses,as well as the viruses capable of infecting L.jaluensis.
基金supported by the Guangzhou National Laboratory(SRPG22-002 to X.W.Chen)the Pearl River Talent Recruitment Program(2019CX01Y422 to X.W.Chen)+1 种基金the Natural Science Foundation of Guangdong Province(2025A1515011018 to J.L.Tang)the Basic and Applied Basic Research Projects of Guangzhou Basic Research Program(2025A04J5492 to J.L.Tang,2023A04J0161 to Q.Yang).
文摘Human bocavirus 1(HBoV1;family:Parvoviridae)causes a wide spectrum of respiratory diseases in children and gastroenteritis in adults.A lack of sensitive cell lines and efficient animal models hinders research on HBoV,including the development of anti-HBoV drugs or vaccines.Although the construction of a wild-type HBoV1 infectious clone has been reported,generating HBoV1 infectious clone carrying foreign reporter genes with suitable insertion sites in its genome while retaining replicative ability remains challenging.Here,HBoV1 infectious clones harboring the 11-amino-acid HiBiT tag at five distinct insertion sites were constructed and evaluated.Only the recombinant HBoV1 carrying the HiBiT tag in the N-terminus of the NS1 protein(HBoV1-HiBiTNS1)displayed comparable characteristics to wild-type HBoV1 as determined via the analysis of viral DNA copy number,NanoLuc activity,viral protein expression,and the formation of replication intermediates.Notably,the replication kinetics of HBoV1-HiBiTNS1 could be examined by monitoring NanoLuc activity,which was noted to be correlated with the viral DNA level.Additionally,we successfully applied HiBiT-tagged HBoV1 for the evaluation of antiviral drug activity and identified ivermectin(EC50=2.27μM)as a potent anti-HBoV1 replication drug.Overall,our study demonstrated that the HBoV1-HiBiTNS1 reporter can serve as a convenient platform for screening candidate drugs targeting HBoV1 replication and may also be useful for investigating the life cycle of the virus.
基金supported by the National Natural Science Foundation of China(grant nos.82002135 and 82172250).
文摘The utilization of enteroviruses engineered with reporter genes serves as a valuable tool for advancing our understanding of enterovirus biology and its applications,enabling the development of effective therapeutic and preventive strategies.In this study,our initial attempts to introduce a NanoLuc luciferase(NLuc)reporter gene into recombinant enteroviruses were unsuccessful in rescuing viable progenies.We hypothesized that the size of the inserted tag might be a determining factor in the rescue of the virus.Therefore,we inserted the 11-amino-acid HiBiT tag into the genomes of enterovirus A71(EV-A71),coxsackievirus A10(CVA10),coxsackievirus A7(CVA7),coxsackievirus A16(CVA16),namely EV-A71-HiBiT,CVA16-HiBiT,CVA10-HiBiT,CVA7-HiBiT,and observed that the HiBiT-tagged viruses exhibited remarkably high rescue efficiency.Notably,the HiBiT-tagged enteroviruses displayed comparable characteristics to the wild-type viruses.A direct comparison between CVA16-NLuc and CVA16-HiBiT recombinant viruses revealed that the tiny HiBiT insertion had minimal impact on virus infectivity and replication kinetics.Moreover,these HiBiT-tagged enteroviruses demonstrated high genetic stability in different cell lines over multiple passages.In addition,the HiBiT-tagged viruses were successfully tested in antiviral drug assays,and the sensitivity of the viruses to drugs was not affected by the HiBiT tag.Ultimately,our findings provide definitive evidence that the integration of HiBiT into enteroviruses presents a universal,convenient,and invaluable method for advancing research in the realm of enterovirus virology.Furthermore,HiBiT-tagged enteroviruses exhibit great potential for diverse applications,including the development of antivirals and the elucidation of viral infection mechanisms.
基金supported by the National Natural Science Foundation of China (31701941 and 31401810)the grants from the earmarked fund for China Agriculture Research System (CARS-26-13)the Agricultural Science and Technology Innovation Program of Chinese Academy of Agricultural Sciences (ASTIP) (CAAS-ASTIP-2018-ZFRI-08)
文摘It has been reported that squash leaf curl China virus(SLCCNV)infects some Cucurbitaceae crops except for melon(Cucumis melo L.).A new disease of melon exhibiting severe leaf curl and dwarfing was observed in Hainan Province of China.In this study,the pathogen was identified as SLCCNV through biological and molecular characterization.The isolate(SLCCNV-HN)possess a bipartite genome,DNA-A(HM566112.1)with the highest nucleotide identity(99%)to SLCCNV-Hn(MF062251.1)pumpkin and SLCCNV-Hn61(AM260205.1)squash isolates from China,whereas DNA-B(HM566113.1)with the highest nucleotide identity(99%)to SLCCNV-Hn(MF062252.1).Phylogenetic analyses based on the full-length SLCCNV-HN DNA-A and-B sequences indicated that SLCCNV-HN melon isolate is clustered with SLCCNV-Hn pumpkin,SLCCNV-Hn61 and SLCCNV-SY squash isolates from southern China,forming an independent cluster.Infectious clone of SLCCNV-HN was constructed and the melon plants were inoculated and the infection rate is 100%,the systemic symptoms in melon showed identical to those of melon plants infected in fields.Additionally,melon plants transmission of this virus by Bemisia tabaci with a transmission rate of 95%(19/20)showed leaf curl and dwarf symptoms 15 days post transmission,thereby fulfilling Koch’s postulates.Analysis of genomic organization and phylogenetic trees indicated that SLCCNV-HN melon isolate belongs to the Begomovirus genus.To the best of our knowledge,this is the first characterization of meloninfecting SLCCNV through its genome,infectious clone and transmission.
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the National Natural Science Foundation of China(Nos.32072386 and 31801700)the Key Research and Development Project of Anhui Province(202004a06020013)the Anhui Postdoctoral Fund(2019B360).
文摘It was rarely reported about strawberry vein banding virus(SVBV)genome sequence in China and most countries worldwide.In this work,we determined the complete genome sequences of two SVBV isolates in China,designated SVBV-AH and SVBV-BJ,that were obtained from naturally infected strawberry samples from Anhui province and Beijing city of China,respectively.The complete genomes of SVBV-AH and SVBV-BJ were 7,862 nucleotides(nts)and 7,863 nts long,respectively,and both constituted with seven genes typical of the caulimoviruses.Alignment of complete nucleotide sequences showed that SVBV-AH and SVBV-BJ shared a significant nucleotide sequence identity of 97.7%of each other and had 85.7%and 86.0%sequence identity related to SVBV from the United States(SVBV-US),respectively.Phylogenetic trees,based on the alignment of complete nucleotide sequences and amino acid sequences of Coat Protein(CP),both showed that SVBV-AH and SVBV-BJ clustered into one branch with all the other SVBV isolates,and other species of caulimoviruses clustered into another tree branch.It illustrated that all the SVBV isolates had an extremely high relationship but had a distant relationship with other species of caulimoviruses.We further confirmed that SVBV-AH infectious clone could cause similar symptoms to SVBVinfected in strawberry under natural conditions.Taken together,our study provided valuable information to elucidate the origin and dissemination of SVBV Chinese isolates,meanwhile providing the necessary vector for studying the gene functions of strawberry.
基金supported by the National Key R&D Program of China(2019YFD1001800)the China Agriculture Research System of MOF and MARA(CARS24-C-04)+1 种基金supported by the Science&Technology Public Welfare Project of Ningbo City,China(202002N3005)K.C.Wong Education Foundation,China。
文摘Cre/loxP,a site-specific recombination system,has been widely used for various purposes,including chromosomal translocations,generation of marker-free transgenic plants,tissue-specific activation of a reporter gene and efficient heterologous gene expression in plants.However,stable or transient expression of Cre recombinase in plants can cause chlorosis or necrosis.Here,we describe a modified Cre/loxP recombination system using a DNA fragment flanked with loxP sites in the same orientation in which necrosis induced by Cre recombinase in Nicotiana benthamiana leaves was alleviated.The modified system was successfully used to create functional GFP-tagged pepper mild mottle virus(PMMoV)and a chimeric virus with coat protein(CP)substitution assembled from separate pro-vector modules.Our results provide a new strategy and flexible technique to construct chimeric virus and infectious clones for plant viruses with large genomes.
基金supported by funds from the scientific and technical program BR218004/0223“Improving measures to ensure biological safety in Kazakhstan:counteracting dangerous and especially dangerous infections”.Part of this work carried out in Russian Federation was supported by internal grant funding from the Scientific Centre for Family Health and Human Reproduction Problems:grant number 121022500179-0.Title:“Molecular,Organismal,and Population Patterns of the Epidemic Process of Anthropozoonotic and Transmissible Infections in the Territory of Northern Asia and Adjacent Areas".
文摘Tick-borne encephalitis virus(TBEV)is a re-emerging pathogen in Kazakhstan,where the increasing risk of its spread underscores the need for improved healthcare preparedness,including the development of local vaccines.However,the absence of reference TBEV strains in the country presented a major challenge.To address this,we generated a prototype strain(Vasilchenko)of the Siberian TBEV genotype,predominant in Kazakhstan,using synthetic genome and molecular infectious clone technology.A DNA-launched TBEV molecular clone was assembled from DNA fragments,enabling virus rescue upon plasmid transfection.During the propagation of the post-transfection virus in cell culture,a single amino acid substitution(E51K)in the envelope protein emerged,resulting in a 100-fold increase in the titer of the mutant variant.In vivo,this mutation significantly attenuated virulence:while wild-type TBEV caused 100%mortality in BALB/c mice,the E51K variant was non-lethal and exhibited reduced viremia,suggesting impaired neuroinvasiveness.To further exploit this attenuated,high-titer virus,we developed a GFP-expressing reporter TBEV variant.Using this reporter system,we demonstrated that favipiravir possesses antiviral activity against TBEV,with inhibitory concentrations within a pharmacologically relevant range.In conclusion,synthetic genomics enabled the generation of a reference TBEV strain to replenish Kazakhstan's collections.The E51K mutation enhances viral replication in vitro while attenuating pathogenicity in vivo,and the derived reporter virus is suitable for antiviral compound screening.
