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
Ebola virus(EBOV)and Marburg virus(MARV),members of the Filoviridae family,are highly pathogenic and can cause hemorrhagic fevers,significantly impacting human society.Bats are considered reservoirs of these viruses b...Ebola virus(EBOV)and Marburg virus(MARV),members of the Filoviridae family,are highly pathogenic and can cause hemorrhagic fevers,significantly impacting human society.Bats are considered reservoirs of these viruses because related filoviruses have been discovered in bats.However,due to the requirement for maximum containment laboratories when studying infectious viruses,the characterization of bat filoviruses often relies on pseudoviruses and minigenome systems.In this study,we used RACE technology to sequence the 30-leader and 50-trailer of Mengla virus(MLAV)and constructed a minigenome.Similar to MARV,the transcription activities of the MLAV minigenome are independent of VP30.We further assessed the effects of polymorphisms at the 50 end on MLAV minigenome activity and identified certain mutations that decrease minigenome reporter efficiency,probably due to alterations in the RNA secondary structure.The reporter activity upon recombination of the 30-leaders and 50-trailers of MLAV,MARV,and EBOV with those of the homologous or heterologous minigenomes was compared and it was found that the polymerase complex and leader and trailer sequences exhibit intrinsic specificities.Additionally,we investigated whether the polymerase complex proteins from EBOV and MARV support MLAV minigenome RNA synthesis and found that the homologous system is more efficient than the heterologous system.Remdesivir efficiently inhibited MLAV as well as EBOV replication.In summary,this study provides new information on bat filoviruses and the minigenome will be a useful tool for high-throughput antiviral drug screening.展开更多
Human respiratory syncytial virus（RSV） is an important pediatric pathogen of lower respiratory tract worldwide. No vaccines and antiviral drugs are available. Herein the use of an RNA polymerase I-driven RSV minigen...Human respiratory syncytial virus（RSV） is an important pediatric pathogen of lower respiratory tract worldwide. No vaccines and antiviral drugs are available. Herein the use of an RNA polymerase I-driven RSV minigenome for analyzing RSV replication and screening anti-RSV drugs was investigated. The RNA polymerase I（Pol I） was used to transcribe RSV minigenome from the constructed plasmid, designated p HM-RSV-Gluc, of minigenome c DNA which comprised trailer region, gene start sequence（GS）, reverse complementary copy of Gaussia luciferase（Gluc） gene, gene end sequence（GE）, and leader region in the direction of 5＇–3＇end and was flanked by promoter and terminator of Pol I. The expression of Gluc was confirmed in p HM-RSV-Gluc transfected HEp-2 cells following RSV infection and had the characteristics of dose-dependent, which provided a rapid, sensitive, and quantitative method for quantifying virus titers and screening antiviral drugs.展开更多
基金supported by grants from the National Natural Science Foundation of China(No.31900146Open Research Fund Program of the State Key Laboratory of Virology of China(No.2020IOV003)Team project of Health Commission of Hubei Province(WJ2019C003)。
文摘Severe fever with thrombocytopenia syndrome virus(SFTSV)is an emerging tick-borne bunyavirus that causes hemorrhagic fever-like disease(SFTS)in humans with a case fatality rate up to 30%.To date,the molecular biology involved in SFTSV infection remains obscure.There are seven major genotypes of SFTSV(C1-C4 and J1-J3)and previously a reverse genetic system was established on a C3 strain of SFTSV.Here,we reported successfully establishment of a reverse genetics system based on a SFTSV C4 strain.First,we obtained the 5’-and 3’-terminal untranslated region(UTR)sequences of the Large(L),Medium(M)and Small(S)segments of a laboratory-adapted SFTSV C4 strain through rapid amplification of cDNA ends analysis,and developed functional T7 polymerase-based L-,M-and S-segment minigenome assays.Then,fulllength cDNA clones were constructed and infectious SFTSV were recovered from co-transfected cells.Viral infectivity,growth kinetics,and viral protein expression profile of the rescued virus were compared with the laboratory-adapted virus.Focus formation assay showed that the size and morphology of the foci formed by the rescued SFTSV were indistinguishable with the laboratory-adapted virus.However,one-step growth curve and nucleoprotein expression analyses revealed the rescued virus replicated less efficiently than the laboratory-adapted virus.Sequence analysis indicated that the difference may be due to the mutations in the laboratory-adapted strain which are more prone to cell culture.The results help us to understand the molecular biology of SFTSV,and provide a useful tool for developing vaccines and antivirals against SFTS.
基金supported by the Key project of the Chinese Academy of Sciences(KJZD-SW-L11 to Z.-L.S.)the Guangzhou Laboratory(SRPG22-001 to Z.-L.S.)+3 种基金the Advanced Customer Cultivation Project of Wuhan National Biosafety Laboratory,Chinese Academy of Sciences(2021ACCP-MS02)the Strategic Priority Research Program of the Chinese Academy of Sciences(XDB0490000 to X.-L.Y.)the Young Topnotch Talent Cultivation Program of Hubei Province(X.-L.Y.)the Youth Innovation Promotion Association of the Chinese Academy of Science(X.-L.Y.).
文摘Ebola virus(EBOV)and Marburg virus(MARV),members of the Filoviridae family,are highly pathogenic and can cause hemorrhagic fevers,significantly impacting human society.Bats are considered reservoirs of these viruses because related filoviruses have been discovered in bats.However,due to the requirement for maximum containment laboratories when studying infectious viruses,the characterization of bat filoviruses often relies on pseudoviruses and minigenome systems.In this study,we used RACE technology to sequence the 30-leader and 50-trailer of Mengla virus(MLAV)and constructed a minigenome.Similar to MARV,the transcription activities of the MLAV minigenome are independent of VP30.We further assessed the effects of polymorphisms at the 50 end on MLAV minigenome activity and identified certain mutations that decrease minigenome reporter efficiency,probably due to alterations in the RNA secondary structure.The reporter activity upon recombination of the 30-leaders and 50-trailers of MLAV,MARV,and EBOV with those of the homologous or heterologous minigenomes was compared and it was found that the polymerase complex and leader and trailer sequences exhibit intrinsic specificities.Additionally,we investigated whether the polymerase complex proteins from EBOV and MARV support MLAV minigenome RNA synthesis and found that the homologous system is more efficient than the heterologous system.Remdesivir efficiently inhibited MLAV as well as EBOV replication.In summary,this study provides new information on bat filoviruses and the minigenome will be a useful tool for high-throughput antiviral drug screening.
基金supported by National Major Scientific and Technological Special Project for ‘‘AIDS and Viral Hepatitis and Other Major Infectious Diseases Prevention and Control’’ during the Twelfth Five-year Plan Period (No. 2013ZX10004-601)
文摘Human respiratory syncytial virus（RSV） is an important pediatric pathogen of lower respiratory tract worldwide. No vaccines and antiviral drugs are available. Herein the use of an RNA polymerase I-driven RSV minigenome for analyzing RSV replication and screening anti-RSV drugs was investigated. The RNA polymerase I（Pol I） was used to transcribe RSV minigenome from the constructed plasmid, designated p HM-RSV-Gluc, of minigenome c DNA which comprised trailer region, gene start sequence（GS）, reverse complementary copy of Gaussia luciferase（Gluc） gene, gene end sequence（GE）, and leader region in the direction of 5＇–3＇end and was flanked by promoter and terminator of Pol I. The expression of Gluc was confirmed in p HM-RSV-Gluc transfected HEp-2 cells following RSV infection and had the characteristics of dose-dependent, which provided a rapid, sensitive, and quantitative method for quantifying virus titers and screening antiviral drugs.
