Yellow fever virus(YFV) is a re-emerging virus that can cause life-threatening yellow fever disease in humans.Despite the availability of an effective vaccine,little is known about the replication mechanism of YFV,and...Yellow fever virus(YFV) is a re-emerging virus that can cause life-threatening yellow fever disease in humans.Despite the availability of an effective vaccine,little is known about the replication mechanism of YFV,and there are still no available specific anti-YFV medicines.Herein,by introducing the Renilla luciferase gene(Rluc) into an infectious clone of YFV vaccine strain 17 D,we generated a recombinant virus 17 D-Rluc.2 A via reverse genetics approaches.The 17 D-Rluc.2 A had similar plaque morphology and comparable in vitro growth characteristics with its parental strain.Importantly,the reporter luciferase was efficiently expressed in 17 D-Rluc.2 A-infected mammalian and mosquito cells,and there was a good linear correlation between intracellular luciferase expression and extracellular infectious virion reproduction.Furthermore,by a combination of the 17 D-Rluc.2 A reporter virus and selective 2’-hydroxyl acylation analyzed by primer extension(SHAPE)technology,the conserved 5’-SLA element was shown to be essential for YFV replication,highlighting the capability of17 D-R1 uc.2 A in the investigation of YFV replication.At last,we demonstrated that two compounds with distinct anti-viral mechanisms can effectively inhibit the viral propagation in 17 D-Rluc.2 A-infected cells,demonstrating its potential application in the evaluation of anti-viral medicines.Taken together,the 17 D-Rluc.2 A serves as a useful tool for the study of YFV replication and anti-YFV medicine development.展开更多
Zika virus(ZIKV) is associated with severe birth defects and Guillain-Barre′ syndrome and no approved vaccines or specific therapies to combat ZIKV infection are currently available. To accelerate anti-ZIKV therapeut...Zika virus(ZIKV) is associated with severe birth defects and Guillain-Barre′ syndrome and no approved vaccines or specific therapies to combat ZIKV infection are currently available. To accelerate anti-ZIKV therapeutics research, we developed a stable ZIKV GFP-reporter virus system with considerably improved GFP visibility and stability. In this system a BHK-21 cell line expressing DC-SIGNR was established to facilitate the proliferation of GFP-reporter ZIKV. Using this reporter virus system, we established a high-throughput screening assay and screened a selected plant-sourced compounds library for their ability to block ZIKV infection. More than 31 out of 974 tested compounds effectively decreased ZIKV reporter infection. Four selected compounds, homoharringtonine(HHT), bruceine D(BD), dihydroartemisinin(DHA) and digitonin(DGT), were further validated to inhibit wild-type ZIKV infection in cells of BHK-21 and human cell line A549.The FDA-approved chronic myeloid leukemia treatment drug HHT and BD were identified as broad-spectrum flavivirus inhibitors. DHA, another FDA-approved antimalarial drug effectively inhibited ZIKV infection in BHK-21 cells. HHT, BD and DHA inhibited ZIKV infection at a post-entry stage. Digitonin was found to have inhibitory activity in the early stage of viral infection. Our research provides an efficient high-throughput screening assay for ZIKV inhibitors. The active compounds identified in this study represent potential therapies for the treatment of ZIKV infection.展开更多
Alphaviruses,which contain a variety of mosquito-borne pathogens,are important pathogens of emerging/reemerging infectious diseases and potential biological weapons.Currently,no specific antiviral drugs are available ...Alphaviruses,which contain a variety of mosquito-borne pathogens,are important pathogens of emerging/reemerging infectious diseases and potential biological weapons.Currently,no specific antiviral drugs are available for the treatment of alphaviruses infection.For most highly pathogenic alphaviruses are classified as risk group-3 agents,the requirement of biosafety level 3(BSL-3)facilities limits the live virus-based antiviral study.To facilitate the antiviral development of alphaviruses,we developed a high throughput screening(HTS)platform based on a recombinant Semliki Forest virus(SFV)which can be manipulated in BSL-2 laboratory.Using the reverse genetics approach,the recombinant SFV and SFV reporter virus expressing eGFP(SFV-eGFP)were successfully rescued.The SFV-eGFP reporter virus exhibited robust eGFP expression and remained relatively stable after four passages in BHK-21 cells.Using a broad-spectrum alphavirus inhibitor ribavirin,we demonstrated that the SFV-eGFP can be used as an effective tool for antiviral study.The SFV-eGFP reporter virus-based HTS assay in a 96-well format was then established and optimized with a robust Z0 score.A section of reference compounds that inhibit highly pathogenic alphaviruses were used to validate that the SFV-eGFP reporter virus-based HTS assay enables rapid screening of potent broad-spectrum inhibitors of alphaviruses.This assay provides a safe and convenient platform for antiviral study of alphaviruses.展开更多
Unveiling the molecular mechanisms underlying rotavirus replication and pathogenesis has been hampered by the lack of a reverse genetics(RG)system in the past.Since 2017,multiple plasmid-based RG systems for simian,hu...Unveiling the molecular mechanisms underlying rotavirus replication and pathogenesis has been hampered by the lack of a reverse genetics(RG)system in the past.Since 2017,multiple plasmid-based RG systems for simian,human,and murine-like rotaviruses have been established.However,none of the described methods have supported the recovery of bovine rotaviruses(BRVs).Here,we established an optimized plasmid-based RG system for BRV culture-adapted strain(BRV G10P[15]BLR)and clinical isolates(BRV G6P[1]C73,G10P[11]HM26)based on a BHK-T7 cell clone stably expressing T7 polymerase.Furthermore,using this optimized RG system,we successfully rescued the reporter virus BRV rC73/Zs,rHM26/Zs and rBLR/Zs,harboring a genetically modified 1.8-kb segment 7 encoding full-length nonstructural protein 3(NSP3)fused to ZsGreen,a 232-amino acid green fluorescent protein.Analysis of the stability of genomic insertions showed that the rC73/Zs and rBLR/Zs replicated efficiently and were genetically stable in seven rounds of serial passaging,while rHM26/Zs can be stabilized only up to the third generation,indicating that the BRV segment composition may influence the viral fitness.In addition,we adopted the recombinant reporter viruses for high-throughput screening application and discovered 12 candidates out of 1440 compounds with potential antiviral activities against rotavirus.In summary,this improved RG system of BRVs represents an important tool with great potential for understanding the molecular biology of BRV and facilitates the development of novel therapeutics and vaccines for BRV.展开更多
基金This work is supported by the National Natural Science Foundation of China(81871632 and 32070183)the Natural Science Foundation of Guangdong Province(2020A1515010656)+1 种基金the Creative Research Group Foster Project of the Sun Yat-sen Universitysupported by the One-Hundred People Project of the Sun Yat-sen University。
文摘Yellow fever virus(YFV) is a re-emerging virus that can cause life-threatening yellow fever disease in humans.Despite the availability of an effective vaccine,little is known about the replication mechanism of YFV,and there are still no available specific anti-YFV medicines.Herein,by introducing the Renilla luciferase gene(Rluc) into an infectious clone of YFV vaccine strain 17 D,we generated a recombinant virus 17 D-Rluc.2 A via reverse genetics approaches.The 17 D-Rluc.2 A had similar plaque morphology and comparable in vitro growth characteristics with its parental strain.Importantly,the reporter luciferase was efficiently expressed in 17 D-Rluc.2 A-infected mammalian and mosquito cells,and there was a good linear correlation between intracellular luciferase expression and extracellular infectious virion reproduction.Furthermore,by a combination of the 17 D-Rluc.2 A reporter virus and selective 2’-hydroxyl acylation analyzed by primer extension(SHAPE)technology,the conserved 5’-SLA element was shown to be essential for YFV replication,highlighting the capability of17 D-R1 uc.2 A in the investigation of YFV replication.At last,we demonstrated that two compounds with distinct anti-viral mechanisms can effectively inhibit the viral propagation in 17 D-Rluc.2 A-infected cells,demonstrating its potential application in the evaluation of anti-viral medicines.Taken together,the 17 D-Rluc.2 A serves as a useful tool for the study of YFV replication and anti-YFV medicine development.
基金partially supported by the National Key R&D Program of China (grant 2018YFC1200602 and 2016YFD0500403 to RHH)。
文摘Zika virus(ZIKV) is associated with severe birth defects and Guillain-Barre′ syndrome and no approved vaccines or specific therapies to combat ZIKV infection are currently available. To accelerate anti-ZIKV therapeutics research, we developed a stable ZIKV GFP-reporter virus system with considerably improved GFP visibility and stability. In this system a BHK-21 cell line expressing DC-SIGNR was established to facilitate the proliferation of GFP-reporter ZIKV. Using this reporter virus system, we established a high-throughput screening assay and screened a selected plant-sourced compounds library for their ability to block ZIKV infection. More than 31 out of 974 tested compounds effectively decreased ZIKV reporter infection. Four selected compounds, homoharringtonine(HHT), bruceine D(BD), dihydroartemisinin(DHA) and digitonin(DGT), were further validated to inhibit wild-type ZIKV infection in cells of BHK-21 and human cell line A549.The FDA-approved chronic myeloid leukemia treatment drug HHT and BD were identified as broad-spectrum flavivirus inhibitors. DHA, another FDA-approved antimalarial drug effectively inhibited ZIKV infection in BHK-21 cells. HHT, BD and DHA inhibited ZIKV infection at a post-entry stage. Digitonin was found to have inhibitory activity in the early stage of viral infection. Our research provides an efficient high-throughput screening assay for ZIKV inhibitors. The active compounds identified in this study represent potential therapies for the treatment of ZIKV infection.
基金supported by the Creative Research Group Program of Natural Science Foundation of Hubei Province (2022CFA021)National Natural Science Foundation of China (81702005).
文摘Alphaviruses,which contain a variety of mosquito-borne pathogens,are important pathogens of emerging/reemerging infectious diseases and potential biological weapons.Currently,no specific antiviral drugs are available for the treatment of alphaviruses infection.For most highly pathogenic alphaviruses are classified as risk group-3 agents,the requirement of biosafety level 3(BSL-3)facilities limits the live virus-based antiviral study.To facilitate the antiviral development of alphaviruses,we developed a high throughput screening(HTS)platform based on a recombinant Semliki Forest virus(SFV)which can be manipulated in BSL-2 laboratory.Using the reverse genetics approach,the recombinant SFV and SFV reporter virus expressing eGFP(SFV-eGFP)were successfully rescued.The SFV-eGFP reporter virus exhibited robust eGFP expression and remained relatively stable after four passages in BHK-21 cells.Using a broad-spectrum alphavirus inhibitor ribavirin,we demonstrated that the SFV-eGFP can be used as an effective tool for antiviral study.The SFV-eGFP reporter virus-based HTS assay in a 96-well format was then established and optimized with a robust Z0 score.A section of reference compounds that inhibit highly pathogenic alphaviruses were used to validate that the SFV-eGFP reporter virus-based HTS assay enables rapid screening of potent broad-spectrum inhibitors of alphaviruses.This assay provides a safe and convenient platform for antiviral study of alphaviruses.
基金supported by the Heilongjiang Provincial Natural Science Foundation of China(grant no.LH2033C107)the National Key Research and Development Program of China(2023YFD1801302)the Central Public-interest Scientific Institution Basal Research Fund(grant no.1610302022010).
文摘Unveiling the molecular mechanisms underlying rotavirus replication and pathogenesis has been hampered by the lack of a reverse genetics(RG)system in the past.Since 2017,multiple plasmid-based RG systems for simian,human,and murine-like rotaviruses have been established.However,none of the described methods have supported the recovery of bovine rotaviruses(BRVs).Here,we established an optimized plasmid-based RG system for BRV culture-adapted strain(BRV G10P[15]BLR)and clinical isolates(BRV G6P[1]C73,G10P[11]HM26)based on a BHK-T7 cell clone stably expressing T7 polymerase.Furthermore,using this optimized RG system,we successfully rescued the reporter virus BRV rC73/Zs,rHM26/Zs and rBLR/Zs,harboring a genetically modified 1.8-kb segment 7 encoding full-length nonstructural protein 3(NSP3)fused to ZsGreen,a 232-amino acid green fluorescent protein.Analysis of the stability of genomic insertions showed that the rC73/Zs and rBLR/Zs replicated efficiently and were genetically stable in seven rounds of serial passaging,while rHM26/Zs can be stabilized only up to the third generation,indicating that the BRV segment composition may influence the viral fitness.In addition,we adopted the recombinant reporter viruses for high-throughput screening application and discovered 12 candidates out of 1440 compounds with potential antiviral activities against rotavirus.In summary,this improved RG system of BRVs represents an important tool with great potential for understanding the molecular biology of BRV and facilitates the development of novel therapeutics and vaccines for BRV.
