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
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.