Many flaviviruses are emerging and reemerging pathogens, such as West Nile virus （WNV）, dengue virus （DENV）, yellow fever virus （YFV）, and Japanese encephalitis virus. Serological assay is the dominant method fo...Many flaviviruses are emerging and reemerging pathogens, such as West Nile virus （WNV）, dengue virus （DENV）, yellow fever virus （YFV）, and Japanese encephalitis virus. Serological assay is the dominant method for diagnosis of flavivirus infections in human. Because antibodies generated during flavivirus infections cross-react with other flavivirus members, plaque reduction neutralization test （PRNT） is the only available assay to determine the infecting flavivirus type. Since PRNT requires culturing raw viruses, it must be performed in biosafety levet-3 or level-4 containment for many flaviviruses, and takes more than ten days to complete. To overcome these problems, we have developed flavivirus viral-like particles （VLPs） that could be used to replace raw viruses in the neutralization assay. The VLPs were prepared by trans packaging a luciferase-reporting replicon with viral structural proteins. This novel assay involves three simple steps： （i） VLPs from a panel of flaviviruses are incubated with flavivirus-infected sera at 37℃ for 1 h; （ii）the neutralized VLPs are used to infect Vero cells; and （iii） the infected cells are measured for luciferase activities at 22 h post-infection. The virus type whose VLP is most efficiently neutralized by the serum specimen （as quantified by the luciferase activities） is the etiologic agent. As a proof-of-concept, we show that a WNV-infected mouse serum neutralized the WNV VLP more efficiently and selectively than the DENV and YFV VLPs. Our results demonstrate that the VLP neutralization assay maintains the ＂gold standard＂ of the classic PRNT; importantly, it shortens the assay time from 〉10 days to 〈1 day, and can be performed in biosafety level-2 facility.展开更多
Several variants of concern(VOCs)have emerged since the WIV04 strain of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)was first isolated in January 2020.Due to mutations in the spike(S)protein,these VOCs ...Several variants of concern(VOCs)have emerged since the WIV04 strain of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)was first isolated in January 2020.Due to mutations in the spike(S)protein,these VOCs have evolved to enhance viral infectivity and immune evasion.However,whether mutations of the other viral proteins lead to altered viral propagation and drug resistance remains obscure.The replicon is a noninfectious viral surrogate capable of recapitulating certain steps of the viral life cycle.Although several SARS-CoV-2 replicons have been developed,none of them were derived from emerging VOCs and could only recapitulate viral genome replication and subgenomic RNA(sgRNA)transcription.In this study,SARS-CoV-2 replicons derived from the WIV04 strain and two VOCs(the Beta and Delta variants)were prepared by removing the S gene from their genomes,while other structural genes remained untouched.These replicons not only recapitulate viral genome replication and sgRNA transcription but also support the assembly and release of viral-like particles,as manifested by electron microscopic assays.Thus,the S-deletion replicon could recapitulate virtually all the post-entry steps of the viral life cycle and provides a versatile tool for measuring viral intracellular propagation and screening novel antiviral drugs,including inhibitors of virion assembly and release.Through the quantification of replicon RNA released into the supernatant,we demonstrate that viral intracellular propagation and drug response to remdesivir have not yet substantially changed during the evolution of SARS-CoV-2 from the WIV04 strain to the Beta and Delta VOCs.展开更多
基金supported by National Institute of Health grants U01 AI061193 and U54-AI057158 (Northeast Biodefense Center).
文摘Many flaviviruses are emerging and reemerging pathogens, such as West Nile virus （WNV）, dengue virus （DENV）, yellow fever virus （YFV）, and Japanese encephalitis virus. Serological assay is the dominant method for diagnosis of flavivirus infections in human. Because antibodies generated during flavivirus infections cross-react with other flavivirus members, plaque reduction neutralization test （PRNT） is the only available assay to determine the infecting flavivirus type. Since PRNT requires culturing raw viruses, it must be performed in biosafety levet-3 or level-4 containment for many flaviviruses, and takes more than ten days to complete. To overcome these problems, we have developed flavivirus viral-like particles （VLPs） that could be used to replace raw viruses in the neutralization assay. The VLPs were prepared by trans packaging a luciferase-reporting replicon with viral structural proteins. This novel assay involves three simple steps： （i） VLPs from a panel of flaviviruses are incubated with flavivirus-infected sera at 37℃ for 1 h; （ii）the neutralized VLPs are used to infect Vero cells; and （iii） the infected cells are measured for luciferase activities at 22 h post-infection. The virus type whose VLP is most efficiently neutralized by the serum specimen （as quantified by the luciferase activities） is the etiologic agent. As a proof-of-concept, we show that a WNV-infected mouse serum neutralized the WNV VLP more efficiently and selectively than the DENV and YFV VLPs. Our results demonstrate that the VLP neutralization assay maintains the ＂gold standard＂ of the classic PRNT; importantly, it shortens the assay time from 〉10 days to 〈1 day, and can be performed in biosafety level-2 facility.
基金supported by grants from the National Key Research and Development Project of China (2020YFC0845900)the China Postdoctoral Science Foundation (2020T130021ZX,2021M693198)
文摘Several variants of concern(VOCs)have emerged since the WIV04 strain of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)was first isolated in January 2020.Due to mutations in the spike(S)protein,these VOCs have evolved to enhance viral infectivity and immune evasion.However,whether mutations of the other viral proteins lead to altered viral propagation and drug resistance remains obscure.The replicon is a noninfectious viral surrogate capable of recapitulating certain steps of the viral life cycle.Although several SARS-CoV-2 replicons have been developed,none of them were derived from emerging VOCs and could only recapitulate viral genome replication and subgenomic RNA(sgRNA)transcription.In this study,SARS-CoV-2 replicons derived from the WIV04 strain and two VOCs(the Beta and Delta variants)were prepared by removing the S gene from their genomes,while other structural genes remained untouched.These replicons not only recapitulate viral genome replication and sgRNA transcription but also support the assembly and release of viral-like particles,as manifested by electron microscopic assays.Thus,the S-deletion replicon could recapitulate virtually all the post-entry steps of the viral life cycle and provides a versatile tool for measuring viral intracellular propagation and screening novel antiviral drugs,including inhibitors of virion assembly and release.Through the quantification of replicon RNA released into the supernatant,we demonstrate that viral intracellular propagation and drug response to remdesivir have not yet substantially changed during the evolution of SARS-CoV-2 from the WIV04 strain to the Beta and Delta VOCs.
