Development,Characterization And Application Of Novel Reverse Genetics Systems For Ebola Virus

Ebola virus(EBOV)is a highly pathogenic agent which causes severe hemorrhagic fever in humans and other primates with a high case-fatality rate.The biggest Ebola outbreak on record is the west Africa epidemic from 2013 to 2016 with almost 30,000 cases and over 11,000 deaths.In the ongoing outbreak in the Democratic Republic of the Congo(DRC)starting from 2018,EBOV has caused a higher case-fatality rate than that of the 2013-2016 outbreak with more than 3,400 cases and over 2,200 deaths,even though a vaccine has been widely applied to more than 300,000 people.With continuous outbreaks of huge impact and no licencsed therapeutics,it necessitates more and deeper understanding of EBOVStudies on EBOV have been hampered because experimentations involving live virus are restricted to Biosafety Level 4(BSL-4)laboratories.To study the virus outside the BSL-4 laboratory,various reverse genetics systems have been developed,including the minigenome system and transcription-and replication-competent virus-like particle(trVLP)system,to recapitulate different steps of EBOV life cycle.The current reverse genetics systems for EBOV,however,all require co-transfection of multiple plasmids expressing viral proteins as well as a viral genome-encoding plasmid often accompanied with an additional plasmid expressing an exogenous polymerase for transcription of viral genome,which not only complicates the handling and experimental settings but also limits the target cell lines to those easily transfectable cells such as HEK293TIn the first part of this study,we improved the minigenome system by developing a minigenome replicon system that can stably replicate and transcribe the EBOV minigenome.The minigenomic RNA harboring a Gaussia luciferase and hygromycin-resistant marker can replicate for months in Huh7-4P cells which stably expresses viral nucleoprotein,polymerase co-factor VP35,transcriptional activator VP30,and polymerase L.Quantification of viral RNA(vRNA),complementary RNA(cRNA),and mRNA levels of the EBOV minigenome demonstrated that the stable replicon has more-active genome replication than previously developed transient-transfection-based minigenome systems,providing a better surrogate system to study EBOV replication Interestingly,minigenome replication in the stable replicon system was insensitive to interferon treatment or RNA interference,suggesting the limit of these two therapeutic strategies.Moreover,RNase digestion of the replicon cell lysates revealed the remarkably stable nature of the vRNA ribonucleoprotein complex,which may help improve the understanding of EBOV persistence in convalescent patients.In the second part of this study,we first developed a novel EBOV trVLP system.Huh7-4P cell line was optimized with phenotypic selection and subcloning to generate Huh7-4PX cell line which expresses optimal levels of viral proteins.EBOV subgenome that encodes viral protein VP40,GP and VP24 could be efficiently replicated and transcribed and the resultant subgenomic trVLP could be continuously propagated in Huh7-4PX cells.Characterization of the trVLP system showed that it could be used as an authentic surrogate of infectious virus to evaluate anti-EBOV drugs and antibodies.We also showed that viral VP40 mutations emerging in the recent DRC outbreak accelerate trVLP propagation.Thus,the novel trVLP system provides a convenient tool for studying EBOV virology under the BSL-2 condition.