Ebola virus (EBOV) causes severe hemorrhagic fever in humans and other primates with a high case fatality rate. No approved drug or vaccine of EBOV is available， which necessitates better understanding of the virus life cycle. Studies on EBOV have been hampered because experimentations involving live virus are restricted to biosafety level 4 (BSL-4) laboratories. EBOV minigenome system has provided researchers with the opportunity to study EBOV under BSL-2 conditions. Here， we developed a novel EBOV minigenome replicon which， to our knowledge， is the first EBOV cell culture system that can stably replicate and transcribe EBOV minigenome. The minigenomic RNA harboring a Luciferase and hygromycin-resista... More
Ebola virus (EBOV) causes severe hemorrhagic fever in humans and other primates with a high case fatality rate. No approved drug or vaccine of EBOV is available， which necessitates better understanding of the virus life cycle. Studies on EBOV have been hampered because experimentations involving live virus are restricted to biosafety level 4 (BSL-4) laboratories. EBOV minigenome system has provided researchers with the opportunity to study EBOV under BSL-2 conditions. Here， we developed a novel EBOV minigenome replicon which， to our knowledge， is the first EBOV cell culture system that can stably replicate and transcribe EBOV minigenome. The minigenomic RNA harboring a Luciferase and hygromycin-resistant marker can replicate for months in a helper cell stably expressing viral NP， VP35， VP30 and L proteins. Quantification of vRNA， cRNA and mRNA levels of EBOV minigenome demonstrated that the stable EBOV replicon had much more active minigenome replication than previously developed transient transfection-based EBOV minigenome systems which recapitulate viral primary transcription more than genome replication. Interestingly， minigenome replication in the stable EBOV replicon cells was insensitive to interferon treatment or RNA interference. Moreover， RNase digestion of the replicon cell lysates revealed the remarkably stable nature of EBOV minigenomic vRNA ribonucleoprotein complex， which may help understand EBOV persistence in convalescent patients. The scope and severity of the recent Ebola outbreak in the Western Africa justified more comprehensive investigation of the causative Risk Group 4 agent Ebola virus (EBOV). Study of EBOV replication and antiviral development can be facilitated by developing a cell culture system that allows for experimentations in the biosafety level 2 conditions. Here， we developed a novel stable EBOV minigenome replicon which， to our knowledge， is the first EBOV cell culture system that can stably replicate and transcribe EBOV minigenome. Compared to previously developed transient transfection-based EBOV minigenome systems， the replicon system had more active genome replication， providing a convenient surrogate system to study EBOV replication. Furthermore， self-replicating minigenomic vRNA in the replicon cells displayed strong stability in response to interferon treatment， RNA silencing and RNase digestion， which may provide an explanation for the persistence of EBOV in survivors.
