上海科技大学知识管理系统

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 (BSL4) laboratories. The EBOV minigenome system has provided researchers with the opportunity to study EBOV under BSL2 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 the EBOV minigenome. The minigenomic RNA harboring a Gaussia luciferase and hygromycin-resistant marker can replicate for months in a helper cell stably expressing viral nucleoprotein (NP), viral protein 35 (VP35), VP30, and L proteins. Quantification of viral RNA (vRNA), cRNA, and mRNA levels of the 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 the EBOV minigenomic vRNA ribonucleoprotein complex, which may help improve understanding of EBOV persistence in convalescent patients.