Development of a New Reverse Genetics System for Ebola Virus

Abstract

Ebola virus (EBOV) is a highly pathogenic negative-stranded RNA virus that has caused several deadly endemics in the past decades. EBOV reverse genetics systems are available for studying live viruses under biosafety level 4 (BSL-4) or subviral particles under BSL-2 conditions. However, these systems all require cotransfection of multiple plasmids expressing viral genome and viral proteins essential for EBOV replication, which is technically challenging and unable to naturally mimic virus propagation using the subviral particle. Here, we established a new EBOV reverse genetics system only requiring transfection of a single viral RNA genome into an engineered cell line that stably expresses viral nucleoprotein (NP), viral protein 35 (VP35), VP30, and large (L) proteins and has been fine-tuned for its superior permissiveness for EBOV replication. Using this system, subviral particles expressing viral VP40, glycoprotein (GP), and VP24 could be produced and continuously propagated and eventually infect the entire cell population. We demonstrated the authentic response of the subviral system to antivirals and uncovered that the VP35 amount is critical for optimal virus replication. Furthermore, we showed that fully infectious virions can be efficiently rescued by delivering the full-length EBOV genome into the same supporting cell, and the efficiency is not affected by genome polarity or virus variant specificity. In summary, our work provides a new tool for studying EBOV under different biosafety levels. IMPORTANCE Ebola virus is among the most dangerous viral pathogens, with a case fatality rate of up to 90%. Since 2013, the two largest and most complex Ebola outbreaks in Africa have revealed the lack of investigation on this notorious virus. A reverse genetics system is an important tool for studying viruses by producing mutant viruses or generating safer and convenient model systems. Here, we developed an EBOV life cycle modeling system in which subviral particles can spontaneously propagate in cell culture. In addition, this system can be employed to rescue infectious virions of homologous or heterologous EBOV isolates using either sense or antisense viral RNA genomes. In summary, we developed a new tool for EBOV research.

A novel BSL-2 Lassa virus reverse genetics system modelling the complete viral life cycle

“…In this study, we successfully developed a novel reverse genetics system to study the complete life cycle of LASV under BSL-2 conditions. A similar strategy has been used to rescue the genome-deficient virions in a stable cell line for other highly pathogenetic viruses, such as Ebola virus (EBOV) [ 36 , 37 ] and SARS-CoV-2 [ 38 ]. Different from previously developed chimeric viruses, all viral genes and cis -acting elements within LASVmg are derived from LASV itself.…”

Development and optimization of biologically contained Marburg virus for high-throughput antiviral screening

“…The key advantage of the systems described here is that they can be used outside of a BSL-4 setting, thereby making research using replication-competent filoviruses available to a much bigger research community. Other virus alternatives that can be used in lower biosafety settings have previously been developed for EBOV and MARV, including minigenome systems, VLP systems and even trVLP systems [8, 9, 35, 36, 37]. While each of these surrogate systems has its advantages, they all have their limitations, including their dependency on repeated transfections or being limited to specific parts of the virus life cycle.…”

Developments in Negative-Strand RNA Virus Reverse Genetics

“…Later, Tianyu Gan et al established a new reverse genetic system for EBOV that only requires a single viral RNA genome to be transfected into a cell line expressing the viral NP, VP35, VP30, and L proteins. This modification enables efficient replication of EBOV ( Figure 5 ) [ 73 ].…”