1. Academic Validation
  2. Development of a New Reverse Genetics System for Ebola Virus

Development of a New Reverse Genetics System for Ebola Virus

  • mSphere. 2021 May 5;6(3):e00235-21. doi: 10.1128/mSphere.00235-21.
Tianyu Gan  # 1 2 Dihan Zhou  # 3 Yi Huang  # 4 Shuqi Xiao 4 Ziyue Ma 1 5 Xiaoyou Hu 1 2 Yimin Tong 1 Huimin Yan 6 2 Jin Zhong 7 2 5
Affiliations

Affiliations

  • 1 Unit of Viral Hepatitis, CAS Key Laboratory of Molecular Virology and Immunology, Institut Pasteur of Shanghai, Center for Biosafety Mega-Science, Chinese Academy of Sciences, Shanghai, China.
  • 2 University of Chinese Academy of Sciences, Beijing, China.
  • 3 The Joint Laboratory for Translational Precision Medicine, Guangzhou Women and Children's Medical Center, Guangzhou, China, and Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China.
  • 4 Wuhan National Biosafety Laboratory, Center for Biosafety Mega-Science, Chinese Academy of Sciences, Wuhan, China.
  • 5 ShanghaiTech University, Shanghai, China.
  • 6 Mucosal Immunity Research Group, State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China hmyan@wh.iov.cn jzhong@ips.ac.cn.
  • 7 Unit of Viral Hepatitis, CAS Key Laboratory of Molecular Virology and Immunology, Institut Pasteur of Shanghai, Center for Biosafety Mega-Science, Chinese Academy of Sciences, Shanghai, China hmyan@wh.iov.cn jzhong@ips.ac.cn.
  • # Contributed equally.
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.

Keywords

Ebola virus; filovirus; negative-stranded RNA virus; reverse genetics.

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