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A simple method for developing an infectious cDNA clone of Japanese encephalitis virus
Hao Zheng1,2, Xuchen Zheng1, Wu Tong1,2
1Department of Swine Infectious Diseases, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, 200241, China.
Virus Genes
|September 26, 2016
Summary
Researchers developed a stable Japanese encephalitis virus (JEV) cDNA clone for easier manipulation. A specific mutation reduced viral growth and neurovirulence, aiding JEV replication and pathogenesis studies.
Area of Science:
- Virology
- Molecular Biology
- Genetics
Background:
- Flavivirus cDNA clones often exhibit genetic instability in bacteria, complicating infectious virus construction.
- This instability hinders research into flavivirus replication and pathogenesis.
Purpose of the Study:
- To develop a stable, full-length cDNA clone for Japanese encephalitis virus (JEV).
- To investigate the role of NS2a frameshifting in JEV replication and neurovirulence.
Main Methods:
- Constructed a stable JEV cDNA clone (pJEHEN) using a pBR322 vector and room temperature propagation.
- Introduced a silent mutation in the NS2a gene to prevent frameshifting, creating clone vJETA.
- Rescued viruses from cDNA clones and compared them to the parent virus (HEN0701) and each other.
Main Results:
- The stable clone pJEHEN rescued a virus (vJEHEN) indistinguishable from the parent virus.
- The vJETA mutant, lacking NS1' expression due to the mutation, showed reduced growth kinetics.
- The vJETA mutant exhibited decreased neurovirulence in mouse models.
Conclusions:
- A stable JEV cDNA cloning method was successfully developed.
- Eliminating NS2a frameshifting impacts JEV replication and neurovirulence.
- This approach facilitates further research into JEV molecular mechanisms.