Temperature-sensitive mutants of Japanese encephalitis virus

Area of Science:

• Virology
• Molecular Biology
• Genetics

Background:

• Japanese encephalitis virus (JEV) is a significant human pathogen responsible for neurological disease.
• Understanding JEV's genetic makeup is crucial for developing effective antiviral strategies.
• Temperature-sensitive (ts) mutants are valuable tools for studying viral replication and gene function.

Purpose of the Study:

• To isolate and characterize temperature-sensitive mutants of Japanese encephalitis virus.
• To define complementation groups and analyze the biochemical phenotypes of these mutants.
• To investigate complementation and interference phenomena in flavivirus mutants.

Main Methods:

• Mutagenesis of cloned wild-type JEV using nucleic acid precursor analogs (5-fluorouracil and 5-azacytidine).
• Selection of mutants exhibiting a significant growth defect at the nonpermissive temperature (41°C) compared to the permissive temperature (33°C).
• Analysis of virus-specific RNA and protein synthesis at the nonpermissive temperature, and assessment of complementation in mixed infections.

Main Results:

• Ten stable temperature-sensitive JEV mutants were isolated, with 5-fluorouracil proving more effective than 5-azacytidine.
• Seven distinct complementation groups were established based on mutant behavior in mixed infections.
• Mutants were classified by their ability to synthesize viral RNA (RNA+ or RNA-) and protein levels (wild-type, low, or undetectable) at the nonpermissive temperature.

Conclusions:

• Complementation occurred at higher levels than previously reported for flaviviruses, with some instances of nonreciprocal complementation and interference.
• The defined complementation groups provide insights into the genetic organization and functional roles of JEV genes.
• These ts mutants serve as a valuable resource for further genetic and biochemical studies of Japanese encephalitis virus.