Infectious transcripts of tick-borne encephalitis virus, generated in days by RT-PCR

Area of Science:

• Virology
• Molecular Biology
• Genetic Engineering

Background:

• Constructing infectious flavivirus clones is challenging due to instability and recombination in bacterial vectors.
• Existing methods for creating genetically engineered viruses are time-consuming and complex.

Purpose of the Study:

• To develop a rapid, simple method for producing infectious genetically engineered tick-borne encephalitis virus.
• To overcome limitations of traditional cloning techniques for RNA viruses.

Main Methods:

• Utilized high-fidelity reverse transcription-polymerase chain reaction to generate overlapping viral cDNA segments.
• Fused cDNA segments via ligation or PCR to create full-length cDNA, followed by RNA transcription.
• Injected full-length RNA intracerebrally into mice to generate infectious virus, introducing a unique restriction site for identification.

Main Results:

• Successfully produced infectious genetically engineered tick-borne encephalitis virus in less than 10 days.
• Introduced a SunI restriction site into the engineered virus, confirming its genetic modification.
• Antigenic and electrophoretic analyses showed the engineered virus was indistinguishable from the parent virus.

Conclusions:

• The developed protocol offers a rapid and efficient method for producing infectious engineered RNA viruses.
• This approach facilitates site-directed mutagenesis without the need for viral DNA cloning.
• The method is broadly applicable to other infectious RNA viruses, significantly reducing production timelines.