Rapid, Seamless Generation of Recombinant Poxviruses using Host Range and Visual Selection

Area of Science:

• Virology
• Molecular Biology
• Genetic Engineering

Background:

• Vaccinia virus (VACV) is a versatile viral vector used in vaccines and oncolytic therapies.
• Existing methods for generating recombinant VACV often involve drug selection or extensive screening.
• Efficient generation of recombinant VACV is crucial for its therapeutic applications.

Purpose of the Study:

• To develop a refined host range selection method for generating recombinant VACV.
• To utilize host antiviral protein kinase R (PKR) and a fluorescent reporter for selection.
• To enable seamless and efficient generation of recombinant VACV without drug selection.

Main Methods:

• A host range selection method was refined using PKR pressure and mCherry-E3L fluorescent fusion.
• The method employs a cassette with the gene of interest and mCherry-E3L, flanked by VACV sequences.
• Homologous recombination promotes the loss of the mCherry-E3L gene after selection.

Main Results:

• The developed method allows for efficient and seamless generation of recombinant VACV.
• This technique is effective across various cell types.
• No drug selection or extensive screening was required for mutant virus identification.

Conclusions:

• This refined method offers an improved approach for generating recombinant VACV.
• The strategy simplifies the production of VACV-based therapeutic vectors.
• The technique is broadly applicable for generating genetically modified VACV.