Cloning of equine herpesvirus type 1 438/77 strain genome as a bacterial artificial chromosome.

The research focused on the cloning of equine herpesvirus type 1 (a virus that causes respiratory and reproductive diseases in horses) strain 438/77’s genome as a bacterial artificial chromosome in an E. coli bacterial model. The team managed to do this without any gene deletions and were able to successfully reconstitute the recombinant virus.

Procedure

• The team began by inserting the mini-F plasmid sequence in the middle of ORF19 and 20 using homologous recombination. This was done after the co-transfection of viral DNA and plasmid (pE19_20/HA) into RK13 cells.
• Next, they extracted circular viral DNA from RK13 cells that were infected with a recombinant virus with Green Fluorescent Protein (GFP) expression, and then injected this into E. coli DH10B cells using electroporation.

Analysis and Reconstitution

• The clone that had the bacterial artificial chromosome (BAC) was screened and analyzed using PCR and RFLP – techniques used to multiply and map genetic material respectively.
• Once the clone had been verified, the process of reconstituting the recombinant virus began through transfusion of the BAC DNA into RK13 cells.

Post Reconstitution Analysis

• After reconstitution, the research team removed the mini-F sequence in the reconstituted virus using homologous recombination between virus DNA and plasmid pE1920XM. This process caused a point mutation in the Xbal site located in ORF19.
• They then compared restriction fragment length polymorphism (RFLP) profiles from the rescued, recovered and wild-type viral genome to ensure there were no unexpected changes from the mutagenesis.
• Lastly, they ran in vitro replication assays to confirm that the bacterial artificial chromosome (BAC)-reconstituted virus mutant’s growth kinetics and plaque formation morphologies/sizes were undistinguishable relative to those of the wild-type virus.