Use of a Poxvirus K3 Ortholog as a Positive Selection Marker for Construction of Recombinant Vaccinia Virus with Modified Host Range.

Vaccinia virus (VACV) demonstrates a wide host range, which is determined by its host range genes including the E3L and K3L. The E3L and K3L deletion mutant VACV (VACVΔE3ΔK3) is only able to replicate in cells defective in PKR and RNase L activity. Interestingly, by expressing a K3 ortholog from another poxvirus, the host range of the VACVΔE3ΔK3 can be fine-tuned to specific host species. Accordingly, we developed a novel method for construction of recombinant VACV using the poxvirus K3 protein as a selection marker. This protocol has the advantage of being fast, cheap, and efficient. More importantly, the recombinant VACV constructed using this protocol is highly attenuated due to the absence of the virulent gene E3L.