VECTORS FOR THE GENETIC MANIPULATION OF AFRICAN SWINE FEVER VIRUS

Plasmid vectors designed to facilitate the genetic manipulation of Afr ican swine fever virus (ASFV) are described. Our results demonstrate t hat the beta-glucuronidase enzyme (GUS) can be used to follow gene exp ression in ASFV-infected cells. Infectious plaques formed by ASFV expr essing GUS are visually detectable, thus providing a simple and highly sensitive method for the selection of ASFV recombinants. These and pr evious results have allowed us to construct two chimeric gene cassette s that constitute the basic tools for the generation of vectors to car ry out the deletion of multiple target sequences from the ASFV genome. These cassettes, formed by: (a) a virus promoter; (b) the coding sequ ence of a reporter gene, either Lac Z or gusA; and (c) a strong signal for the 3' end formation of ASFV mRNAs, can be easily isolated by end onuclease restriction from their corresponding plasmid vectors. A gene ral insertion/coexpression plasmid vector, pEPV2, has also been constr ucted. pEPV2 facilitates the insertion of foreign genes, together with the Lac Z reporter, into the thymidine kinase locus of ASFV. The func tionality of pEPV2 has been tested by generating a recombinant ASFV ex pressing the luciferase gene. The vectors presented in this report con stitute the first reported set of tools for the genetic manipulation o f ASFV.