ELSEVIER Journal of Biotechnology 40 (1995) 121-131 Vectors for the genetic manipulation of African swine fever virus Ramh Garcia, Fernando Almazhn, Javier Maria Rodriguez ‘, Marta Alonso 2, Eladio Viiiuela, Jose Francisco Rodriguez * zyxwvutsrqponmlkjihgfedcbaZ Centro de Biologfa Molecular “Sever0 Ochoa” (CSIC-UAM), Facultad de Ciencias, lJniL>ersidad Autdnoma, Cantohlanco. 28049 M adrid, Spain Received 19 January 1995; accepted 1 March 1995 zyxwvutsrqponmlkjihgfedcbaZYXWVUTS Abstract Plasmid vectors designed to facilitate the genetic manipulation of African swine fever virus (ASFV) are described. Our results demonstrate that the /?-glucuronidase enzyme (GUS) can be used to follow gene expression in ASFV-infected cells. Infectious plaques formed by ASFV expressing GUS are visually detectable, thus providing a simple and highly sensitive method for the selection of ASFV recombinants. These and previous results have allowed us to construct two chimeric gene cassettes that constitute the basic tools for the generation of vectors to carry out the deletion of multiple target sequences from the ASFV genome. These cassettes, formed by: (a) a virus promoter; (b) the coding sequence 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 endonuclease restriction from their corresponding plasmid vectors. A general insertion/coexpression plasmid vector, pEPV2, has also been constructed. pEPV2 facilitates the insertion of foreign genes, together with the Lac Z reporter, into the thymidine kinase locus of ASFV. The functionality of pEPV2 has been tested by generating a recombinant ASF’V expressing the luciferase gene. The vectors presented in this report constitute the first reported set of tools for the genetic manipulation of ASFV. Keywords: African swine fever virus; Genetic manipulation; Vector; GUS; Lac Z; Luciferase 1. Introduction African swine fever virus (ASFV), the only known member of an, as yet, unnamed family of DNA viruses, is the etiological agent of an eco- nomically important disease of swine. In addition to its ability to infect different species of suids, ASFV infects soft ticks of the Ornithodoros genus (reviewed by Viiiuela, 1987; Wilkinson, 1989). The genome of ASFV is formed by a single molecule of double-stranded DNA ranging in size from 170 to 190 kilobases (kb) depending upon the virus strain (Viiiuela, 1987). The complete nucleotide sequence of the genome from the BA71V strain of the virus has been determined * Corresponding author. ’ Present address: Institute of Virology and Environmental Microbiology, Mansfield Road, Oxford, OX1 3SR, UK. ’ Present address: Centro de Investigaciones BioMgicas (CSIC), VelLzquez 144, 28006 Madrid, Spain. and has revealed the existence of more than 150 putative genes (unpublished results). The func- tion of most of the virus-encoded proteins is completely unknown. 0168-1656/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SSDI 0168-1656(95)00037-2