Genetic manipulation of African swine fever virus: Construction of recombinant viruses expressing the β-galactosidase gene

Abstract

Homologous recombination is shown to be specifically induced in Vero cells by infection with African swine fever (ASF) virus. The frequency of recombination induced by ASF virus infection between cotransfecting plasmids is comparable to that found after infection with the prototype poxvirus, vaccinia virus. The induction of recombination is accompanied by replication of the plasmid templates in the ASF virus-infected cells. An ASF virus insertion/expression plasmid vector containing the Escherichia coli reporter gene β-galactosidase (β-gal) fused to a viral promoter sequence was constructed. Recombination between homologous sequences present in both the plasmid vector and the virus genome led to the generation of recombinant viruses expressing the β-gal gene. Visual screening of β-gal + plaques allowed the isolation and plaque purification of recombinant ASF viruses. The characterization of a β-gal + virus isolate showed that the β-gal gene had been stably inserted into the thymidine kinase locus of the virus genome, thus demonstrating that controlled genetic manipulation of ASF virus can be achieved by homologous recombination in infected cells.