title: Novel approach for the generation of recombinant African swine fever virus from a field isolate using GFP expression and 5-bromo-2'-deoxyuridine selection.
abstract: Generation of African swine fever virus (ASFV) recombinants has so far relied mainly on the manipulation of virus strains which had been adapted to growth in cell culture, since field isolates do not usually replicate efficiently in established cell lines. Using wild boar lung cells (WSL) which allow for propagation of ASFV field isolates, a novel approach for the generation of recombinant ASFV directly from field isolates was developed which includes the integration into the viral thymidine kinase (TK) locus of an ASFV p72-promoter driven expression cassette for enhanced green fluorescent protein (EGFP) embedded in a 16 kbp mini F-plasmid into the genome of the ASFV field strain NHV. This procedure enabled the monitoring of recombinant virus replication by EGFP autofluorescence. Selection for the TK-negative (TK(-)) phenotype of the recombinants on TK(-) Vero (VeroTK(-)) cells in the presence of 5-bromo-2'-deoxyuridine (BrdU) led to efficient isolation of recombinant virus due to the elimination of TK(+) wild type virus by BrdU-phosporylation in infected VeroTK(-) cells. The recombinant NHV-dTK-GFP produced titres of both cell-associated and secreted viral progeny in WSL cells similar to parental NHV indicating that insertion of large heterologous sequences into the viral TK locus and EGFP expression do not impair viral replication in these cells. In summary, a novel method has been developed for generation of ASFV recombinants directly from field isolates, providing an efficacious method for further manipulations of wild-type virus genomes.
authors.affiliation: Friedrich-Loeffler-Institut, Südufer 10, 17493 Greifswald-Insel Riems, Germany.
authors.forename: Raquel
authors.initials: R
authors.lastname: Portugal
authors.forename: Carlos
authors.initials: C
authors.lastname: Martins
authors.forename: Günther M
authors.initials: GM
authors.lastname: Keil
mesh_terms: D000358:African Swine Fever Virus / Q000235:genetics* / Q000254:growth & development; D000818:Animals; D000998:Antiviral Agents / Q000378:metabolism*; D001973:Bromodeoxyuridine / Q000378:metabolism*; D002478:Cells, Cultured; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D010957:Plasmids; D011995:Recombination, Genetic; D012641:Selection, Genetic; D013194:Staining and Labeling / Q000379:methods; D034421:Sus scrofa; D014773:Virology / Q000379:methods*
chemical_list: D000998:Antiviral Agents; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D001973:Bromodeoxyuridine