Journal of Virological Methods 183 (2012) 86–89 Contents lists available at SciVerse ScienceDirect Journal of Virological Methods j ournal homepage: www.elsevier.com/locate/jviromet Short communication Novel approach for the generation of recombinant African swine fever virus from a field isolate using GFP expression and 5-bromo-2!-deoxyuridine selection a b a, Raquel Portugal , Carlos Martins , Günther M. Keil ∗ a Friedrich-Loeffler-Institut, Südufer 10, 17493 Greifswald-Insel Riems, Germany b Laboratório de Doenc¸ as Infecciosas, CIISA, Faculdade de Medicina Veterinária, Technical University of Lisbon, Lisbon, Portugal a b s t r a c t Article history: Generation of African swine fever virus (ASFV) recombinants has so far relied mainly on the manipulation Received 4 August 2011 of virus strains which had been adapted to growth in cell culture, since field isolates do not usually Received in revised form 14 March 2012 replicate efficiently in established cell lines. Using wild boar lung cells (WSL) which allow for propagation Accepted 21 March 2012 of ASFV field isolates, a novel approach for the generation of recombinant ASFV directly from field isolates Available online 4 April 2012 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 Keywords: mini F-plasmid into the genome of the ASFV field strain NHV. This procedure enabled the monitoring of African swine fever virus recombinants recombinant virus replication by EGFP autofluorescence. Selection for the TK-negative (TK−) phenotype Field isolate of the recombinants on TK− Vero (VeroTK−) cells in the presence of 5-bromo-2!-deoxyuridine (BrdU) Green fluorescent protein + Thymidine kinase led to efficient isolation of recombinant virus due to the elimination of TK wild type virus by BrdU- BrdU selection 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. © 2012 Elsevier B.V. All rights reserved. African swine fever virus (ASFV), or as proposed recently African remain largely unknown which underlines the need to develop swine fever asfivirus (Van Regenmortel et al., 2010) is classified as strategies to facilitate the study and manipulation of this complex the sole member of the family Asfarviridae, genus Asfivirus (Dixon virus. et al., 2005). The size of the double stranded DNA genome varies Generation of ASFV recombinants has relied mainly on the between 170 and 190 kbp, depending on the virus isolate. The mutagenesis of cell-culture adapted viruses since field isolates – African swine fever virus (ASFV) infects all members of the Suidae with the exception of COS-1 cells (Hurtado et al., 2010) – do not family. In domestic pigs and wild boars it causes African swine fever grow well in cultured cells. On the other hand, working with pri- (ASF), a highly contagious hemorrhagic disease with high mortality mary swine macrophages, the natural host cells of ASFV, has proven rates for which no efficacious vaccine is available (for review see to be difficult. In this report the use of a new cell line derived from Tulman et al., 2009). Therefore it constitutes a major threat for pig wild boar lung cells (WSL, provided by the Collection of Cell Lines husbandry worldwide, highlighted particularly by the recent intro- in Veterinary Medicine, FLI Insel Riems, Germany) is described, duction of ASFV into Caucasian countries (Rowlands et al., 2008; which is suitable for efficient propagation of several ASFV field Costard et al., 2009; Rahimi et al., 2010) and its ongoing spread in isolates (unpublished results), in a novel approach for generation the affected area. of recombinant ASFV directly from the field isolate NHV, a non- The ASFV genome contains approximately 150 open read- fatal, non-haemadsorbing ASFV strain, isolated from a pig infected ing frames (ORFs) coding for proteins with functions at both chronically (Vigário et al., 1974) which provided the basis for a use- the cellular and the viral replication and morphogenesis levels ful and reliable infection model for studies on the mechanisms of (Yánez˜ et al., 1995) which account for the high complexity of the protective immunity (Leitao et al., 2001). To this end, heterologous virus-host interactions. Pathogenesis and virulence determinants sequences encompassing the gene for enhanced green fluorescent protein (EGFP) were integrated into the thymidine kinase (TK) locus of NHV Cells infected with the TK-negative, EGFP-positive recom- binant virus could be detected easily by fluorescence microscopy. ∗ Corresponding author. Tel.: +49 38351 71272; fax: +49 38351 71151. E-mail address: Guenther.Keil@fli.bund.de (G.M. Keil). Subsequently, viral mutants were selected positively on a 0166-0934/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jviromet.2012.03.030 R. Portugal et al. / Journal of Virological Methods 183 (2012) 86–89 87 TK-negative Vero cell line using BrdU to eliminate TK-positive wild type virus. For generation of recombinant ASFV, a transfer plasmid con- taining the EGFP ORF under transcriptional control of the promoter from the gene encoding vp72 (López-Otín et al., 1990), the major viral structural protein, and flanked by segments of the viral TK gene, was constructed (Fig. 1). The resulting plasmid pASFV-dTK- EGFP-BAC-Lox had been designed initially for cloning of the ASFV genome as a bacterial artificial chromosome. It contains the viral TK-spanning locus from nt 43,995 to nt 50,739 with the same 316 bp deletion within the TK ORF as described by Moore et al. (1998). The TK ORF flanking sequences are both about 3 kbp in size to provide longer sequence segments for homologous recombination as used in previous constructs to target the same genomic region (Moore et al., 1998). To generate recombinants, 4 ␮g of plasmid pASFV-dTK-EGFP- BAC-Lox were transfected into semi-confluent WSL cells in 6-well 6 plates (approximately 10 cells per well) using the FuGene HD transfection reagent as recommended by the supplier (Roche, Mannheim, Germany). The medium was removed 5 h after trans- fection and the cells were infected with NHV at an MOI of 2. The inoculum was removed 1 h after adsorption. Cells were washed with culture medium and incubated further in fresh medium for 3 days, when autofluorescing foci of rounded and granulated cells indicated productive replication of recombinant virus. Infected cells from these foci were collected by aspiration and re-inoculated onto WSL cells after one 70 C freeze/thaw cycle. Cells from aut- − ◦ ofluorescing foci were harvested as mentioned above and used for infection of bromodeoxyuridine (BrdU)-resistant Vero (VeroTK−) cells in presence of 50 ␮g/ml BrdU for positive selection of recom- binants. VeroTK− cells were selected using a strategy employed by Bello et al. (1987) for MDBK cells and Kit et al. (1966) for HeLa cells and kindly provided by Roland Riebe, FLI, Insel Riems, Germany. Appearance of autofluorescent cells was monitored daily. At 7 dpi cultures were harvested and after 2 freeze/thaw cycles aliquots of Fig. 1. Construction of recombination plasmid pASFV-dTK-EGFP-BAC Lox. (A) the virus/cell suspension were again added to Vero TK− cells and Schematic representation of the ASFV genome region containing the TK ORF. incubated in the presence of 50 ␮g/ml BrdU. Nucleotide numbers are given in kilobases (kb), names and direction of transcrip- tion of contained ORFs are indicated. The location of the TK-ORF (K196R) is shown in Fluorescent foci which consisted of only a few cells and thus bold. (B) Plasmid constructions. The left (TK-L) and right (TK-R) segments of the ASFV were considerably smaller than foci in WSL cell cultures, were col- TK gene and respective flanking sequences were amplified by PCR from infected lected and passaged again on Vero TK cells in medium containing − macrophages DNA. Primers used were TK-L+ (GTG GGC GTA TAG ATA AGG ATA TC) 50 ␮g/ml BrdU. After two further rounds of positive selection, GFP- and TK-L (TAA GGT ACC GTG TTT TAA TAG TTT TGT CTC GGG TG) amplifying a − 3207 bp fragment from nt 43,995 to nt 47,201 (TK-L), and primers TK-R+ (TGA CCC expressing infected cells were freeze/thawed twice and used for GGG CGT AAG AAC GCA GAC AAG ACG C) and TK-R (CCT GCT CGT GTT ACT TAT the infection of WSL cells to obtain high titre stocks of recombi- − GAA AC) amplifying a 3236 bp fragment from nt 47,504 to nt 50,739 (TK-R). All nant virus. Large fluorescent foci readily developed and finally led nucleotide numbers are from GenBank accession # U18466.1. Both amplicons were to the isolation of the ASFV recombinant NHV-dTK-GFP. To test sequentially cloned into plasmid vector pSP73 (Promega) using established standard for homogeneity of the recombinant virus preparation, WSL cul- procedures. TK-L and TK-R were blunt ended with Klenow polymerase. TK-L was then cleaved with Acc65I and inserted into pSP73 cleaved with Acc65I and EcoRV. tures on coverslips were inoculated with approximately 100 PFU. TK-R was cleaved with SmaI, and cloned into the TK-L containing plasmid after cleav- At 4 days p.i., cells were fixed and stained for detection of ASFV age with SmaI to yield pspASFV-dTK, containing the viral TK-spanning locus from nt infected cells by indirect immunofluorescence using mouse mon- 43,995 to nt 50,739 with a 304 bp deletion from 47,202 to 47,503.