Journal of Virological Methods 183 (2012) 86–89
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Journal of Virological Methods
journal 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
Raquel Portugal a , Carlos Martins b , Günther M. Keil a,∗
a
b
Friedrich-Loeffler-Institut, Südufer 10, 17493 Greifswald-Insel Riems, Germany
Laboratório de Doenças Infecciosas, CIISA, Faculdade de Medicina Veterinária, Technical University of Lisbon, Lisbon, Portugal
a b s t r a c t
Article history:
Received 4 August 2011
Received in revised form 14 March 2012
Accepted 21 March 2012
Available online 4 April 2012
Keywords:
African swine fever virus recombinants
Field isolate
Green fluorescent protein
Thymidine kinase
BrdU selection
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 p72promoter 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 BrdUphosporylation in infected VeroTK− cells. The recombinant NHV-dTK-GFP produced titres of both cellassociated 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
swine fever asfivirus (Van Regenmortel et al., 2010) is classified as
the sole member of the family Asfarviridae, genus Asfivirus (Dixon
et al., 2005). The size of the double stranded DNA genome varies
between 170 and 190 kbp, depending on the virus isolate. The
African swine fever virus (ASFV) infects all members of the Suidae
family. In domestic pigs and wild boars it causes African swine fever
(ASF), a highly contagious hemorrhagic disease with high mortality
rates for which no efficacious vaccine is available (for review see
Tulman et al., 2009). Therefore it constitutes a major threat for pig
husbandry worldwide, highlighted particularly by the recent introduction of ASFV into Caucasian countries (Rowlands et al., 2008;
Costard et al., 2009; Rahimi et al., 2010) and its ongoing spread in
the affected area.
The ASFV genome contains approximately 150 open reading frames (ORFs) coding for proteins with functions at both
the cellular and the viral replication and morphogenesis levels
(Yáñez et al., 1995) which account for the high complexity of the
virus-host interactions. Pathogenesis and virulence determinants
∗ Corresponding author. Tel.: +49 38351 71272; fax: +49 38351 71151.
E-mail address: [email protected] (G.M. Keil).
0166-0934/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.jviromet.2012.03.030
remain largely unknown which underlines the need to develop
strategies to facilitate the study and manipulation of this complex
virus.
Generation of ASFV recombinants has relied mainly on the
mutagenesis of cell-culture adapted viruses since field isolates –
with the exception of COS-1 cells (Hurtado et al., 2010) – do not
grow well in cultured cells. On the other hand, working with primary swine macrophages, the natural host cells of ASFV, has proven
to be difficult. In this report the use of a new cell line derived from
wild boar lung cells (WSL, provided by the Collection of Cell Lines
in Veterinary Medicine, FLI Insel Riems, Germany) is described,
which is suitable for efficient propagation of several ASFV field
isolates (unpublished results), in a novel approach for generation
of recombinant ASFV directly from the field isolate NHV, a nonfatal, non-haemadsorbing ASFV strain, isolated from a pig infected
chronically (Vigário et al., 1974) which provided the basis for a useful and reliable infection model for studies on the mechanisms of
protective immunity (Leitao et al., 2001). To this end, heterologous
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 recombinant virus could be detected easily by fluorescence microscopy.
Subsequently, viral mutants were selected positively on a
R. Portugal et al. / Journal of Virological Methods 183 (2012) 86–89
TK-negative Vero cell line using BrdU to eliminate TK-positive wild
type virus.
For generation of recombinant ASFV, a transfer plasmid containing 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-dTKEGFP-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-EGFPBAC-Lox were transfected into semi-confluent WSL cells in 6-well
plates (approximately 106 cells per well) using the FuGene HD
transfection reagent as recommended by the supplier (Roche,
Mannheim, Germany). The medium was removed 5 h after transfection 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 autofluorescing 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 recombinants. 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
the virus/cell suspension were again added to Vero TK− cells and
incubated in the presence of 50 !g/ml BrdU.
Fluorescent foci which consisted of only a few cells and thus
were considerably smaller than foci in WSL cell cultures, were collected and passaged again on Vero TK− cells in medium containing
50 !g/ml BrdU. After two further rounds of positive selection, GFPexpressing infected cells were freeze/thawed twice and used for
the infection of WSL cells to obtain high titre stocks of recombinant virus. Large fluorescent foci readily developed and finally led
to the isolation of the ASFV recombinant NHV-dTK-GFP. To test
for homogeneity of the recombinant virus preparation, WSL cultures on coverslips were inoculated with approximately 100 PFU.
At 4 days p.i., cells were fixed and stained for detection of ASFV
infected cells by indirect immunofluorescence using mouse monoclonal antibody C18 directed against the early viral protein vp30
(kindly provided by Linda Dixon, Pirbright, UK). Fig. 2A shows that
all foci with green autofluorescence (left picture) were recognized
also by the anti-vp30 antibodies (red fluorescence, right picture).
No non-autofluorescing foci were observed among more than 100
infected cell foci, indicating that the recombinant virus stock was
essentially free from contaminating parental NHV virus. This conclusion was supported by the results of PCR assays (Fig. 2B) which
revealed that no wild type virus genomes were detectable in NHVdTK-GFP infected WSL cells.
To test whether TK-deletion and EGFP-expression affect in vitro
replication of the recombinant, WSL cells were infected with NHVdTK-EGFP or parental NHV. At the time points indicated in Fig. 3,
titres of cell-associated virus and infectivity released into the culture medium were determined by titration on WSL cells. As shown,
NHV-dTK-GFP produced similar or even slightly higher titres of
both cell associated and secreted viral progeny in comparison to
87
Fig. 1. Construction of recombination plasmid pASFV-dTK-EGFP-BAC Lox. (A)
Schematic representation of the ASFV genome region containing the TK ORF.
Nucleotide numbers are given in kilobases (kb), names and direction of transcription of contained ORFs are indicated. The location of the TK-ORF (K196R) is shown in
bold. (B) Plasmid constructions. The left (TK-L) and right (TK-R) segments of the ASFV
TK gene and respective flanking sequences were amplified by PCR from infected
macrophages DNA. Primers used were TK-L+ (GTG GGC GTA TAG ATA AGG ATA TC)
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
GGG CGT AAG AAC GCA GAC AAG ACG C) and TK-R− (CCT GCT CGT GTT ACT TAT
GAA AC) amplifying a 3236 bp fragment from nt 47,504 to nt 50,739 (TK-R). All
nucleotide numbers are from GenBank accession # U18466.1. Both amplicons were
sequentially cloned into plasmid vector pSP73 (Promega) using established standard
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.
TK-R was cleaved with SmaI, and cloned into the TK-L containing plasmid after cleavage with SmaI to yield pspASFV-dTK, containing the viral TK-spanning locus from nt
43,995 to nt 50,739 with a 304 bp deletion from 47,202 to 47,503. Plasmid pspASFVdTK was then used for the integration of the synthetic sequence GGT ACC GTA TAC
GCG GCC GCA TAA CTT CGT ATA ATG TAT GCT ATA CGA AGT TAT CCC GGG, which
contains a loxP site (shown in bold) flanked by recognition sequences for Acc65I,
BstZ17Iand NotI, and for SmaI (printed in italics). A p72-EGFP expression cassette
was afterwards also integrated, containing the ASFV vp72 promoter region (LópezOtín et al., 1990) from nt 218 to nt 430 (GenBank accession # M34142.1) fused to
the EGFP ORF from plasmid pEGFP-N1 (Clontech), resulting in pASFV-dTK-EGFPLox. The 6385 bp plasmid pMBO131 (kindly provided by W. Fuchs, FLI) was cloned
into the blunt-ended NotI site of pASFV-dTK-EGFP-Lox, resulting in the final transfer plasmid construct pASFV-dTK-EGFP-BAC-Lox. Only relevant restriction enzyme
cleavage sites are indicated. Segments and plasmids are not drawn to scale. All PCR
reaction conditions are available upon request.
NHV, indicating that insertion of the foreign sequences into the
viral TK locus and expression of EGFP do not impair viral replication and release of infectious virions in WSL cells. It should be
noted that the increased virus yield, although statistically not significant, might reflect an adaptation process for virus replication in
88
R. Portugal et al. / Journal of Virological Methods 183 (2012) 86–89
Fig. 2. Homogeneity of ASFV recombinant NHV-dTK-GFP. (A) WSL cells, grown on coverslips, were infected with an appropriate dilution of NHV-dTK-GFP stock virus and
fixed 4 days p.i. with 3% paraformaldehyde in PBS for 20 min, permeabilized with 0.2% Triton X-100 in 3% paraformaldehyde/PBS for 10 min, washed 4 times for 5 min with
PBS and incubated with anti-vp30 monoclonal antibody C18 for 1 h at room temperature. After 4 washes with PBS for 5 min each, bound antibodies were visualized by
incubation with Alexa Fluor 555-conjugated goat anti-mouse serum (Invitrogen, Karlsruhe, Germany) for 1 h. The coverslips were mounted on microscope slides with 1,4diazabicyclo[2.2.2]octane (DABCO) in PBS/glycerol and fluorescing cells were photographed and pictured using a Zeiss Axioskop fluorescence microscope with CCD camera
and AxioVision software, respectively. GFP autofluorescence is shown in the left panel, bound Alexa Fluor 555 is shown in the right panel. (B) PCR amplification with primers
CTT ATT CAT TGC ATT TAC ATG CTC G and ACA ACA TGT TAC GTA CAG TTC AC which target TK ORF sequences flanking the p72EGFP-BAC-Lox insert (see Fig. 1) on whole-cell
DNA extracted from WSL cells infected with wild type NHV (lane 1), non-infected WSL cells (lane 2), whole-cell DNA extracted from WSL cells infected with NHV-dTK-GFP
(lane 3), no-template control (lane 4). M: 1 kbp ladder (Invitrogen). PCR reaction conditions are available upon request.
Fig. 3. Infectious replication of NHV-dTK-GFP and wild type NHV in WSL cells is comparable. WSL cultures were infected with wild type NHV (closed circles) or NHV-dTK-GFP
(closed triangles) at an MOI of 0.5. Cultures were washed with medium after 1 h adsorption. At the times indicated, culture supernatants were collected and adherent cells
were washed with medium which was added to the respective supernatants. Fresh medium corresponding to the total supernatant volume was added to the adherent cells.
Cell cultures and supernatants were stored at −70 ◦ C until titration. Virus titres were determined after 2 freeze/thaw cycles (cell associated virus) by inoculation of serial
dilutions on WSL cell monolayers in 96-well tissue culture plates. Values are means of three independent determinations. Standard deviations are indicated.
R. Portugal et al. / Journal of Virological Methods 183 (2012) 86–89
cell culture. Since NHV replicates only inefficiently in Vero TK− cells
which might also contribute to cell culture adaptation, attempts are
currently being made to select a TK-deficient variant of the WSL
cell line which, however is highly sensitive to BrdU already at a
concentration of 0.5 !g/ml.
The ASFV TK locus has been used previously for generation of
recombinant viruses from cell culture adapted isolates since TK
is non-essential for viral growth in cultured cells (García et al.,
1995; Brun et al., 1999; Garcia-Escudero et al., 1998). Disruption of the viral TK locus from pathogenic ASFV isolates partially
adapted to Vero cells has been reported to impair viral growth
in macrophages after a low MOI inoculation (0.01) but not after
infection at a MOI of 10–20, and to decrease pathogenicity for
swine (Moore et al., 1998). Whether NHV-dTK-EGFP exhibits
a similar phenotype in macrophages and in vivo needs to be
analyzed.
In summary, a novel method was developed for the generation of ASFV recombinants directly from field isolates, providing a
method for further manipulations of such wild type virus genomes.
Acknowledgments
We thank Thomas C. Mettenleiter for valuable comments and
suggestions on the manuscript, Roland Riebe for supplying WSL
and Vero TK-cells, and Linda Dixon and Walter Fuchs for providing
materials and Anette and Craig Beidler for proofreading. This work
was funded through the EU-Project # 211691, FP7-KBEE-2007-13-05.
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