Recovery of infectious virus from full-length cowpox virus (CPXV) DNA cloned as a bacterial artificial chromosome (BAC)
© Roth et al; licensee BioMed Central Ltd. 2011
Received: 24 June 2010
Accepted: 13 September 2010
Published: 11 January 2011
Transmission from pet rats and cats to humans as well as severe infection in felids and other animal species have recently drawn increasing attention to cowpox virus (CPXV). We report the cloning of the entire genome of cowpox virus strain Brighton Red (BR) as a bacterial artificial chromosome (BAC) in Escherichia coli and the recovery of infectious virus from cloned DNA. Generation of a full-length CPXV DNA clone was achieved by first introducing a mini-F vector, which allows maintenance of large circular DNA in E. coli, into the thymidine kinase locus of CPXV by homologous recombination. Circular replication intermediates were then electroporated into E. coli DH10B cells. Upon successful establishment of the infectious BR clone, we modified the full-length clone such that recombination-mediated excision of bacterial sequences can occur upon transfection in eukaryotic cells. This self-excision of the bacterial replicon is made possible by a sequence duplication within mini-F sequences and allows recovery of recombinant virus progeny without remaining marker or vector sequences. The in vitro growth properties of viruses derived from both BAC clones were determined and found to be virtually indistinguishable from those of parental, wild-type BR. Finally, the complete genomic sequence of the infectious clone was determined and the cloned viral genome was shown to be identical to that of the parental virus. In summary, the generated infectious clone will greatly facilitate studies on individual genes and pathogenesis of CPXV. Moreover, the vector potential of CPXV can now be more systematically explored using this newly generated tool.
Cowpox virus (CPXV) belongs to the family Poxviridae, a family of large double- stranded DNA viruses replicating in the cytoplasm. It is endemic in Eurasia and reportedly transmitted from rodents as the reservoir host to other mammals. CPXV causes disease in felids, zoo animals, man and, despite its name, only very rarely in cattle. Not only recent outbreaks in humans but also its close relationship to other important members of the genus such as variola virus have drawn attention to this virus as a model, especially regarding its impressive abilities to evade host immunity [1–3].
Bacterial artificial chromosomes (BAC), first published by Shizuya et al. in 1992 , allow the stable maintenance of large plasmids of up to 300 kbp in size at low copy numbers in Escherichia coli. The genome of a herpesvirus, human cytomegalovirus (HCMV), was the first full-length viral genome cloned as a BAC in 1997 . Infectious genomes cloned as BAC have proven a useful tool in molecular virology, both for investigations into viral pathogenesis as well as for applications in vaccination and gene therapy, because mutations can be introduced with ease using recombination or transposon systems of E. coli.
With respect to poxviruses, the cloning and successful recovery of infectious BAC was published for vaccinia virus strains Western Reserve [7, 8] and for modified vaccinia virus Ankara (MVA), an attenuated vaccinia virus strain that is replication-deficient in most cell types . Here, we show the generation and characterization of the first infectious clone of a cowpox virus that contains the complete genome of CPXV strain Brighton Red (BR). The BAC allows the application of bacterial mutagenesis procedures including minimal modifications and markerless approaches. With the help of the transcriptional machinery of fowlpox virus (FWPV), infectious CPXV could be recovered in cell culture. Since it is desirable to avoid any foreign sequences in reconstituted viruses, we modified the parental CPXV BAC into a self-excisable construct by introducing a duplication of flanking sequences in inverse orientation into the mini-F cassette, an approach that was previously shown for infectious herpesvirus and poxvirus genomes . Self-excision of mini-F sequences was efficient upon reconstitution in eukaryotic cells and virus progeny derived from cloned genomes therefore is genetically and phenotypically indistinguishable from parental CPXV BR.
Materials and methods
Cell lines and viruses
African green monkey Vero 76 cells (Collection of Cell Lines in Veterinary Medicine, Friedrich-Loeffler Institute, Greifswald-Insel Riems, Germany) were kept in Eagle's minimal essential medium (MEM, Biochrom, Berlin, Germany) supplemented with 5-10% fetal bovine serum (FBS, Biochrom). Primary chicken embryo cells (CEC) were prepared from 11-day-old embryos according to standard procedures and cultured in MEM containing 10% FBS .
CPXV strain Brighton Red (AF428758), kindly provided by Dr Philippa Beard, University of Edinburgh, UK, was propagated on Vero cells and FWPV (Nobilis-PD, strain WP, Intervet, Boxmeer, The Netherlands; kindly provided by Dr D. Lüschow, Freie Universität Berlin, Germany), on CEC.
Bacteria and plasmid constructs
Plasmids for generation of the CPXV BAC pBRf
Primers used in this study
oligo #046 forward
oligo #047 reverse
oligo #048 forward
Fse I site underlined
oligo #049 reverse
Fse I site underlined
oligo #237 forward
oligo #238 reverse
oligo #239 forward
oligo #240 reverse
oligo #264 reverse
oligo #265 forward
Plasmids for generation of self-excisable CPXV BAC pBR
The transfer plasmid for introduction of an inverse duplication of the TK flanks into the mini-F sequence present in full-length CPXV.BR BAC (pBRf) was based on plasmid pEPMCS-in-Belo as described previously . The transfer construct contains mini-F-derived sequence flanks A and B of approximately 830 bp in length as well as the kanamycin resistance gene aphAI with an adjoining I-Sce I restriction site. The aphAI-I-Sce I cassette is flanked by duplication sequences utilized for E. coli recombineering, I-Ceu I restriction sites frame the complete construct. The TK fragment was cut out of plasmid #5 using BamH I and Pst I restriction sites and ligated into the multiple cloning site of pEPMCS-in-Belo (plasmid #10). The duplication was inserted into the mini-F cassette in inverse orientation relative to original viral sequences in pBRf.
Bacteria and transformation
Regular plasmids were grown and maintained in E. coli Top10 cells (Invitrogen). For generation of an infectious CPXV full length clone, pBRf, electrocompetent E. coli DH10B cells (MegaX, Invitrogen) were electroporated with viral DNA at 1300 V and 100 Ω resistance (Biorad, München, Germany). For generation of pBR, two-step Red recombination was applied after transfer of pBR into E. coli GS1783 cells . All recombination procedures for two-step en passant recombination were performed exactly as described previously .
Preparation of viral and plasmid DNA, restriction fragment length polymorphism (RFLP) analysis and Southern blotting
Viral DNA for electroporation and generation of pBRf was isolated from cells infected with BR.TK- after addition of 100 μM IβT (isatin-β-thiosemicarbazone, Chemos GmbH, Regenstauf, Germany) at 3 h post infection (pi). Cells were harvested by scraping at 48 h pi and the cell pellet was incubated in lysis buffer (0.02 M Tris HCl (pH 8.0); 0.01 M EDTA; 0.75% SDS; 0.65 mg/mL proteinase K) at 55°C for 5 h. Nucleic acids were extracted by addition of supersaturated NaCl to a final concentration of 2 M and precipitated with isopropanol (final concentration: 50%). The same procedure without addition of IβT was followed for the extraction of viral DNA for PCR analysis and determination of restriction fragment length polymorphisms (RFLP).
Plasmid or BAC DNA for cloning and standard applications such as restriction enzyme digests was extracted by the alkaline lysis method, variably including or omitting phase separation and removal of cellular debris with phenol-chloroform . For whole genome sequencing, large scale BAC DNA purifications were carried out using the plasmid midi kit (Qiagen, Hilden, Germany) following the manufacturer's instructions. For determination of RFLP, DNA cut with various restriction enzymes was separated by electrophoresis for 14 h at 35 V in 0.8% agarose gels using 1× TAE buffer (40 mM Tris; 20 mM acetic acid; 1 mM EDTA; 50× TAE sock solution pH 8.4).
Southern blotting with a digoxigenin (DIG) labeled TK PCR probe was done using the DIG PCR Labeling Kit and the DIG Nucleic Acid Detection Kit (Roche, Mannheim, Germany) exactly following the manufacturer's instructions.
Reconstitution of infectious virus
Transfections for the generation of recombinant virus as well as for recovery of virus from BAC DNA were carried out with approximately 2 μg of purified plasmid or BAC DNA and 4 μL FuGene (Roche) according to the manufacturer's instructions. For virus reconstitution, 1 × 106 Vero cells seeded in one well of a 6-well plate were infected with 100-1 000 plaque-forming units (pfu) of FWPV at 1 to 5 h before or after transfection.
A DNA library for sequencing with Genome Sequencer FLX (Roche) was prepared from enriched and purified BAC DNA according to an established protocol . The resulting dsDNA library was then bound to library capture beads and, subsequently, a single-stranded template DNA (ssDNA) library was recovered. The ssDNA library was prepared for sequencing and sequenced according to the manufacturer's instructions. The obtained raw sequence data were assembled using the GS assembler software Newbler (v. 2.0.00.22; Roche). Assembled contigs were further analyzed with VectorNTI Advance 11 (Invitrogen).
Characterization of virus growth properties
Single- and multi-step growth kinetics of reconstituted viruses were carried out on Vero cells using multiplicities of infection (MOI) of 3 or 0.01, respectively. Cells were infected, washed three times with PBS 90 min after infection, and supplied with 1 mL of fresh cell culture medium. The 0 h time point was taken immediately after addition of new medium. Cell culture supernatant was harvested, subjected to low-speed centrifugation (8 min, 6 000 × g) to remove cells and cellular debris, and 1 mL of fresh medium per well were added. Both the cellular fraction and cleared supernatants were treated by freezing (-70°C) and thawing (37°C) three times before centrifugation and followed by quantification in plaque assays.
End point titers were determined in a similar manner. Virus was added to cells at an MOI of 3, 0.1 or 0.01, and cells were harvested at 24, 48 and 72 h after the washing procedure that was done 90 min after addition of virus as described above.
For plaque size determinations, 1 × 106 Vero cells were infected with 100 pfu per well of a 6-well plate and overlaid 90 min pi with carboxymethyl cellulose (CMC, final concentration: 0.8% in MEM with 3.5% FBS). Plaque images were taken at 48 h pi using the Zeiss Axiovert 25 (Carl Zeiss, Jena, Germany) equipped with a CCD camera (AxioCam MRm, Zeiss), processed with the Axiovision software (Zeiss), and analyzed using ImageJ software http://rsb.info.nih.gov/ij/. Statistical analyses of virus growth kinetics and plaque areas were done using the Student's t-test http://www.graphpad.com.
Generation of recombinant CPXV BR.TK- harboring mini-F vector sequences
Generation of an infectious full-length clone of CPXV BR (pBRf) and reconstitution of recombinant virus (vBRf) from cloned DNA
After confirming the integrity of the full-length pBRf clone, virus was successfully reconstituted from DNA using FWPV as helper virus. BAC DNA was transfected into 1 × 106 Vero cells that were infected with 100-1 000 PFU of FWPV several hours before or after infection. Formation of GFP-expressing virus plaques showed that virus derived from pBRf, termed vBRf, was fully replication-competent. Integrity of the complete vBRf genome including the mini-F sequences was proven by demonstration of the expected restriction fragment patterns and Southern blot analysis (see below). We therefore concluded that the complete CPXV.BR genome was successfully established in E. coli and that fully replication-competent CPXV was reconstituted from viral DNA cloned as a BAC.
Construction of self-excisable CPXV BAC pBR and generation of recombinant vBR virus
Nucleotide sequence of pBR
List of nucleotide sequence differences of CPXV.BR and cloned pBR when compared to the published BR sequence.
A. Changes in coding sequences
Gene (according to NC_003663)
Modification (nt position)
Present in parental virus
C to A (5 038)
C to T (21 015)
Glu to Lys, not in ankyrin repeats
ins. T (36 656)
split protein, resembles situation in other orthopox viruses
glutaredoxin (GRX) family
C to T (76 314)
Asp to Asn, polymorphic region of the protein
protein G1 (Peptidase)
T to C (87 650)
Asn to Asp, like in 91-3
T to C (97 522)
silent, PCR error
polyA polymerase small SU
silent, PCR error
poxvirus early transcription factor (VETF)
Insertion : T (142 924)
C-terminal amino acid sequence changed from RAQIN to KSTNKLNN
C to T (189 440)
Ser to Phe, polymorphic region of the protein
C to T (213 275)
Pro to Ser, polymorphic region of the protein
G to T (229 788)
B. Changes in non-coding sequences
Modification (nt position)
Insertion : TG (8 899-8 900)b
poly-TG repeat, polymorphic in viral DNA
C to T (58 974)
Insertion : T (65 922)
Insertion : CA (225 926-225 927)b
poly-CA repeat, polymorphic in viral DNA
Insertion : TG (8 899-8 900)b
poly-TG repeat, polymorphic in viral DNA
Phenotypic characterization of recombinant CPXV.BR derived from pBRf or pBR
We here describe the first BAC clone containing the complete genomic sequence of CPXV.BR. Furthermore we report an infectious clone in which bacterial sequences are lost via homologous recombination events in eukaryotic cells and therefore present a BAC that, upon reconstitution in cell culture, does not contain any vector or marker sequences and therefore is genetically indistinguishable from the parental virus.
For generation of the infectious clone, the thymidine kinase locus was chosen based on data from other orthopox viruses, which had shown the nonessential nature of the gene when virus is propagated in cultured cells . Our results on the generation of a TK-negative CPXV.BR virus, achieved through the insertion of bacterial vector sequences into the TK locus, lend support to the non-essential function of this gene in CPXV. Furthermore, detailed analysis of the growth characteristics showed that even vBRf, in which the TK is disrupted by the 9 kbp insertion of the mini-F vector, does not behave differently from parental, wild-type BR.
Despite the unaltered growth properties and the fact that use of GFP-expressing recombinant CPXV.BR is very useful for some applications, we further sought to develop the infectious clone in a fashion that would render the geno- and phenotype of viruses reconstituted from the infectious clone absolutely identical to that of parental CPXV.BR. We previously reported that loss of vector sequences is made possible by the occurrence of two antiparallel recombination events mediated by a sequence duplication within mini-F vector sequences . Such recombination in the case of transfection of pBR indeed led to self-excision of the mini-F cassette upon vBR reconstitution, which was shown by the loss of sequences coding for GFP and was confirmed not only by the absence of autofluorescence but also by PCR, RFLP and Southern blotting of virus progeny resulting from pBR transfection.
Genetic identity of the generated CPXV.BAC was proven by RFLP, Southern blot analyses and finally pyrosequencing of the entire cloned genome. Parts of the inverted terminal repeat regions (nt positions 0-2 546 and 232 280-234 825) could not be analyzed due to the high content of A and T rich repeats and the highly repetitive character of the sequences. Remaining sequence differences between the generated BAC and the published sequence available for BR (genbank_accession#NC_003663) were shown to be caused by alterations already present in the parental wild-type CPXV.BR used for cloning or representing errors that occurred following PCR amplification of the TK flanks. Our sequencing results indicate that CPXV.BR isolates differ minimally in their genetic material, an alternative explanation remaining that sequencing errors account for the discrepancies. However, all sequence modifications are located within the less conserved terminal part of the genome, where higher mutation rates due to host-pathogen co-evolutionary events are expected and frequently seen . The fact that congruence was observed regarding the phenotype in vitro was shown by observing plaque sizes and growth kinetics suggests that both generated BAC clones after reconstitution in cultured cells in fact behave in a fashion that is practically identical to that of wild-type virus and present a number of advantages. pBRf, expressing GFP under a poxvirus promoter, produces labeled virus after reconstitution and mutants can easily be tracked by fluorescence microscopy, whilst pBR results in a virus that does not contain any foreign sequences. Therefore, if one generates mutant genomes based on pBR, only the desired mutation will be present and otherwise represent a wild-type background, which will exclude unintended side effects by marker sequences especially in in vivo experiments.
In summary, both BAC clones presented here enable the use of fast and easy bacterial mutagenesis methods for the generation of cowpox virus mutants. In our initial mutagenesis attempts of pBR and pBRf, we observed genomic stability as was shown for the other available orthopoxvirus full-length DNA clones. Therefore, the CPXV.BR BAC represents a toolbox that will simplify pathogenesis studies of a recently emerging virus not only relevant to several species of animals but also exhibiting zoonotic potential. Other members of the family Poxviridae have already proven to show excellent performance as vectors for vaccination or gene therapy purposes, applications that are greatly simplified by the use of cloned viral genomes such as the CPXV BAC generated here.
We should like to thank Dr Philippa Beard, University of Edinburgh, UK, for providing cowpoxvirus strain Brighton Red (BR), Dr M Cottingham at the Jenner Institute, Oxford, UK, for providing the mini-F sequence derivate of pBeloBAC11, and Dr D. Lüschow for the FWPV isolate used for virus recovery. SJ Roth was supported by a grant from the Studienstiftung des Deutschen Volkes and the study was supported by unrestricted funds made available to N Osterrieder by the Freie Universität Berlin.
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