- Short Report
- Open Access
Cloning of Bovine herpesvirus type 1 and type 5 as infectious bacterial artifical chromosomes
© Tobler et al; licensee BioMed Central Ltd. 2009
- Received: 1 July 2009
- Accepted: 14 October 2009
- Published: 14 October 2009
Bovine herpesviruses type 1 (BoHV1) and type 5 (BoHV5) are two closely related pathogens of cattle. The identity of the two viruses on the amino acid level averages 82%. Despite their high antigenetic similarities the two pathogens induce distinctive clinical signs. BoHV1 causes respiratory and genital tract infections while BoHV5 leads to severe encephalitis in calves.
The viral genomes of BoHV1 and BoHV5 were cloned as infectious bacterial artificial chromosomes (BACs). First, recombinant viruses carrying the genetic elements for propagation in bacteria were generated. Second, DNA from these recombinant viruses were transferred into prokaryotic cells. Third, DNA from these bacteria were transferred into eukaryotic cells. Progeny viruses from BAC transfections showed similar kinetics as their corresponding wild types.
The two viral genomes of BoHV1 and BoHV5 cloned as BACs are accessible to the tools of bacterial genetics. The ability to easily manipulate the viral genomes on a molecular level in future experiments will lead to a better understanding of the difference in pathogenesis induced by these two closely related bovine herpesviruses.
- Bacterial Artificial Chromosome
- Recombinant Virus
- MDBK Cell
- Infected Cell Protein
- Bacterial Genetic
Background and aim of this study
Bovine herpesviruses type 1 (BoHV1) and type 5 (BoHV5) belong to the subfamily Alphaherpesvirinae and are closely related pathogens of cattle, with coding capacity of more than seventy open reading frames (ORF's) . Despite their high identity (82%) on the amino acid (aa) level leading to a similar antigenetic repertoire , these two viruses induce distinctive clinical signs. BoHV1 causes respiratory and genital tract symptoms including infectious rhinotracheitis, pustular vulvovaginitis, and abortion . BoHV5 causes severe encephalitis in calves and experimentally in rabbits and mice [4–7]. In order to have an approach to elucidate the differences in the pathogenesis of these two viruses on a molecular level, we have set out to clone the entire genomes of BoHV1 (strain Jura) and BoHV5 (strain N569) as bacterial artificial chromosomes (BACs). As such, these cloned genomes will become accessible to the tools of bacterial genetics , allowing facilitated generation of recombinant viruses in future experiments. In fact, BoHV1 genomes of three different strains were previously cloned as BACs by Mahony et al. (strain V155) , Trapp et al. (strain Schönböken) , and Liu et al. (strain Cooper) . However, in contrast to the approach reported by Mahony et al. and Trapp et al., we introduced the heterologous sequences flanked by loxP sites into a intergenic region of BoHV1 genome. The benefit of this strategy is given by the fact that none of the viral DNA coding sequences are disrupted. The heterologous sequences can be excised on demand by Cre recombinase, which depicts another advantage over the previously used methods. Unlike the construct of Mahoney et al.  our BoHV1 BAC clone harbours GFP coding sequence as part of the heterologous sequences, which easily enables the monitoring of virus plaque formation using fluorescent microscopy. The BoHV1 BAC reported by Liu et al.  has similar genetic features as our BoHV1 BAC though we cloned the strain we investigated most in our lab and we report the cloning of BoHV5 as BAC, which is not reported yet.
The cloning of BoHV genomes as BACs can be divided into three steps: First, genetic elements required for DNA replication and selection in bacteria were inserted by homologous recombination into the virus genomes. Second, circular viral DNA was extracted from infected cells and transferred into bacteria. Third, viruses were reconstituted upon transfection of BAC-DNA into eukaryotic cells.
Generation of recombinant viruses in eukaryotic cells
Transfer and propagation of viral DNA in bacteria
A typical feature of herpesvirus genomes is their circular conformation early in infection . Since circularity of DNA is a prerequisite for the propagation of plasmids in bacteria, such DNA was isolated from infected cells. In brief, confluent MDBK cells grown in 60 mm plates were infected at a multiplicity of infection (moi) of 5 with rBoHV1 or rBoHV5. Two (rBoHV1) or four (rBoHV5) hours post infection (hpi) the monolayer was washed once with PBS, then rinsed using 10 mM EDTA, 10 mM Tris-HCI, pH 8.0 buffer, subsequently the cells were scraped from the tissue culture dish into 0.7 ml of the same buffer. Proteinase K and SDS were added to final concentrations of 0.25 g/l and 0.6%, respectively. Three microliters of circular viral DNA were taken to electroporate E. coli (ElectroMAX™ DH10B™, Invitrogen) using 0.1 cm-cuvettes in a BioRad Gene Pulser (1.8 kV voltage, 200 Ω impedance, 25 μF capacity). The electroporated bacteria were grown on Luria-Bertani (LB)-plates supplemented with 12.5 μg/ml chloramphenicol. Colonies were picked and expanded in liquid LB medium supplemented with 12.5 μg/ml chloramphenicol.
DNA was extracted from bacterial cultures and analysed by restriction enzyme analysis. The resulting BACs were designated fBoHV5 BAC and fBoHV1 BAC, respectively. The restriction enzyme patterns confirmed the integrity of the viral DNA and the insertion of the replicon sequence at the desired location (Figure 1A to 1B). Of note, the unique short segment (US) of the genome did not invert relative to the long segment (UL) in the BAC DNA. The herpes-genomic DNA as part of the BACs were frozen in the following isomeric arrangement: UL (circ through BICP0), internal repeat, US (US1.67 through US9), terminal repeat (Figure 1C).
Viral growth curve analysis
Briefly, the growth curves and the final titers were similar for all viruses, although wt BoHV5 and rBoHV5 reached generally, though not significantly, higher titers than wt BoHV1 and rBoHV1 mutant. Furthermore, the amounts of cell-free virus slightly exceeded the amount of cell-associated virus. However, the viruses with retained BAC cassette had similar kinetics to the respective wt BoHV viruses.
Deletion of BAC cassettes in rBoHV's by cre mediated recombination
MDBK cells were cotransfected with rBoHV1 or rBoHV5 viral DNA and a Cre recombinase expressing vector p116.006 . Three days post transfection five viral plaques were randomly collected and plaque purified three times as described above. Finally, viral DNA from non-eGFP fluorescent progeny was extracted and characterized by restriction enzyme analysis in order to verify the deletion of the BAC. Upon reconstitution of BAC DNA, Unique short (US) segment does again invert relative to Unique long (UL) segment (Figure 1B).
Thus, these cloned genomes will be useful tools towards elucidating the differences in the pathogenesis of BoHV1 and BoHV5, using bacterial genetics for genome manipulation .
This work was supported by grant 3100A0-12498 from the Swiss National Science foundation to MA, by grant 560012 from the University of Zurich to KT, and by grant 3100-058758.99/1 from the Swiss National Science Foundation to CF. We thank Eva Loepfe for excellent technical support, Carlos Abril and Monika Engels for enlightening discussions.
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