Cloning of Bovine herpesvirus type 1 and type 5 as infectious bacterial artifical chromosomes

Abstract

Background: 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.

Findings: 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.

Conclusion: 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.

Figure 1

Strategy for cloning the full-length genomes of wt BoHV5 and wt BoHV1 as BACs and restriction enzyme analyses (REA). Panel A: Restriction maps of BoHV5 (BamHI; left) and BoHV1 (HindIII; right). Division in unique long (U_L), unique short (U_S), internal repeat (IR) and terminal repeat (TR) is shown on top, fragments are lettered alphabetically and sizes are indicated in kbp. Left ends of genomes are additionally blown up to map the site of BAC backbone insertion between CIRC (circ) and BICP27 (27). Upon insertion into BoHV5, BamHI fragment A is replaced by fragments A1, A2, and A3. Likewise, insertion into BoHV1, HindIII fragment N is replaced by fragments N1, N2 and N3. Panel B: Restriction enzyme digests of wt BoHV, rBoHV, fBoHV and rBoHVΔBAC. Fragments of interest are labelled as in panel A: Panel C: Possible two orientations of U_S within fBoHV5 and fBoHV1 respectively. HindIII map of both orientations for BoHV1 is part of panel A, NdeI of both orientations for BoHV5 is given above the REA. fBoHV5 BAC #5 and fBoHV1 BAC #3 pointed with red circles were used for further experiments.

Figure 2

Growth kinetics of rBoHV5 and rBoHV1 mutants versus wt BoHV5 and wt BoHV1. MDBK cells were infected at moi of 0.01 PFU with different viruses and harvested at various times post inoculation as indicated. The virus yields at each time point were determined by titration. Panels A and B: wt BoHV5 (full squares) and rBoHV5 (open squares). Panels C and D: wt BoHV1 (full circles) and rBoHV1 (open circles). Cell-free (supernatant, panels A and C) and cell-associated virus (pellets, panels B and D) were titrated separately. The x-axis represents the time scale post infection. Virus titers (y-axis) are expressed as TCID₅₀/ml.

Figure 3

Recombinant viruses carrying the BAC-backbone and eGFP expression cassette. MDBK cells were infected with BAC derived rBoHV5 (panels A and B) or rBoHV1 (panels C and D). Panels A and C are captured under normal light. Panels B and D are GFP-filtered micrographs.