Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 11 (2)

Genomic Characterization of Orf Virus Strain D1701-V ( Parapoxvirus) and Development of Novel Sites for Multiple Transgene Expression

Affiliations

Genomic Characterization of Orf Virus Strain D1701-V ( Parapoxvirus) and Development of Novel Sites for Multiple Transgene Expression

Hanns-Joachim Rziha et al. Viruses.

Abstract

The Orf virus (ORFV; Parapoxvirus) strain D1701 with an attenuated phenotype and excellent immunogenic capacity is successfully used for the generation of recombinant vaccines against different viral infections. Adaption for growth in Vero cells was accompanied by additional major genomic changes resulting in ORFV strain variant D1701-V. In this study, restriction enzyme mapping, blot hybridization and DNA sequencing of the deleted region s (A, AT and D) in comparison to the predecessor strain D1701-B revealed the loss of 7 open reading frames (ORF008, ORF101, ORF102, ORF114, ORF115, ORF116, ORF117). The suitability of deletion site D for expression of foreign genes is demonstrated using novel synthetic early promoter eP1 and eP2. Comparison of promoter strength showed that the original vegf-e promoter Pv as well as promoter eP2 display an up to 11-fold stronger expression than promoter eP1, irrespective of the insertion site. Successful integration and expression of the fluorescent marker genes is demonstrated by gene- and insertion-site specific PCR assays, fluorescence microscopy and flow cytometry. For the first time ORFV recombinants are generated simultaneously expressing transgenes in two different insertion loci. That allows production of polyvalent vaccines containing several antigens against one or different pathogens in a single vectored ORFV vaccine.

Keywords: ORFV; Orf virus; Vero cell adaptation; attenuation; gene deletion; parapoxvirus; recombinant ORFV; viral vector.

Conflict of interest statement

The authors declare no conflict of interests

Figures

Figure 1
Figure 1
Changes of D1701-V DNA restriction fragment patterns. DNA of D1701-50BK (lanes BK), D1701-B (lanes B) and D1701-V (lanes V) was digested with HindIII or EcoRI and separated in a horizontal 0.8% agarose gel for 36 h. The digested DNAs were run in one identical gel, however, for clearer representation of the individual DNA fragments the photograph of the ethidium-bromide stained gel was split into the shown parts (A) and (C), respectively. (B) Demonstrates the X-ray after Southern blot hybridization of the gel with the 32P-labelled HindIII-fragment G of D1701-B. Those DNA fragments changed after Vero cell culture passaging are marked by a star.
Figure 2
Figure 2
Map locations of the deleted parts of the D1701-V genome. The genomic map of the HindIII-, EcoRI- and KpnI-fragments of D1701-B (A,B) and D1701-V (C,D) is depicted. ITR indicate the inverted terminal repeats at the genomic termini. In (B) and (C) the sequenced right-hand terminus of D1701-B and D1701-V is shown enlarged. The regions AT and D deleted in D1701-V are indicated by dashed brackets. Those DNA fragments affected by the genomic deletions are marked by stars. (D) The black rectangles indicate the deleted regions A, AT and D in D1701-V; in addition, the earlier described insertion site V (vegf-e gene) is indicated.
Figure 3
Figure 3
Open reading frames affected by the 3 deletions in D1701-V. The ORFs of the genomic regions inclosing deletions A (A), AT (B) and D (C) are depicted. The ORFV genes are numbered according to Delhon et al. [61], the deleted parts are shown with black boxes and black arrows. The gene maps were constructed using SnapGene Viewer software.
Figure 4
Figure 4
Legend: Characterization of the ORFV recombinant D1701-V-D12-mCherry. (A) PCR demonstrating correct transgene insertion. (a) Map section of plasmid pD12-mCherry illustrates location of the used primers also described in Table 1. The left (Del2-L) and the right (Del2-R) homology arm for recombination, early promoter eP1 and eP2 and the mCherry gene are shown. (b) Using primer pair Ch-FR the mCherry-specific amplicon of 0.59 kbp in size was obtained with DNA from red virus plaques (lanes 1 and 2) and with control plasmid pD12-Cherry (lane 4) but not in non-infected cells (lane 3). (c) Shows the result of the D locus-specific PCR Del2-FR2. Using DNA from D12-Cherry infected Vero cells (lane 2) the specific 1.4 kbp amplicon was obtained, whereas DNA from cells containing the parental D1701-V (lane 1) results in a 0.39 kbp amplicon due to the absence of gene insertion. DNA from non-infected cells (lane 3) was used as negative control. (d) Demonstrates the results of the PCR with the primers Del2-F and Ch-R. The specific amplicon of 1.1 kbp was obtained with DNA from D12-Cherry infected cells (lanes 1 and 2) or with plasmid pD12-mCherry (lane 5). DNA from non-infected cells (lane 3) and without template DNA (lane 4) were used as negative controls. In lanes M size marker (1 kbp ladder or 100 bp ladder, NEB) were separated. On the left the sizes are given in kbp. (B) Expression of mCherry allows identification of red fluorescent virus plaques of the ORFV recombinant D1701-V-D12-mCherry (D12-Cherry), which became clearly visible 12 h (a) and 24 h (b) after infection. Microscopic magnification 40×. (C) Western blot analysis of Vero cells infected with the recombinant D12-Cherry. (a) The mCherry was detectable with a specific antiserum during 4 to 72 h after infection but not in non-infected (lane ni) or in D1701-V infected (lane V) Vero cells. In infected cells treated with AraC to arrest ORFV gene expression in the early phase mCherry expression was detectable, too. Panel (b) demonstrates expression of the late major envelope protein (ORF059) with monoclonal antibody 4D6 in cells infected with the recombinant as well as with parental D1701-V but not in recombinant virus cells treated with AraC or in non-infected cells. (c) The protein load of each sample was tested using a specific antiserum against cellular β-actin. The sizes are indicated on the right.
Figure 5
Figure 5
Characterization of ORFV recombinant D1701-V-D1-GFP-2-Cherry. (A) Insertion site- and insert-specific PCR. (Aa) Illustrates location of primers in plasmid pD1-AcGFP-2-mCherry, which are used for locus D-specific PCR (Del2-F and DEl2-R2), for mCherry- (Ch-F and Ch-R) and for AcGFP-specific PCR (GFP-F and GFP-R); eP1 and eP2 denote the early promoters. (Ab) The Del2-FR2 PCR with DNA from parental D1701-V infected Vero cells results in 0.39 kbp amplicon (lanes 1 and 2) due to the lack of gene insertion. After infection with the recombinant D1-GFP-2-Cherry the specific 2.28 kbp amplicon (lane 3) was obtained and with recombinant D12-Cherry the specific 1.43 kbp PCR product was amplified (lane 4). No specific amplification was found with DNA from non-infected cells (lane 5) and H2O as another control (lane 6). Lane M shows separation of size markers (1 kbp ladder, NEN Biolabs). (Ac) Correct insertion of AcGFP and mCherry gene in DNA of recombinant D1-GFP-2-Cherry was proven by PCR using the following primer pair combinations: GFP-F and Ch-R (lane 1, 1.57 kbp amplicon), Del2-F and GFP-R (lane 2, 0.98 kbp amplicon), GFP-F and Del2-R2 (lane 3, 1.88 kbp amplicon), Ch-F and Del-2R2 (lane 4, 0.90 kbp amplicon). (B) Confocal microscopy Vero cells were infected overnight with the recombinant D1-GFP-2-Cherry (moi 1.0), fixed and inspected for green fluorescence of AcGFP (a), red fluorescence of mCherry (b) and both merged (yellow) in combination with phase contrast microscopy (c). Microscopic magnification 40×. (C) Flow cytometry Viability (Aqua Life Dead) and fluorescent protein expression in Vero cells 24 hours after infection (moi of 2.0) with ORFV recombinant D1-GFP-2-Cherry (a,b), with ORFV recombinant D12-Cherry (c,d) and as a negative control with D1701-V (e,f). In (b), (d) and (f) the X-axis shows the number of GFP (FITC) -positive cells and the Y-axis the number of mCherry (PE)-positive cells. The overlay of the histograms of (b), (d) and (f) is plotted in (g) for direct comparison of mCherry-positive cells. The mean mCherry fluorescence intensity (MFI) is given in the inset.
Figure 5
Figure 5
Characterization of ORFV recombinant D1701-V-D1-GFP-2-Cherry. (A) Insertion site- and insert-specific PCR. (Aa) Illustrates location of primers in plasmid pD1-AcGFP-2-mCherry, which are used for locus D-specific PCR (Del2-F and DEl2-R2), for mCherry- (Ch-F and Ch-R) and for AcGFP-specific PCR (GFP-F and GFP-R); eP1 and eP2 denote the early promoters. (Ab) The Del2-FR2 PCR with DNA from parental D1701-V infected Vero cells results in 0.39 kbp amplicon (lanes 1 and 2) due to the lack of gene insertion. After infection with the recombinant D1-GFP-2-Cherry the specific 2.28 kbp amplicon (lane 3) was obtained and with recombinant D12-Cherry the specific 1.43 kbp PCR product was amplified (lane 4). No specific amplification was found with DNA from non-infected cells (lane 5) and H2O as another control (lane 6). Lane M shows separation of size markers (1 kbp ladder, NEN Biolabs). (Ac) Correct insertion of AcGFP and mCherry gene in DNA of recombinant D1-GFP-2-Cherry was proven by PCR using the following primer pair combinations: GFP-F and Ch-R (lane 1, 1.57 kbp amplicon), Del2-F and GFP-R (lane 2, 0.98 kbp amplicon), GFP-F and Del2-R2 (lane 3, 1.88 kbp amplicon), Ch-F and Del-2R2 (lane 4, 0.90 kbp amplicon). (B) Confocal microscopy Vero cells were infected overnight with the recombinant D1-GFP-2-Cherry (moi 1.0), fixed and inspected for green fluorescence of AcGFP (a), red fluorescence of mCherry (b) and both merged (yellow) in combination with phase contrast microscopy (c). Microscopic magnification 40×. (C) Flow cytometry Viability (Aqua Life Dead) and fluorescent protein expression in Vero cells 24 hours after infection (moi of 2.0) with ORFV recombinant D1-GFP-2-Cherry (a,b), with ORFV recombinant D12-Cherry (c,d) and as a negative control with D1701-V (e,f). In (b), (d) and (f) the X-axis shows the number of GFP (FITC) -positive cells and the Y-axis the number of mCherry (PE)-positive cells. The overlay of the histograms of (b), (d) and (f) is plotted in (g) for direct comparison of mCherry-positive cells. The mean mCherry fluorescence intensity (MFI) is given in the inset.
Figure 6
Figure 6
(A) PCR to demonstrate correct insertion of AcGFP in the V locus and mCherry in locus D. (a) The map location of primers is depicted, the homology arms vegf-L and vegf-R as well as Del2-F and Del2-R, the early promoters Pv, ep1 and eP2 are schematically drawn. (b) The results of the PCR with the indicated primer combinations are separated in 0.8% agarose gels with templates isolated from (lanes 1) V-GFP-D12-Cherry infected cells, (lanes 2) V-gfp infected cells, (lanes 3) non-infected cells and (lanes 4) non-template reaction. The amplicon sizes are indicated in kbp, DNA size markers are separated in lanes M1 (100 bp ladder) and lane M2 (1 kbp ladder). (B) Confocal images showing plaque formation of Vero cells 24 and 48 hours after infection with moi 2.0 of D1701-V-AcGFP-D12-mCherry (V-GFP-D12-Cherry). Concurrent expression of AcGFP (green) and mCherry (red) are demonstrated in each infected cell (merged). Microscopic magnification 10× (24 h) and 20× (48 h). (C) Flow cytometric determination of viability (Aqua Life Dead) and fluorescent protein expression 24 hours after infection (moi of 2.0) with V-GFP-D12-Cherry (a,b), with D12-Cherry (c,d) and as a negative control with D1701-V (e,f). The dot plots (b), (d) and (f) show on X-axis the number of AcGFP-positive cells and on Y-axis the number of mCherry-positive cells. The overlay of the mCherry histograms of (b), (d) and (f) is plotted in (g) for direct comparing the number of mCherry-positive cells; the mCherry mean fluorescence intensity (MFI) is given for each virus-infected cells in the inset.
Figure 6
Figure 6
(A) PCR to demonstrate correct insertion of AcGFP in the V locus and mCherry in locus D. (a) The map location of primers is depicted, the homology arms vegf-L and vegf-R as well as Del2-F and Del2-R, the early promoters Pv, ep1 and eP2 are schematically drawn. (b) The results of the PCR with the indicated primer combinations are separated in 0.8% agarose gels with templates isolated from (lanes 1) V-GFP-D12-Cherry infected cells, (lanes 2) V-gfp infected cells, (lanes 3) non-infected cells and (lanes 4) non-template reaction. The amplicon sizes are indicated in kbp, DNA size markers are separated in lanes M1 (100 bp ladder) and lane M2 (1 kbp ladder). (B) Confocal images showing plaque formation of Vero cells 24 and 48 hours after infection with moi 2.0 of D1701-V-AcGFP-D12-mCherry (V-GFP-D12-Cherry). Concurrent expression of AcGFP (green) and mCherry (red) are demonstrated in each infected cell (merged). Microscopic magnification 10× (24 h) and 20× (48 h). (C) Flow cytometric determination of viability (Aqua Life Dead) and fluorescent protein expression 24 hours after infection (moi of 2.0) with V-GFP-D12-Cherry (a,b), with D12-Cherry (c,d) and as a negative control with D1701-V (e,f). The dot plots (b), (d) and (f) show on X-axis the number of AcGFP-positive cells and on Y-axis the number of mCherry-positive cells. The overlay of the mCherry histograms of (b), (d) and (f) is plotted in (g) for direct comparing the number of mCherry-positive cells; the mCherry mean fluorescence intensity (MFI) is given for each virus-infected cells in the inset.
Figure 7
Figure 7
Comparison of strength of the promoters Pv, eP1 and eP2 by flow cytometry. Vero cells were infected in triplicates with the indicated ORFV recombinants for 22 h and the mean fluorescence intensity (MFI) was measured by flow cytometry. The numbers in parentheses behind the name of each recombinant indicate the percentage of positive cells determined by flow cytometry. The numbers in the bars denote the multiple of the particular lowest MFI set to 1.0. (A) Activities of eP1 and eP2 in insertion site D directing expression of AcGFP gene. The lowest MFI was found with promoter eP1 (recombinant D1-GFP) and set to 1.0, whereas expression from eP2 was 11.1- or 9.5-fold stronger after infection with V12-Chery-D12-GFP or D12-GFP, respectively. (B) Again, the lowest promoter activity was demonstrated from eP1 (recombinant D1-GFP and D1-GFP-2-Cherry) but 4.2-fold higher expression strength by promoter PV. (C) Expression of mCherry in insertion site V under control of Pv (recombinant V-Cherry) yielded the lowest MFI (set to 1.0) but was 3.1-fold higher when controlled by eP2 (recombinant V12-Cherry). Expression in the D locus showed that the eP2 led to 1.9 higher (recombinant V-GFP-D12-Cherry) or 1.7 higher (recombinant D12-Cherry) MFI. (D) The effect of a tandem arrangement of 5 core elements of Pv (recombinant 5xPv-Cherry) on the expression of mCherry in the V locus was compared to that of the single Pv (V-Cherry; set to 1.0). Vero cells were infected in the absence or in the presence of AraC (+ AraC).
Figure 8
Figure 8
Comparison of gene deletions in the left hand end of ORFV genomes The gene deletions mapped in the left terminus of the genome of the different cell culture passaged ORFV variants NZ2var [72] and Orf-11 [73] are depicted in comparison to the prototype ORFV wild-type (wt) strain NZ2 and to the D1701 derivatives as explained in the text. The earlier first designations for the ORFV genes are given in the upper line, EEV means that this ORF010 encodes an ORFV envelope protein. ITR denotes the inverted terminal region. The framed question mark given for D1701 virus sequenced by McGuire (D1701-McG) indicates the lack of sequence data left to the ORF007. The squared brackets mark the deleted part in each virus variant.

Similar articles

See all similar articles

Cited by 1 article

References

    1. Chan W.M., McFadden G. Oncolytic Poxviruses. Annu. Rev. Virol. 2014;1:119–141. doi: 10.1146/annurev-virology-031413-085442. - DOI - PMC - PubMed
    1. Draper S.J., Cottingham M.G., Gilbert S.C. Utilizing poxviral vectored vaccines for antibody induction-progress and prospects. Vaccine. 2013;31:4223–4230. doi: 10.1016/j.vaccine.2013.05.091. - DOI - PubMed
    1. Gomez C.E., Najera J.L., Krupa M., Perdiguero B., Esteban M. MVA and NYVAC as vaccines against emergent infectious diseases and cancer. Curr. Gene Ther. 2011;11:189–217. doi: 10.2174/156652311795684731. - DOI - PubMed
    1. Walsh S.R., Dolin R. Vaccinia viruses: Vaccines against smallpox and vectors against infectious diseases and tumors. Expert Rev. Vaccines. 2011;10:1221–1240. doi: 10.1586/erv.11.79. - DOI - PMC - PubMed
    1. Jacobs B.L., Langland J.O., Kibler K.V., Denzler K.L., White S.D., Holechek S.A., Wong S., Huynh T., Baskin C.R. Vaccinia virus vaccines: Past, present and future. Antivir. Res. 2009;84:1–13. doi: 10.1016/j.antiviral.2009.06.006. - DOI - PMC - PubMed

Publication types

Feedback