Figure 1.
A) gDcpgD106 peptide sequence and predicted amino-acid product. In red CapHV-1 gD whereas in black the gD106 tag. B) Diagram (not to scale) showing the pCMV-gDcpgD106 construct: In blue the cytomegalovirus enhancer promoter (CMV), in red the CapHV-1 gD, in black the gD106 tag and in white the bovine growth hormone polyadenylation signal (PA). C) western immune-blotting of pCMV-gDcpgD106 transfected HEK cells lysates at different time post transfection (24, 48 and 72 hours). Untransfected HEK cells as negative control (−) and pIgKE2gD-TM transfected HEK cells lysate as a positive control (+).
Figure 2.
A) Diagram showing (not to scale) the targeting by heat-inducible homologous recombination in SW102 containing pBAC-BoHV-4-A-TK-KanaGalK-TK, where the Kana/GalK cassette was removed and replaced with the CMV-gDcpgD106 expression cassette. B) Representative colonies tested by HindIII restriction enzyme analysis, agar gel electrophoresis, and Southern blotting performed with a probe for the CMV promoter sequence, where the 2,612 bp band is present only for the targeted clones bat not for the untargeted control. C) Stability of the pBAC-BoHV-4-A-gDcpgD106ΔTK plasmid in E. coli SW102 cells. D) Representative fluorescent microscopic images of plaques formed by viable reconstituted recombinant BoHV-4-A-gDcpgD106ΔTK after DNA electroporation into BEK cells or in BEK cells expressing cre recombinase. Magnification, ×10. E) Replication kinetics of BoHV-4-A-gDcpgD106ΔTK growth on cre-expressing cells, compared with those of the parental BoHV-4-A isolate. The data presented are the means ± standard errors of triplicate measurements (P>0.05 for all time points as measured by Student's t test).
Figure 3.
A) Diagram not on scale of BoHV-4LucΔTK delivering the CMV-Luc expression cassette. CMV promoter is represented by the blue box, the luciferase ORF by the yellow box whereas the polyadenylation signal by the white box. B) Representative image of the site of BoHV-4LucΔTK injection, under the goat tail (red arrow), and the skin explant visualized by in vivo image analysis. C) Representative image of flasks containing the BoHV-4LucΔTK infected or uninfected goat skin explant organotypic cultures and visualized by in vivo image analysis.
Figure 4.
A) Diagram not on scale of BoHV-4EGFPΔTK delivering the CMV-EGFP expression cassette and BoHV-4-A-gDcpgD106ΔTK delivering the CMV-gDcpgD106 expression cassette. CMV promoter is represented by the blue box, the enhanced green fluorescent protein ORF by the green box, the caprine gD ORF by the red box, the gD106 tag by the grey box, whereas the polyadenylation signal by the white box. B) Representative phase contrast and fluorescence images of BoHV-4EGFPΔTK infected goat subcutaneous primary cell cultures at 24 hours post infection. C) Representative phase contrast and fluorescence images of BoHV-4EGFPΔTK infected goat subcutaneous primary cell cultures at 48 hours post infection and showing cytopathic effects. D) BoHV-4EGFPΔTK titer at 24 and 48 hours post infection as log10 TCID50/ml. The data presented are the means ± standard errors of triplicate measurements (P>0.05 for all time points as measured by Student's t test). E) Western immunoblotting of BoHV-4-A-gDcpgD106ΔTK infected goat subcutaneous epithelial and stromal primary cell cultures lysates. 5, 10 and 20 are the different amount as µl of the lysates.
Figure 5.
A) Kinetics of the humoral immune responses of goats immunized with BoHV-4-A-gDcpgD106ΔTK and BoHV-4-A-CMV-IgK-gE2gD-TM (B). Anti-BoHV-1 [Bovine herpesvirus 1 (grey bars)], anti-BVDV [Bovine viral diarrhea virus (red bars)] and anti-CapHV-1 [Caprine herpesvirus 1 (black bars)] serum-neutralizing antibodies (SN) as measured by serum neutralization test. SN antibodies were expressed as the reciprocal of the highest dilution of the serum that inhibited the development of virus-induced CPE in MDBK cells. Virus neutralization titers of >2 (log2) were considered positive. Each value represents the mean response of five rabbits ± the standard error of the mean (*, P<0.005 as measured by Student's t test or one-way analysis of variance). T1, T2, T3, T4, T5, T6, T7, T8 and T26 is the time (T) expressed as week (1,2,3,4,5,6,7,8 and 26) post inoculation. S(T26) is the sentinel animal at 26 weeks. T0 is the pre immune sera.
Figure 6.
Representative image of typical CpHV-1 induced vaginal lesions in unvaccinated goat (D) respect to vaccinated goats (A, B, C).
Figure 7.
Clinical scores of vaccinated and control goats following CpHV-1 vaginal challenge.
Figure 8.
Virus titers in vaginal swabs of vaccinated and control goats after CpHV-1 challenge.