Figure 1.
Generation of a Bo10 STOP BoHV-4 mutant.
A. Schematic representation of the strategy followed to produce the recombinant BoHV-4 strains. The Bo10 STOP BoHV-4 mutant was derived from a cloned BoHV-4 BAC by galK-counterselection method. The Bo10 coding sequence was disrupted by inserting stop codons near the end of the coding sequence for its predicted signal peptide (Bo10 STOP). The mutation incorporated two BamHI and one EcoRI restriction sites. B. Verification of the molecular structure. Viral DNA was digested with EcoRI, resolved by agarose gel electrophoresis, and hybridized with a 32P-labeled probe, corresponding to nucleotides 65,696–66,595 of the BoHV-4 V.test strain genome. Black triangle shows the restriction fragment that contains the WT Bo10 gene. The Bo10 Del fragment is higher due to eGFP insertion [33], however, it is much less visible due to deletion of most of the ORF. The 16,882-bp wild-type (WT) band becomes 14,033-bp (open triangle) for the Bo10 STOP mutant. The 2,885 bp band is not visible because it only hybridizes with a few nucleotides of the probe. In WT BAC, Bo10 STOP BAC and Bo10 STOP BAC Rev strains, the 4,870 bp band becomes 9,964 bp and 3,416 bp due to BAC cassette insertion as described previously [30]. Marker sizes in Kbp are indicated on the left. C. Detection of the Bo10 encoded gp180 protein by the anti-Bo10-c15 serum. Purified virions (5*105 virions per lane) were subjected to western blotting with anti-Bo10-c15 serum as described in the Material and Methods. The position of a molecular mass (MM) standard (in kDa) is shown on the left.
Figure 2.
In vivo persistence of Bo10- mutants.
Groups consisting of 3 rabbits were mock-infected or infected with 108 PFU of BoHV-4 WT V.test, Bo10 Del and Bo10 Rev strains (A and C) or infected with 107 PFU of BoHV-4 WT V.test and Bo10 STOP excised strains (B, D and E). A–D. Real-Time PCR relative quantification of BoHV-4 genomes. DNA was extracted from the PBMC (A and B) at the different times post-inoculation and from the spleen (C and D) 64 days post-inoculation. Data are expressed as the number of BoHV-4 ORF8 gene copies per 100 ng of total DNA. In A and B, the data presented are the average ± SEMs and were analyzed by 2ways ANOVA and Bonferroni posttests. In C and D, each point shows the genome copies for one rabbit. The data were analyzed by 1way ANOVA and Bonferroni posttests or Student t-test. No significative difference was observed. E. Spleens from the different rabbits were analyzed individually for reactivable BoHV-4 by infectious-center assay. Each point shows the infectious centers for one rabbit. The data were analyzed by Student t-test. No significative difference was observed.
Figure 3.
Neutralization of the BoHV-4 Bo10 mutants.
BoHV-4 WT V.test, Bo10 Del, Bo10 Rev and Bo10 STOP virions were incubated with sera of 4 different rabbits infected with BoHV-4 V.test strain. After incubation (2 h, 37°C) the viruses were plaque assayed for infectivity on MDBK cells. BoHV-4 titers are expressed relative to virus without antibody. The data presented are the average ± SEMs for 4 measurements and were analyzed by 2way ANOVA and Bonferroni posttests, * p<0.05, ** p<0.01, *** p<0.001.
Figure 4.
Sequence analysis of the Bo10 encoded proteins of nine BoHV-4 strains.
A. BoHV-4 Bo10 encoded proteins alignment. Predicted Bo10 transmembrane protein encoded by nine different BoHV-4 strains were aligned (ClustalX; [88]). Predicted peptide signals [89] and transmembrane regions were highlighted in pale and dark grey respectively. Non-conserved residues were printed in red. Serine (S) and threonine (T) residues of the N-terminal ectodomain were highlighted in orange, asparagine (N) residues of the N-terminal ectodomain were highlighted in green. Open and filled circles indicate potential O- and N-glycosylation sites respectively (using NetNglyc 1.0 and NetOglyc 3.1 algorithms [37]) that are predicted for each of these residues in all the different strains that display such residue at that position. B. Prediction of N-glycosylation sites for the complete BoHV-4 V.test gp180 protein sequence using the NetNglyc 1.0 algorithm. The shaded regions indicate the signal peptide and transmembrane region. The red line indicates significative threshold. C. Prediction of O-glycosylation sites for the complete BoHV-4 V.test gp180 protein sequence using the NetOglyc 3.1 algorithm. The shaded regions indicate the signal peptide and transmembrane region. The red line indicates significative threshold.
Figure 5.
Glycosylation of BoHV-4 gp180 and importance of BoHV-4 glycans in neutralization evasion.
A. Purified BoHV-4 WT V.test strain virions were deglycosylated after denaturation. /, no enzyme; N-, protein N-glycanase; O-, sialidase A + β1-4 Galactosidase + O-glycanase; N O-, protein N-glycanase + sialidase A + β1-4 Galactosidase + O-glycanase. Each was then immunoblotted for gp180 with anti-Bo10-c15 serum as described in the Material and Methods. The position of a MM standard (in kDa) is shown on the left. B. Intact MDBK cell-derived BoHV-4 V.test strain WT virions were deglycosylated without denaturation. /, no enzyme, N-, protein N-glycanase, O-, sialidase A + O-glycanase, N-O- protein N-glycanase + sialidase A + O-glycanase. Each was then tested for neutralization by serum of rabbits immunized by the BoHV-4 V.test strain. After incubation (2 h, 37°C) the viruses were plaque assayed for infectivity on MDBK cells. BoHV-4 titers are expressed relative to virus without antibody. The data presented are the average ± SEMs for 4 measurements and were analyzed by 2way ANOVA and Bonferroni posttests, ** p<0.01, *** p<0.001. Statistical significance was only shown for O- treatment. Equivalent data were obtained in two further experiments.
Figure 6.
Antigenicity of infected cells.
MDBK cells were infected (2 PFU/cell, 36 h) with WT (solid black lines), Bo10 Del (red lines), Bo10 Rev (dotted black lines), WT BAC (grey lines), Bo10 STOP (green lines) or Bo10 STOP Rev (dotted grey lines) of BoHV-4 V.test, and then analyzed by flow cytometry. The filled histogram shows uninfected cells. MAb 29 and 35 recognize gB, mAb 16 recognizes gL, and mAb 33 recognizes gH/L. Fixed (PFA 4%, 4°C for 30 min) and permeabilized (saponin 0.1%) cells were used as control of protein expression.
Figure 7.
Antigenicity of purified virions.
A. and B. Immuno-electron microscopy. Purified BoHV-4 WT V.test or Bo10 Del virions were processed for immune-electron microscopy as described in Material and Methods. These samples were stained for gB with mAb 35 followed by secondary goat anti-mouse IgG-10 nm gold labeled. Pictures of individual virions were then taken and gold particles were counted on each virion. The data presented are the average ± SEMs for 20 different virions and were analyzed by Student’s t-test, *** p<0.001 (A). Pictures of representative virions are showed (B). C. Purified BoHV-4 WT V.test and Bo10 Del virus stocks were compared for gB content per PFU by immunoblotting for gB (with mAb 35) as described in the Material and Methods. The position of a MM standard (in kDa) is shown on the left. D. Purified BoHV-4 WT V.test, Bo10 Del, Bo10 Rev, WT BAC, Bo10 STOP and Bo10 STOP Rev virions (105 PFU per lane) were compared for particles content per PFU by immunoblotting with anti BoHV-4 V.test polyserum as described in the Material and Methods. The position of a MM standard (in kDa) is shown on the left. E. Antigenicity of bound purified virions. MDBK cells were exposed to BoHV-4 V.test WT, Bo10 Del, Bo10 Rev, WT BAC, Bo10 STOP and Bo10 STOP Rev virions (20 PFU/cell, 2 h, 4°C), then directly fixed with acetone 95%/water 5%. The cells were then stained for gB with mAb 29 and 35, for gL with mAb 16 and for gH/gL with mAb 33. The pictures were taken with a confocal laser microscope. Images are shown with a pseudo-color glow scale (White, highest to black, lowest intensity, blue represents overexposed pixels).
Figure 8.
Sensitivity of Bo10- mutants to anti gL directed neutralization.
A. BoHV-4 WT V.test, Bo10 del, Bo10 Rev and Bo10 STOP virions were incubated with the gL-specific neutralizing mAb 16. After incubation (2 h, 37°C) the viruses were plaque assayed for infectivity on MDBK cells. BoHV-4 titers are expressed relative to virus without antibody. The data presented are the average ± SEMs for triplicate measurements and were analyzed by 1way ANOVA and Bonferroni posttests, ** p<0.01, *** p<0.001. B. BoHV-4 WT V.test, Bo10 del, Bo10 Rev and Bo10 STOP virions were incubated with increasing amounts of the gL-specific neutralizing mAb 16, and then assayed for infectivity on MDBK cells. BoHV-4 titers are expressed relative to virus without antibody. Equivalent data were obtained in two further experiments.
Figure 9.
Immunogenicity of BoHV-4 lacking gp180.
Rabbit anti-BoHV-4 V.test WT antibody response. Groups consisting of 3 rabbits were infected with 107 PFU of BoHV-4 WT V.test and Bo10 STOP excised strains. A. Sera were collected at different times post-infection and the titre of anti-BoHV-4 antibodies was estimated by ELISA as described in the Material and Methods. Each value represents the mean +/- SD of the data obtained for the three rabbits of each group. The serum of a mock infected rabbit was taken as control. The data were analyzed by 2way ANOVA and Bonferroni posttests. No significative difference was observed between groups. B. Specific anti gB, gH and gL antibody responses were investigated by staining unfixed 293T cells expressing GPI-linked forms of gL, the gB extracellular domain or the gH extracellular domain. These staining were performed with sera collected 63 days post-infection. The data were analyzed by Student t-test. No significative difference was observed between WT and Bo10 STOP sera. C. BoHV-4 WT V.test, Bo10 Del, Bo10 Rev and Bo10 STOP virions were incubated with sera of WT or Bo10 STOP infected rabbits collected 63 days post-infection (3 sera per group). After incubation (2 h, 37°C) the viruses were plaque assayed for infectivity on MDBK cells. BoHV-4 titers are expressed relative to virus without antibody. The data presented are the average ± SEMs for 3 measurements and were analyzed by 2way ANOVA and Bonferroni posttests. No significative difference was observed between WT and Bo10 STOP sera.
Figure 10.
Glycans on BoHV-4 gp180 allow virus neutralization.
A. Sensitivity of WT, Bo10 Del and Bo10 Rev strains to Jacalin mediated neutralization. BoHV-4 WT V.test, Bo10 Del and Bo10 Rev virions were incubated with increasing amounts of O-glycan specific lectin jacalin (1 h, 37°C) and then assayed for infectivity on MDBK cells. BoHV-4 titers are expressed relative to virus without lectin. The data presented are the average ± SEMs for 3 measurements and were analyzed by 2way ANOVA and Bonferroni posttests, *** p<0.001. B. Sensitivity to anti-Gal antibodies induced complement dependent neutralization of BoHV-4 WT, Bo10 Del, Bo10 Rev and Bo10 STOP strains. Virions of the different strains derived from MDBK cells were assayed for their sensitivity to neutralization by horse serum supplemented with increasing amounts of mAb M86 raised against Galα1-3Gal. Horse sera (heat inactivated or not by incubation at 56°C for 30 min) were tested at the final concentration of 10% (vol/vol). After incubation (2 h, 37°C) the viruses were plaque assayed for infectivity on MDBK cells. BoHV-4 titers are expressed relative to virus without antibody.