Gallid herpesvirus 3 SB-1 strain as a recombinant viral vector for poultry vaccination

Fig. 1. a–d Infected cells stained against SB-1 with anti gB Y5.9 antibody (IgG1) followed by Alexa Fluor 568 staining. e–h infected cells stained separately against IBDV VP2 with anti VP2 antibody HH7 (IgG1) followed by Alexa Flour 488 staining. The nuclei of the cells are stained with DAPI and shown in blue (scale bar = 30 µm). a and e show CEF cells infected with parental pSB-1 virus, b and f show CEF cells infected with pSB-1-UL3/4VP2 recombinant virus, c and g show CEF cells infected with pSB-1-UL10/11VP2 recombinant virus and d and h show CEF cells infected with pSB-1-UL21/22VP2 recombinant virus. i–l show plaque formation in CEF cells infected with pSB-1 (i), CEF cells infected with pSB-1-UL3/4VP2 (j), pSB-1-UL21/22VP2 (l) and pSB-1-UL10/11VP2 (k) recombinant viruses (scale bar = 200 µm)

Fig. 2. Growth curve for SB-1 recombinant viruses in vitro. Time course experiment was performed in triplicate with each time point was infected independently. Each replicate was titrated in duplicate. PFU in 1 ml of infected cell suspension was calculated for each time point; SB-1(), SB-1-UL3/4VP2 (), SB-1-UL10/11VP2 () and SB-1-UL21/22VP2 (). A significant difference at time points 72 and 120 h (p = 0.0031 and p = 0.0001, respectively) was observed for SB-1-UL3/4VP2 when compared to its parental virus. Similarly, a significant difference was observed between the titre of SB-1 and SB-1-UL21/22VP2 time points 72 and 120 h (p = 0.0094 and p = 0.0001, respectively). In comparison with the other two recombinant viruses, SB-1-UL10/11VP2 showed a significant difference at time points 72, 96 and 120 h post infection (p = 0.0194, p = 0.0284 and p = 0.0001, respectively)

Fig. 3. a Experiment 1—Titre of neutralising antibodies in the sera of chickens vaccinated with VAXXITEKHVT+IBD (), SB-1-UL3/4VP2 (), SB-1-UL21/22VP2 () or SB-1-UL10/11VP2 () vaccine viruses. Serum samples were collected at weeks 2, 3 and 4 post-vaccination. The mean titre in each group is shown as a horizontal bar. b Experiment 2—Titre of neutralising antibodies in week 3 serum of chickens vaccinated with VAXXITEKHVT + IBD (), SB-1-UL3/4VP2 (), SB-1-UL21/22VP2 () or SB-1-UL10/11VP2 (). Individual values are shown. Group names are given on the X-axis

Fig. 4. a Clinical score in birds vaccinated with VAXXITEKHVT+IBD, SB-1-UL3/4VP2, SB-1-UL21/22VP2 and SB-1 following infection with104.3 TCID50 of IBDV strain UK661. Clinical scores are shown for individual birds. A rapid increase in the clinical score after 45 h post challenge was observed in SB-1 group and increased to its highest level at 55 h post challenge when affected birds were euthanized for humane reasons. b Percentage survival for the vaccinated birds challenged with IBDV UK661. Birds inoculated with pSB-1 were euthanized for humane reasons or died 55 h post IBDV infection. VAXXITEKHVT+IBD (), SB-1(), SB-1-UL3/4VP2 (), SB-1-UL21/22VP2 ()

Fig. 5. Gene expression analysis for a UL21 and UL22 in cells infected with SB-1-UL21/22VP2, b UL10 and UL11 in cells infected with SB-1-UL10/11VP2, and c UL3 and UL4 in cells infected with SB-1-UL3/4VP2 (c). Fold change expression was presented as 2−ΔΔCt on the Y-axis of each graph. Expression of VP2 from the 3 insertion loci are shown (d) data are presented as ΔCt on the Y-axis. The groups are shown on the X axis. A significant difference in the expression of VP2 was observed in cells which were infected with SB-1-UL3/4VP2 compared to the cells infected with SB-1-UL21/22VP2 or SB-1-UL10/11VP2 (p = 0.0298 or p = 0.0035, respectively)