Recombinant Marek’s disease virus type 1 provides full protection against very virulent Marek’s and infectious bursal disease viruses in chickens

Figure 1. Characterization of the rescued parental MDV vaccine virus (rMDV) and the wild-type virus (MDV). (A) The cytopathic effects induced by rMDV and MDV. Bar length, 400 μm. (B) Electron microscopy detection of rMDV and MDV in infected CEFs. Bar length, 200 nm. Arrows represent the MDV virions detected in the cell nucleus in infected cells. (C) The restriction pattern of the rMDV and MDV genomic DNA digested with HindIII. (D) The growth properties of rMDV and MDV in cell cultures. Data presented are the means ± standard deviations (S.D.) from three independent experiments.

Figure 2. Construction of recombinant fosmids inserted the VP2 gene of IBDV. (A) The genomic structure of MDV, and the five fosmids derived from MDV1 vaccine strain 814 used for MDV regeneration. Numbers represent the fosmid names and the location of each fosmid fragment in the genome of strain 814. (B–D) The schematic diagrams of recombinant fosmids inserted the VP2 gene of IBDV within the UL41 (B), US2 (C) and US10 (D) genes.

Figure 3. Characterization of the recombinant MDVs expressing VP2 gene. (A) Confirmation of the successful expression of VP2 by indirect immunofluorescence assay. (B) Detection of the expression of VP2 with western blotting. Chicken β-actin was detected as the internal control. (C) Comparison of VP2 expression from different viruses with FACS analysis. Results presented as the mean fluorescence intensity (MFI) of the VP2 positive cells in 10,000 events captured during the procedure. (D) Negative-stain electron microscopy detection of the IBDV subviral particles expressed by rMDV-VP2. Bar length, 40 nm. (E) PCR amplification of the VP2 gene from the recombinant viruses passaged 20 times in CEFs. (F) Detection of VP2 expression from the recombinant viruses passaged 20 times in CEFs with indirect immunofluorescence assay. (G) Comparison of the replication kinetics of the recombinant viruses. Data presented are the means ± standard deviations (S.D.) from three independent experiments. Statistical significance was set at P < 0.05.

Figure 4. Antibody responses against IBDV in chickens. (A) Detection of IBDV antibody titres of immunized animals by ELISA. Sera were collected weekly and detected using a commercial IBDV Antibody test kit (IDEXX, Westbrook, Maine). (B) Detection of viral neutralization antibodies against IBDV in chickens after 28 days of vaccination. Data presented are the means ± standard deviations (S.D.) from 10 birds in each group. Statistical significance was set at P < 0.05.

Figure 5. Protective efficacy of the recombinant vaccines against vvIBDV challenge. (A) Survival rate of chickens challenged with vvIBDV within an observation period of 10 days. (B) The mean symptomatic index scores for chickens after vvIBDV challenge. (C) The bursa: body-weight index (BBIX) values of chickens died and survived after challenge. (D) The histopathologic bursal lesion score (HBLS) of chickens died and survived after challenge. The dead birds were dissected at the day when they died and the surviving birds were sacrificed and analyzed after the observation period at day 10 post challenge. Data presented are the means ± standard deviations (S.D.) from 15 birds in each of the vaccinated group and 10 birds in the healthy control group. Statistical significance was set at P < 0.05.