Figure 1.
Schematic illustration of the construction strategies of the recombinant viruses.
(A) The organization of the left terminus of the EHV-1 RacH genome showing that ORF1 and ORF2 are absent. UL: unique long; US: unique short; IR: internal repeat; TR: terminal repeat. (B) A fragment released from transfer plasmid pEP-VP2 by I-CeuI digestion was used to recombine with RacH genome, resulting in an intermediate kanamycin (aphAI cassette)-resistant BAC clone. After I-SceI digestion, kanamycin was removed in the following step of en passant mutagenesis (in box) to generate VP2-expressing virus. (C) With another round of en passant mutagenesis, VP5 gene with an IRES sequence upstream were inserted in between VP2 and BGH polyA, and a final construct expressing both VP2 and VP5 (D) was generated.
Figure 2.
Expression of the transgenes and in vitro growth properties.
(A) RK13 cells were infected with parental rRacH1, rH_VP2 or rH_VP2_5 at an m.o.i of 0.0001. Two days post infection, cells were fixed and incubated with anti-VP2 mAb 13C10 or anti-EHV-1 gp2 mAb 3B12, followed by Alexa Fluor 568-conjugated goat anti-mouse IgG. Fluorescence signal was inspected under the inverted fluorescence microscope. Bar indicates 50 µm. (B) Cell lysates infected by rRacH1, rH_VP2, rH_VP2_5 or BTV-8 were separated by 10% SDS-PAGE and analysed by Western blot. Expression of VP2 and VP5 was detected using primary antibody 13C10 and sheep anti-BTV-8 hyperimmune sera, respectively. EHV-1 MCP was used as a control and detected with mAb 3G4. (C) RK13 cells were infected by the individual virus at an m.o.i of 0.0001 and overlaid. Three days post infection, plaques were photographed and the areas were measured. For each virus, at least 50 plaques were measured. The relative plaque area was compared to that of rRacH, which was set as 100%. * P<0.001. (D) The single-step growth kinetics of these viruses was analysed. RK13 cells were infected by the viruses at an m.o.i of 5. Extracellular and intracellular virus titres were determined at the indicated time points.
Figure 3.
Neutralising antibody response was induced by the recombinant viruses.
Three-week old female Balb/c mice were prime/booster immunised with rRacH1, rH_VP2 or rH_VP2_5. At the indicated days (0, 14, 21, 28, 35), mice were bled and the antibody was examined using standard serum neutralisation test. IN: intranasal; SC: subcutaneous.
Figure 4.
Protective efficacy of the recombinant vaccines against BTV-8 challenge in IFNAR−/− mice.
(A) Average group weights after challenge. (B) Survival rate of IFNAR−/− mice after BTV-8 challenge infection. (C) BTV-8 RNA in spleen samples taken 14 days after challenge was quantified using real-time RT-PCR targeting segment 5. The average segment 5 RNA copies per mg of spleen are shown. (D) BTV-8 virus titers in spleen samples were determined by end-point titration of supernatants on Vero cells and shown as average TCID50 per mg of spleen. Differences in virus titres between the groups were statistically evaluated with a Kruskal-Wallis rank sum test.
Figure 5.
Detection of VP7 antibody from survived mice using ELISA.
At the end of the experiment, antibody levels to BTV-8 VP7 were determined from blood samples of the surviving mice (rH_VP2_5, BTVPUR AlSap™ 8, and the environmental groups) with the ID Screen BT Competition ELISA. The OD values of samples were evaluated by comparing them to the kit negative control. All samples with an OD of up to 50% of the negative control are antibody-positive. Samples with higher ODs are considered doubtful (50% to 60%) or negative (over 60%).
Table 1.
Oligonucleotides used in this study.