Figure 5

Virus-neutralizing titer assay. The virus-neutralizing titers were determined in sera pooled from immunized mice of different groups 2 weeks post immunization. The sera (diluted 1:5 in MEM medium) were heat-activated for 30 min at 56°C, then serially diluted two-fold in 96-well flat bottom plates and incubated with 0.1 μg of purified ORFV diluted in 50 μl MEM for 90 min at 37°C. All sera were run in triplicate. Subsequently, 100 μl of OFTu cell suspension were added to each well at a concentration of 45,000 cells/well. The plates were incubated for 5 days at 37°C in a 5% CO₂ incubator. The titer is expressed as ND50 and calculated using the Reed and Muench method. As shown in Figure 5, the assay of virus-neutralizing titers performed at 2 weeks post immunization indicates a significant difference between four vaccine plasmids groups (including pcDNA 3.1-ORFV 011 group, pcDNA 3.1-ORFV 059 group, pcDNA-ORFV 011 plus pcDNA-ORFV 059 group and pcDNA-ORFV 011/ORFV 059 group) and control groups (including PBS and pcDNA3.1(+) group) (**P < 0.01). Among the four vaccine plasmids groups, the virus-neutralizing titers in the pcDNA-ORFV 011/ORFV 059 group was significantly higher than that of pcDNA-ORFV 011 plus pcDNA-ORFV 059 group (*P < 0.05). A group: PBS; B group: pcDNA3.1(+) vector; C group: pcDNA3.1-ORFV011; D group: pcDNA3.1-ORFV059; E group: pcDNA3.1-ORFV011 plus pcDNA3.1-ORFV059; F group: pcDNA3.1-ORFV011/ORFV059.