ABSTRACT
New-emerging variants of Pseudorabies virus (PRV) compromise the protection provided by current vaccines and cause the death of all ages of vaccinated pigs since 2011. New vaccines based on current circulating PRV strain are needed to control the spread of disease since the variants are antigenically different from classical strains of virus. In this study, a TK/gE/gI triple gene-deleted PRV derived from current circulating field isolate was generated by using bacterial artificial chromosome techniques, and the rescued virus showed similar growth properties in vitro to its parent strain but reduced plaque size. To evaluate it as vaccine candidate, 9 day-old pigs were vaccinated and challenged with a virulent PRV variant. The results showed that vaccination can generate high level of protective gB-specific antibodies after vaccination and provide complete protection to the viral challenge. By contrast, the unvaccinated piglets all died within 6 days after viral challenge. Therefore, the TK/gE/gI triple gene-deleted PRV could be a promising vaccine candidate to control the wide spreading of PR variants in China.
Subject(s)
Herpesvirus 1, Suid/immunology , Pseudorabies Vaccines/immunology , Pseudorabies/prevention & control , Swine Diseases/prevention & control , Animals , Antibodies, Viral/blood , China , Gene Deletion , Herpesvirus 1, Suid/genetics , Pseudorabies Vaccines/administration & dosage , Pseudorabies Vaccines/genetics , Pseudorabies Vaccines/isolation & purification , Survival Analysis , Swine , Vaccines, Attenuated/administration & dosage , Vaccines, Attenuated/genetics , Vaccines, Attenuated/immunology , Vaccines, Attenuated/isolation & purification , Vaccines, Synthetic/administration & dosage , Vaccines, Synthetic/genetics , Vaccines, Synthetic/immunology , Vaccines, Synthetic/isolation & purificationABSTRACT
We designed two pairs of primers and their corresponding TaqMan probes according to gH, gE gene of PRV. By optimizing the probe's concentration, Mg2+ concentration, primers concentration and sample DNA extraction, real-time fluorescent quantitative PCR (FQ-PCR) which can quickly identity field virus and vaccine virus of PRV was established. According to our results, the dynamic range of the FQ-PCR assay is between 10 x 10(1) copies/microL and 10 x l0(8) copies/microL, and the detection limit of FQ-PCR is 1.0 x 10(1) copies/microL, which is 100 fold higher than that of conventional PCR. We detected 60 doubtful tissue samples using the FQ-PCR assay, serum neutralization and conventional PCR. In conclusion, the FQ-PCR method is rapid, sensitive, specific and accurate, and can be used to detect field strains of PRV rapidly. The closed-tube format of the assay minimized the risk of contamination of subsequent reaction and the assay can be performed in 2 h or less. Development of real-time quantitative PCR provides the basis for the early and rapid detection and analyzing quantitatively the infectious degree of PRV.