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Introduction: Homologous recombination is an effective way to generate recombinant viruses for vaccine research such as pseudorabies virus (PRV) and adenovirus. Its efficiency can be affected by the integrity of viral genome and the linearization sites. Methods: In the study, we described a simple approach to isolate the viral DNA with high genomic integrity for large DNA viruses and a time-saving method to generate recombinant PRVs. Several cleavage sites in the PRV genome were investigated by using the EGFP as a reporter gene for identification of PRV recombination. Results: Our study showed that cleavage sites of XbaI and AvrII are ideal for PRV recombination which showed higher recombinant efficiency than others. The recombinant PRV-EGFP virus can be easily plaque purified in 1-2 weeks after the transfection. By using PRV-EGFP virus as the template and XbaI as the linearizing enzyme, we successfully constructed the PRV-PCV2d_ORF2 recombiant virus within a short period by simply transfecting the linearized PRV-EGFP genome and PCV2d_ORF2 donor vector into BHK-21 cells. This easy and efficient method for producing recombinant PRV might be adapted in other DNA viruses for the generation of recombinant viruses.
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BACKGROUND: Virus neutralization test (VNT) is widely used for serological survey of classical swine fever (CSF) and efficacy evaluation of CSF vaccines. However, VNT is a time consuming procedure that requires cell culture and live virus manipulation. C-strain CSF vaccine is the most frequently used vaccine for CSF control and prevention. In this study, we presented a neutralizing monoclonal antibody (mAb) based competitive enzyme-linked immunosorbent assay (cELISA) with the emphasis on the replacement of VNT for C-strain post-vaccination monitoring. RESULTS: One monoclonal antibody (6B211) which has potent neutralizing activity against C-strain was generated. A novel cELISA was established and optimized based on the strategy that 6B211 can compete with C-strain induced neutralizing antibodies in pig serum to bind capture antigen C-strain E2. By testing C-strain VNT negative pig sera (n = 445) and C-strain VNT positive pig sera (n = 70), the 6B211 based cELISA showed 100% sensitivity (95% confidence interval: 94.87 to 100%) and 100% specificity (95% confidence interval: 100 to 100%). The C-strain antibody can be tested in pigs as early as 7 days post vaccination with the cELISA. By testing pig sera (n = 139) in parallel, the cELISA showed excellent agreement (Kappa = 0.957) with VNT. The inhibition rate of serum samples in the cELISA is highly correlated with their titers in VNT (r2 = 0.903, p < 0.001). In addition, intra- and inter-assays of the cELISA exhibited acceptable repeatability with low coefficient of variations (CVs). CONCLUSIONS: This novel cELISA demonstrated excellent agreement and high level correlation with VNT. It is a reliable tool for sero-monitoring of C-strain vaccination campaign because it is a rapid, simple, safe and cost effective assay that can be used to monitor vaccination-induced immune response at the population level.
