RECOMBINATION-NEGATIVE, IMMUNE-ENHANCED, AND CLINICALLY-ATTENUATED PRRSV AS A VACCINE PLATFORM

<b>Project Methods</b><br> The major focus of the current project is to develop a platform for RNA recombination-negative vaccine candidates for PRRSV. The project contains four objectives, and experimental methods for each objective are described as follows.In Objective 1, IFN suppression-negative and NF-κB activation-negative PRRSV will be further characterized and optimized as the backbone virus for the construction of RNA recombination-resistant virus. Viral mechanisms for IFN suppression will further be identified. Target mechanisms include the PML degradation, ubiquitination, and SUMOylation of viral proteins, and the methods include gene expression in cells, proinflammatory cytokine assays, reporter assays, quantitative RT-PCR assays, viral reverse genetics, and generation and propagation of mutant viruses.Objective 2 will design for synthetic transcription regulatory sequence (TRS) network and construction of a TRS network-reprogrammed virus using the IFN suppression-negative and NF-κB activation-negative mutant virus as the backbone. Leader-body junction and body TRS for viral structural genes will be determined by RT-PCR and sequencing using viral mRNA collected from virus-infected cells. The synthetic TRS network will be designed using a software program to predict the secondary structure of the viral genome. A TRS-network reprogrammed mutant virus will be generated by reverse genetics.In Objective 3, the recombination-negative virus will be characterized in cells for genetic stability.Recombination resistance will be determined by co-infection with the wild-type virus. Plaque assays will be conducted, and each plaque will be sequenced to examine recombination phenotype.In Objective 4, the genetic stability of recombination-negative virus will be examined in pigs. Groups of pigs will be coinfected with the reprogrammed virus and wild-type virus or a commercial vaccine virus. Clinical signs, viremia, and antibodies will be examined. Genomic RNA recombination will be examined by plaque assay and sequencing. The data will be statistically analyzed using SAS and ANOVA.