RT-qPCR genotyping assays for differentiating Rift Valley fever phlebovirus strains

Rift Valley fever phlebovirus (RVFV) is an emerging, mosquito-borne, zoonotic pathogen. Real time RT-qPCR genotyping (GT) assays were developed to differentiate between two RVFV wild-type strains (128B-15 and SA01-1322) and a vaccine strain (MP-12). The GT assay uses a one-step RT-qPCR mix, with two different RVFV strain-specific primers (either forward or reverse) with long or short G/C tags and a common primer (either forward or reverse) for each of the 3 genomic segments. The GT assay produces PCR amplicons with unique melting temperatures that are resolved in a post PCR melt curve analysis for strain identification. Furthermore, a strain specific RT-qPCR (SS-PCR) assay was developed to allow for specific detection of low titer RVFV strains in mixed RVFV samples. Our data shows that the GT assays are capable of differentiating L, M, and S segments of RVFV strains 128B-15 versus MP-12, and 128B-15 versus SA01-1322. The SS-PCR assay results revealed that it can specifically amplify and detect a low titer MP-12 strain in mixed RVFV samples. Overall, these two novel assays are useful as screening tools for determining reassortment of the segmented RVFV genome during co-infections, and could be adapted and applied for other segmented pathogens of interest.