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Research Project: Rift Valley Fever Pathogenesis and Epidemiology

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

item BALARAMAN, VELMURUGAN - Kansas State University
item GAUDREAULT, NATASHA - Kansas State University
item TRUJILLO, JESSIE - Kansas State University
item INDRAN, SABARISH - Kansas State University
item Wilson, William
item RICHT, JUERGEN - Kansas State University

Submitted to: Journal of Virological Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/14/2023
Publication Date: 2/16/2023
Citation: Balaraman, V., Gaudreault, N.N., Trujillo, J.D., Indran, S.V., Wilson, W.C., Richt, J.A. 2023. RT-qPCR genotyping assays for differentiating Rift Valley fever phlebovirus strains. Journal of Virological Methods. 315(2023). Article 114693.

Interpretive Summary: The emerging zoonotic pathogen, Rift Valley fever phlebovirus (RVFV) can cause lethal disease in livestock and humans. There are no licensed mitigation tools (therapeutic or vaccines) for humans and only a conditional licensed vaccine for animals in the US. Here, we describe genotyping (GT) assays developed to differentiate genes from two wild-type strains and a vaccine strain of Rift Valley fever phleboviruses. The assay can genotype up to 13 samples in a 96-well plate format in 3 hours. An additional assay was developed as a screening assay for the detection of low quantities of RVFV strains in mixed RVFV samples.

Technical Abstract: Rift Valley fever phlebovirus (RVFV) is an emerging, mosquito-borne, zoonotic pathogen. Real time RT65 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.