RT-qPCR genotyping assay for Rift Valley Fever virus with the capability of low copy resolution in mixed virus populations.

Authors: BALARAMAN, VELMURUGAN - Kansas State University; GAUDREAULT, NATASHA - Kansas State University; INDRAN, SABARISH - Kansas State University; TRUJILLO, JESSIE - Kansas State University; Wilson, William - Bill; RICHT, JUERGEN - Kansas State University

Submitted to: American Association of Veterinary Laboratory Diagnosticians
Publication Type: Abstract Only
Publication Acceptance Date: 7/17/2018
Publication Date: 10/19/2018
Citation: Balaraman, V., Gaudreault, N., Indran, S., Trujillo, J., Wilson, W.C., Richt, J. 2018. RT-qPCR genotyping assay for Rift Valley Fever virus with the capability of low copy resolution in mixed virus populations.. American Association of Veterinary Laboratory Diagnosticians. http://www.aavld.org/.

Interpretive Summary: .

Technical Abstract: Rift Valley Fever virus (RVFV) is an emerging, mosquito borne, zoonotic pathogen, that poses a serious threat to livestock and humans. RVFV can cause death and abortion storms in domestic ruminants including camels. In humans, RVFV causes febrile illness that can lead to hemorrhagic fever, encephalitis and death. Detection methods for the virus are based on the presence of serum antibodies, virus isolation and detection of viral RNA via RT-qPCR. However, these methods generally do not offer strain differentiation, which may be important to distinguish between the various circulating strains and genetic reassortants that may emerge. A Single Nucleotide Polymorphism (SNP) - based RT-qPCR with melt curve analysis was developed to differentiate genotypes of RVFV wildtype and vaccine strains (Kenya 128B-15 and MP-12, respectively). The RT-qPCR assay uses a one-step RT-PCR mix, with RVFV strain-specific forward primers and universal reverse primer sets for each genomic segment. The primer sets were designed to produce PCR amplicons with strain-differentiating melting temperatures that are resolved in a post PCR melt curve analysis. Genotypes identified by melt curve analysis were further confirmed by restriction enzyme analysis and Sanger sequencing. This method specifically differentiated L, M and S segments of wildtype Kenya 128B-15 strain from MP-12 vaccine strain. To increase sensitivity for low copy variants, a secondary blocking PCR assay was developed to allow for preferential amplification of low copy RVFV strains in a mixed genotype samples. For this purpose, primers specific to the high copy variant (wild type 128B-15) were 3’ phosphorylated to prevent extension of 128B-15 genomic sequences during cDNA synthesis. Sensitivity of this blocking assay was confirmed by performing the RT-qPCR assay with varying concentrations of mixed 128B-15 and MP-12 samples. The custom blocking assay, coupled with the multiple gene SNP-based RT-qPCR with melt curve analysis can be used for rapid genotyping of RVFV strains to support research and molecular epidemiological studies.