Diagnostics for Rift Valley fever virus

Abstract

Rift Valley fever virus (RVFV) is a mosquito-borne, zoonotic Phlebovirus that is a significant threat to ruminants and humans. RVFV is categorized as an overlap Select Agent by the Department of Health and Human Services and US Department of Agriculture. Therefore, the study of RVFV’s pathogenesis and the development of novel diagnostic tools for the prevention and control of outbreaks and virus spread is crucial. RVF is endemic to sub-Saharan Africa but has spread beyond the continent to the Arabian Peninsula indicating the competence of the virus to emerge in new areas. Thus, the high likelihood of RVF’s spread to other non- endemic countries also spurs the need for development and implementation of rapid diagnostic tests and surveillance programs. In the US, RVFV is a Select Agent, requiring BSL-3 enhanced containment practices for research work. First, we developed a method for the detection of RVFV RNA by reverse transcriptase real-time PCR (RT-qPCR) using non-infectious, formalin- fixed, paraffin-embedded tissues (FFPET). The results from FFPET RT-qPCR were compared to prior results for fresh-frozen tissues (FFT) RT-qPCR, as well as immunohistochemistry and histopathology completed on the same FFPET blocks. We developed a novel technique using a rapid and low cost magnetic bead extraction method for recovery of amplifiable RVFV RNA from FFPET. FFPET RT-qPCR can serve as an alternative tissue-based diagnostic test, which does not require a BSL-3 research facility. Second, we assessed the diagnostic accuracy and precision of a recombinant RVFV nucleoprotein based competitive ELISA (cELISA) assay to detect RVFV antibodies. The cELISA results were compared to the virus neutralization test, the gold standard serological assay for RVFV. This prototype cELISA is easy to implement, sensitive, specific, and safe test for the detection of antibodies to RVFV in diagnostic and surveillance applications. RVF is an important transboundary disease that should be monitored on a regular basis. The diagnostic tests developed and validated in this thesis could be used in endemic or non-endemic countries for the early detection of RVF and assist with the implementation of countermeasures against RVFV.