Reverse Transcriptase Real Time PCR Detection of Rift Valley Fever Virus RNA in Formalin‐Fixed, Paraffin‐Embedded Tissues

Abstract

Rift Valley Fever Virus (RVFV) is a mosquito‐borne zoonotic pathogen endemic in parts of Africa and the Arabian Peninsula. RVFV causes acute febrile disease with mass abortion and death in ruminants and occasional encephalitis and death in humans. In the US, RVFV is a BSL‐3Ag Select Agent and a foreign animal disease, requiring special containment laboratories for research work. Research goals include the study of RVFV pathogenesis and the development of novel diagnostic tools and disease prevention methods such as vaccines. Ongoing surveillance plays a critical role in prevention and control of outbreaks and virus spread. Formalin‐fixed, paraffin‐embedded tissues (FFPET) are non‐infectious diagnostic specimens, routinely used for histopathology and immunohistochemistry (IHC), however use of this diagnostic specimen for molecular detection of pathogen RNA is underinvestigated. The objective of this work is to develop and validate a protocol for use of FFPET for the sensitive and specific detection of RVFV RNA.Study samples were fresh‐frozen tissues (FFT) and FFPET (liver, spleen, lung, kidney) collected from sheep and calves experimentally infected with two RVFV strains (Kenya 2006 and Saudi Arabia 2001). Deparaffinization of paraffin whorls (5‐5μm thick), tissue lysis with proteinase K, and reversal of nucleic acid cross linking in heated alkaline buffer was performed in a single tube using commercial buffers (Qiagen). RNA was purified using automated magnetic bead extraction on the Taco mini (GeneReach) for FFPET or the Applied Biosystems reagents on the Kingfisher (FFT). Multiplex Reverse Transcriptase Real Time PCR (RT‐qPCR) for RVFV L, M and S gene segments was used to detect RVFV RNA using Quanta qScript XLT One‐Step RT‐qPCR ToughMix (FFPET) or AgPath RT‐qPCR mix (FFT). IHC for RVFV antigen was done with a mouse monoclonal anti‐RVFV nucleoprotein antibody and avidin‐biotin complex detection.To date, 18 FFPET samples have been tested. For samples that were both IHC and FFT RT‐qPCR positive (n=13), FFPET results were 100% positive. For FFT samples (n=5) that were not tested by RT‐qPCR but were IHC positive, the FFPET RVFV RT‐qPCR results were also positive. Testing of an additional 46 FFPE samples is presently underway.Using this newly developed single tube FFPET technique for nucleic acid recovery, followed by a rapid, low cost magnetic bead nucleic acid extraction allows for highly sensitive detection of RNA specific for RVFV. This novel method could strengthen routine diagnostics, surveillance and molecular epidemiological studies that are currently limited by the strict biosecurity requirements for RVFV samples. Moreover, this methodology could also be expanded for detection of RNA targets for additional high impact pathogens or for transcriptional studies that must relie on archival FFPET.Support or Funding InformationThis work was funded by startup funds provided to Dr. Davis by the Department of Diagnostic Medicine and Pathobiology, College of Veterinary Medicine, Kansas State University and uses archival animal study materials from research funded by grants from the Department of Homeland Security Center of Excellence for Emerging and Zoonotic Animal Diseases (CEEZAD), Grant No. 2010‐ST061‐AG0001, USDA Agricultural Services project 3020‐32000‐005‐00D and the Kansas Bioscience Authority.