In Situ Hybridization for Detection of Rift Valley Fever Virus

Rift Valley fever virus (RVFV) is a mosquito‐borne zoonotic arbovirus with a tripartite genome consisting of a small, a medium (M), and a large (L) segment. This RNA virus causes frequent epidemics in Africa and the Arabian Peninsula. Outbreaks of Rift Valley fever typically result in abortion storms, the deaths of young ruminants and less often the deaths of adult ruminants. As a zoonotic virus, RVFV can also cause symptoms ranging from a mild flu‐like illness to encephalitis and death in humans. As such, RVFV‐infected tissues must be handled with caution. Formalin‐fixed, paraffin‐embedded (FFPE) tissues offer a safer format for researchers and diagnosticians testing for the presence of this virus. Currently, immunohistochemistry (IHC) serves as a confirmatory diagnostic test in South Africa alongside RT‐qPCR on fresh frozen tissue. However, PCR does not enable the ability to visualize the virus within a histological context and IHC has limitations in its ability to detect viral antigen in suboptimally preserved samples. These limitations create a need for more powerful confirmatory diagnostic tools. Here we demonstrate the use of RNAscope ® in situ hybridization (ISH) technology for the detection of RVFV viral RNA (vRNA) in FFPE tissues. Samples tested included a broad set of previously published, confirmed positive field samples from sheep naturally infected during the 2010 South African outbreak. Prior, a DNA probe was designed to target the nucleotides between 20‐997 of the RVFV L segment (NC_014397.1) and the assay was validated on experimentally infected tissues. Here we tested this pan‐RVFV ISH assay on naturally infected liver, spleen, kidney, lung, thyroid, brain, skeletal and cardiac muscle, lymph node, salivary gland, adrenal gland, gastrointestinal tract, and male and female reproductive organs. We also compared ISH results to other tests, a previously established triplex FFPE RT‐qPCR targeting all three gene segments as well as IHC targeting the RVFV Gn glycoprotein. Finally, we were able to dual label samples with pan‐RVFV ISH and IHC for analysis of both RVFV RNA and protein distribution as well as RVFV RNA and cell type markers, providing finer grained detail regarding cellular tropism. Pan‐RVFV ISH appropriately labeled vRNA and this signal correlated with visible lesions in the various organs tested. Additionally, vRNA was detected in macrophages in the lymph nodes, a result previously seen by IHC. The positive ISH results mirrored those obtained by FFPE RT‐qPCR at the tissue block level. In comparison to IHC, ISH subjectively appears to produce a stronger and more reliable signal. This was observed in multiple organs where histopathology was present but RVFV antigen IHC was negative. We believe that this highly sensitive and specific ISH assay has potential as a RVFV confirmatory diagnostic test and research applications. Support or Funding Information This work was funded by the Department of Diagnostic Medicine, College of Veterinary Medicine, Kansas State University; the Department of Paraclinical Sciences, University of Pretoria, USDA Agricultural Research Services, and APHIS. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .