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Early detection of emerging viruses by next generation in situ hybridization

Final Report Summary - WILD SCOPE (Early detection of emerging viruses by next generation in situ hybridization)

Summary description of the project objectives:
The main goal of the project is to develop, validate and apply a new generation in situ hybridazation (ISH) technique, RNAscope®, for early detection of (re)emerging viruses in formalin-fixed, paraffin-embedded (FFPE) tissues from wildlife. The first part of the project focuses on setting up specific RNAscope® tests for the detection of the relevant viruses in FFPE tissues. The developed specific RNAscope® tests will be tested on virus-positive tissues from controlled infection experiments for each selected virus. Finally, validation in real settings will be done in comparison with the traditional serology, PCR and random sequencing methods in order to assess the performance of each tool, alone or combined.

Description of the work performed since the beginning of the project:
Two viruses were first selected for their epidemiological relevance. Progress has been done in two different viruses: MERS coronavirus (MERS-CoV) in camels (Camelus dromedarius) and hepacivirus in bank voles (Myodes glareolus). MERS-CoV in camels was chosen because epidemiologic and surveillance data on MERS-CoV strongly point toward a role for dromedary camels as a reservoir for zoonotic transmission of the virus. Hepacivirus was included in the project for being a newly discovered virus in a wildlife species that had the potential to serve as a rodent model for hepatitis C virus (HCV) infection in humans.

Regarding MERS-CoV, the first study was to analyze the presence of MERS-CoV in FFPE respiratory tissues from zoo camels in continental Europe and free-living dromedary camels from the Canary Islands and Morocco. The main objective was to test if FFPE camel tissues could serve to retrospectively detect MERS-CoV presence first by TaqMan® real-time reverse transcriptase PCR (RT-PCR) and further test positive samples by RNAscope® ISH. Different zoological institutions, Universities and free-lance veterinary pathologists in Europe and Morocco were asked to provide FFPE respiratory tissues in paraffin blocks from camelid species. Only tissues belonging to the respiratory tract or lymph nodes were included in the study. Six FFPE tissues (lung and nose) from 2 experimentally MERS-CoV infected camels were used as positive controls for MERS-CoV detection and PCR validation. FFPE tissues chosen from the provided collection and positive controls were further used for RNA isolation and TaqMan® RT-PCR assay for the detection of MERS-CoV.

Hepacivirus in bank voles was included in the Wild Scope project for being a newly discovered virus. The relevance of this pathogen-host pair is its potential to serve as a laboratory model for HCV infection in humans. The pathogenesis caused by Hepacivirus in bank voles is being studied in more detail, in order to study the similarities of the liver infection with HCV in humans. In order to correlate the lesions observed in the liver with the presence of HepaC virus particles, a specific RNAscope® probe was designed to detect this newly discovered virus. The technique was further optimized for bank vole tissues. Finally ISH was performed on livers and other organs from experimentally infected animals with positive results.

Description of the main results achieved so far:
Camel MERS-CoV: MERS-CoV RT-PCR was validated with the positive controls, which resulted positive in the analysis. Since field samples resulted negative, ISH was not subsequently performed in the FFPE tissues. Camel samples tested by RNAscope® resulted negative, thus no further investigation was done. Regarding bank vole hepacivirus, the developed ISH allowed the detection of the virus in FFPE bank vole liver tissues.

Expected final results and their potential impact and use (including the socioeconomic impact and the wider societal implications of the project so far):
This work constitutes the first report on the use of RT-PCR for the detection of MERS-CoV in FFPE sections. The described assay is a useful tool for the examination of archival FFPE tissues, and allows for both prospective and retrospective evaluation of tissue samples for the presence of MERS-CoV and other potentially relevant viruses. For Hepacivirus, the RNAscope® ISH opens the potential of a technique that produces clinically relevant information regarding cellular and tissue context to be used for an animal model of HCV.