Wild Scope · Early detection of emerging viruses by next generation in situ hybridization
FP7 — People (Marie Curie Actions)
- Duration
- 2014-07-01 → 2016-06-30
- EU contribution
- €175,975
- Participants
- 1
- Scheme
- MC-IEF
Lines connect the coordinator with its partners.
Results in brief
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.
Data: CORDIS, © European Union
Project objective
Europe is a recognized hotspot for emerging infectious diseases (EIDs). A total of 72% of zoonotic EID events have a wildlife origin. Those countries that conduct disease surveillance of their wild animal populations are therefore more likely to detect potential EID events at an early stage and to swiftly implement countermeasures. Detecting viruses in wildlife samples is currently done using serology, immunohistochemistry (IHC), or PCR among others. Although these techniques have their merits, they also have significant disadvantages: they either have a low specificity or require logistics that are not feasible or at best are difficult to apply in wildlife sampling. Although the proposed novel technique of in situ hybridization (ISH) has some shortcomings that have to be taken into account, it does not have these disadvantages. It provides an opportunity to profile multiple messenger RNA transcripts at a single cell level, and enables the detection of single-copy genes. As compared to PCR, ISH produces clinically relevant information regarding cellular and tissue context. Importantly, ISH can be applied to formalin-fixed, paraffin-embedded (FFPE) tissue sections, which easily can be transported and stored without biohazard or cold-chain constraints; this is a major advantage in wildlife disease surveillance. The objectives of this research are, first, to set up specific ISH techniques for the detection of viruses in FFPE tissues, including members of the families Asfarviridae, Bunyaviridae, Coronaviridae, Flaviviridae, Rhabdoviridae, Orthomyxoviridae, and Paramyxoviridae. This list is open-ended in case new viruses emerge in Europe. Second, to validate and use the ISH in a selected group of viruses and in real disease surveillance settings in the southern and eastern European borders and in EU surrounding countries. In summary, the proposed research will be of importance for the EU to help better protect itself against the incursion of emerging pathogens.
Original text from CORDIS.
Participants
- ERASMUS UNIVERSITAIR MEDISCH CENTRUM ROTTERDAM · RotterdamCoordinatorNetherlands
Links
Data: CORDIS, © European Union