TELE-Vir: Point-of-incidence toolbox for emerging virus threats

It is crucial to address new emerging virus threats and to better control virus outbreaks. The SARS-CoV-2 pandemic and the reoccurrence of emerging high virus pathogens, as for example Ebola- and West Nile virus, highlighted the importance of rapid diagnostics and improved methods to detect viruses, as early detection can assist interventive measures in order to avoid intensive outbreaks. Therefore, novel advanced virus detection methods are warranted to be prepared for future pandemics and next dangerous pathogen identification.

In the TELEVIR project, we developed a point of incidence (POI) toolbox, for metagenomic virus detection in the field that will contribute to the identification of known viruses as well as of emerging virus threats. Metagenomic next-generation sequencing (mNGS) has the ability to identify pathogens in as hypothesis-free manner compared to targeted strategies (Fomsgaard et al., J. Clin. Virol. Plus, 2(4), 100120 (2022)). This is of advantage to conventional diagnostic tests, since different pathogens can produce similar symptoms, which makes it difficult to choose for a specific (targeted) test. Moreover, out POI toolbox includes methods that can be transferred into a field setting to detect any kind of virus, in animals and humans, even in geographical areas without available laboratory infrastructure. In work package (WP) 3 we established a POI protocol that includes sample pre-treatment, nucleic acids extraction, isothermal random amplification followed by non-targeted sequencing using the Oxford Nanopore Technology (ONT) system. It requires a minimum of laboratory equipment to be field employable and is relatively fast. WP2 supported this approach by:

1. improving and adapting the “surveillance oriented” INSaFLU-TELEVIR platform to handle sequence data (ONT, Illumina and Ion Torrent) of any virus (from reads to quality control, mutations detection, consensus generation, classification, alignments, genotype-phenotype” screening, phylogenetics, integrative phylogeographical and temporal analysis) and
2. by developing, benchmarking and implementing a novel pathogen detection module, from reads and quality control for the identification of both RNA and DNA viruses.

The POI toolbox was successfully tested in proof-of-concept studies by the TELEVIR members (WP4), for example under different conditions in the field in Norway, South France and West Africa or by testing diverse sample material frozen and fresh material from in vivo laboratory studies to compare our POI protocol to well established detection methods. Sequencing data obtained from the partners performing the wet-lab activities were analysed with the INSaFLU-TELEVIR platform, as a complement to the multiple sample/virus tests performed during the tool development, benchmarking and final refinement.  We could detect a broad range of RNA as well as DNA viruses in different sample materials, proving a good symbiosis between the developed POI protocol and the INSaFLU- TELEVIR platform, towards the deployment of a “start-to-end” framework that can potentiate strengthened timely metagenomics virus detection and routine genomics surveillance.

In summary, the TELEVIR POI toolbox allows for an easy, rapid and user-friendly in situ identification with immediate characterisation and hence a very fast response and improved decision-making during an outbreak or a potential outbreak of an emerging or re-emerging virus threat, which will aid in the overall outbreak preparedness in Europe.

Project Outputs and Outcomes

• To be able to react fast to new virus outbreaks the point of incidence (POI) protocol was established for unbiased detection of RNA and DNA viruses, that comprises sample pre-treatment, nucleic acids extraction, isothermal random amplification followed by library preparation for non-targeted sequencing using the Oxford Nanopore Technology (ONT) system. The POI protocol requires a minimum of laboratory equipment to be field employable.
• We established an open and user-oriented “start-to-end” bioinformatics framework for metagenomics virus detection and routine genomic surveillance, the INSaFLU-TELEVIR platform. This web-based (also locally installable) platform handles read sequence data (ONT, Illumina and Ion Torrent) of “any” virus, enabling the identification of both RNA and DNA viruses, while providing multiple surveillance-oriented features towards mutations detection, consensus generation, lineage/genotype classification, alignments, “genotype-phenotype” screening, phylogenetics, and integrative phylogeographical and temporal analysis.
• The POI toolbox consist of the INSaFLU-TELEVIR platform and the POI protocol and was successfully tested in proof of concept/field studies.
• The recent virus pandemic made it clear that there is a strong need to identify new emerging virus threats. International stakeholders should recognize the potential of the POI toolbox to rapidly detect any virus in diseased humans and animals in the field. This tool can be especially relevant for regions with low laboratory infrastructure or with a high biodiversity.