Next-generation diagnostics: virus capture facilitates a sensitive viral diagnosis for epizootic and zoonotic pathogens including SARS-CoV-2 : [Preprint]

Wylezich, Claudia GND; Sten Calvelage; Schlottau, Kore GND; Ziegler, Ute GND; Pohlmann, Anne GND; Höper, Dirk GND; Beer, Martin GND

Background: The detection of pathogens in clinical and environmental samples using high-throughput sequencing (HTS) is often hampered by large amounts of background information, which is especially true for viruses with small genomes. Enormous sequencing depth can be necessary to compile sufficient information for identification of a certain pathogen. Generic HTS combining with in-solution capture enrichment can markedly increase the sensitivity for virus detection in complex diagnostic samples. Methods: A virus panel based on the principle of biotinylated RNA-baits was developed for specific capture enrichment of epizootic and zoonotic viruses (VirBaits). The VirBaits set was supplemented by a SARS-CoV-2 predesigned bait set for testing recent SARS-CoV-2 positive samples. Libraries generated from complex samples were sequenced via generic HTS and afterwards enriched with the VirBaits set. For validation, an internal proficiency test for emerging epizootic and zoonotic viruses (African swine fever virus, Ebolavirus, Marburgvirus, Nipah henipavirus, Rift Valley fever virus) was conducted. Results: The VirBaits set consists of 177,471 RNA-baits (80-mer) based on about 18,800 complete viral genomes targeting 35 epizootic and zoonotic viruses. In all tested samples, viruses with both DNA and RNA genomes were clearly enriched ranging from about 10-fold to 10,000-fold for viruses including distantly related viruses with at least 72% overall identity to viruses represented in the bait set. Viruses showing a lower overall identity (38% and 46%) to them were not enriched but could nonetheless be detected based on capturing conserved genome regions. The internal proficiency test supports the improved virus detection using the combination of HTS plus targeted enrichment but also point to the risk of carryover between samples. Conclusions: The VirBaits approach showed a high diagnostic performance, also for distantly related viruses. The bait set is modular and expandable according to the favored diagnostics, health sector or research question. The risk of carryover needs to be taken into consideration. The application of the RNA-baits principle turned out to be user-friendly, and even non-experts (without sophisticated bioinformatics skills) can easily use the VirBait workflow. The rapid extension of the established VirBaits set adapted to actual outbreak events is possible without any problems as shown for SARS-CoV-2.

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Wylezich, Claudia / Calvelage, Sten / Schlottau, Kore / et al: Next-generation diagnostics: virus capture facilitates a sensitive viral diagnosis for epizootic and zoonotic pathogens including SARS-CoV-2. [Preprint]. 2020.

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