Norovirus genomic surveillance: benchmarking, baseline survey, and outbreak investigation. Norovirus genomic surveillance: benchmarking, baseline survey, and outbreak investigation

Norovirus (NoV) is the leading cause of gastroenteritis and outbreaks may occur through contaminated food sources. As oysters have a long history as vectors of human norovirus, this European Food Safety Authority (EFSA) project aims to accomplish NoV genomic surveillance and to assess the variability of different sequencing approaches. The geographic location of sample acquisition, apart from being carried out at the European level, remains confidential. The EFSA norovirus consortium 2021-22 comprises of researchers from: 1) Erasmus Medical Center (EMC), the Netherlands 2) French Research Institute for Exploitation of the Sea (IFREMER), France 3) Centre for Environment, Fisheries and Aquaculture Science (CEFAS), United Kingdom 4) Technical University of Denmark (DTU), Denmark Two sequencing approaches, a metagenomics-based and a metabarcoding-based (amplicon targeted) were employed for NoV genomic surveillance. Samples of NoV found in oysters, its environmental niche, and stool samples, come from three different sequencing sets - Baseline Survey (BLS) samples, Outbreak-related (OB) samples, and two synthetic Test Set samples (TSS1 and TSS2). Synthetic sample sequencing allows for benchmarking the accuracy, reliability, and reproducibility of the methods. BMS is the abbreviation for bivalve molluscan shellfish, which includes oysters and mussels that were sequenced in this project. The files name and sample alias reflect the collecting institute, the sequencing set, and the genotypes (for metabarcoding samples). For metagenomics sequencing, viral nucleotides were captured by the VirCapSeq-VERT probe set. This probe set enriches for all vertebrate viruses. The metagenomics sequencing is, in theory agnostic, and offers the advantage of unbiased detection of NoV, including the detection of novel NoV recombinants. For metabarcoding, well-studied regions of the viral genome that are relevant for NoV genotyping were selected. Portions of the genes encoding the RNA-dependent RNA polymerase (RdRp), the capsid protein (VP1), and a longer target (400-500 nt) comprising an overlap spanning RdRp and VP1 gene, were selected. Comparing the assembled sequences to established databases (NCBI and the Norovirus Typing Tool 2.0) allows for the characterization of the diversity of the NoV genotypes. Such genotyping efforts help monitor seasonal outbreaks, track the evolution of NoV in Europe, and keep a close check on the contamination levels of oysters.