Metavirome Sequencing to Evaluate Norovirus Diversity in Sewage and Related Bioaccumulated Oysters

Abstract

Metagenomic sequencing is a promising method to determine the virus diversity in environmental samples such as sewage or shellfish. However, to identify the short RNA genomes of human enteric viruses among the large diversity of nucleic acids present in such complex matrices, method optimization is still needed. This work presents methodological developments focused on norovirus, a small ssRNA non-enveloped virus known as the major cause of human gastroenteritis worldwide and frequently present in human excreta and sewage. Different elution protocols were applied and Illumina MiSeq technology were used to study norovirus diversity. A double approach, agnostic deep sequencing and a capture-based approach (VirCapSeq-VERT) was used to identify norovirus in environmental samples. Family-specific viral contigs were classified and sorted by SLIM and final norovirus contigs were genotyped using the online Norovirus genotyping tool v2.0. From sewage samples, 14 norovirus genogroup I sequences were identified of which six were complete genomes. For norovirus genogroup II, nine sequences were identified and three of them comprised more than half of the genome. In oyster samples bioaccumulated with these sewage samples, only the use of an enrichment step during library preparation allowed successful identification of nine different sequences of norovirus genogroup I and four for genogroup II (>500 bp). This study demonstrates the importance of method development to increase virus recovery, and the interest of a capture-based approach to be able to identify viruses present at low concentrations.

Novel opportunities for NGS-based one health surveillance of foodborne viruses

“…Among the different steps in metagenomic sequence analysis, one of the major challenges is the assembly of reads into full genomes or long contigs. Most metagenomics studies conducted on food performed de novo assembly, which allows agnostic treatment of the data without prior knowledge of the viral species to be found, and favors the discovery of new sequences [34,36,37,[40][41][42]. However, the bioinformatics strategy used to analyze sequencing data may greatly affect the results [40].…”

“…With these techniques, the list of novel noroviruses has expanded especially for animals [32,33]. We and others have followed this agnostic metagenomics strategy, sequencing the whole virome of foods linked to foodborne outbreaks [34,35], samples from the production chain [36][37][38], or artificially contaminated samples to allow method validation [39][40][41]. One study also assessed a common, simple workflow for RNA or DNA metagenomics of various matrices (cultured-cell supernatants, stool, tissue and food) for the detection of any kind of pathogen (virus, bacteria and parasites) [42].…”

“…One study also assessed a common, simple workflow for RNA or DNA metagenomics of various matrices (cultured-cell supernatants, stool, tissue and food) for the detection of any kind of pathogen (virus, bacteria and parasites) [42]. In complex matrices, the abundance of reads assigned to human viruses was very low [34,36,37,[39][40][41][42], or even null for certain viruses [42], reflecting the very low levels of viral contamination in food. Yet, this strategy applied on berries linked to HAV and norovirus outbreaks allowed the sequencing of a nearly-complete HAV IB genome [35] and portions of the norovirus genome closely related to the virus identified in patients [34], respectively.…”

“…Another strategy to enhance the recovery of human viruses in complex matrices is the capture-based metagenomics (ViroCap), where nucleic acid libraries are enriched in viral sequences using probes targeting all known vertebrate viruses [54]. We successfully applied this technique for the sequencing of norovirus and other human enteric viruses in environmental and shellfish samples [41]. For all of the above, a critical element in the primer/ probe design is the choice of a reference virus set that is sufficiently diverse to be able to "catch" all strains from this genetically highly diverse genus.…”

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

“…Target enrichment with complex samples was already applied for viral [5][6][7], bacterial [8], parasitical [9] and fungal [10] pathogens to compile full or nearly full genomes for follow-up analyses. For diagnostic reasons, however, the combination of probes or RNA baits of several pathogens into one set, depending on the scope, for example virus, microbiological or syndromic diagnostics, is beneficial.…”