Detection and Sequencing of Multiple Human Norovirus Genotypes from Imported Frozen Raspberries Linked to Outbreaks in the Province of Quebec, Canada, in 2017

Abstract

Human noroviruses are among the main causes of acute gastroenteritis worldwide. Frozen raspberries have been linked to several norovirus food-related outbreaks. However, the extraction of norovirus RNA from frozen raspberries remains challenging. Recovery yields are low and PCR inhibitors limit the sensitivity of the detection methodologies. In 2017, 724 people from various regions of the Province of Quebec, Canada, were infected by noroviruses and the outbreak investigation pointed to frozen raspberries as a putative source. A new magnetic silica bead approach was used for the extraction of viruses from different outbreak samples. The RNA extracts were tested by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and five samples were confirmed positive for norovirus by RT-qPCR amplicon sequencing. A multiplex long-range two-step RT-PCR approach was developed to amplify norovirus ORF2 and ORF3 capsid genes from the positive frozen raspberry RNA extracts and other sequencing strategies were also explored. These capsid genes were sequenced by Next-Generation Sequencing. Phylogenetic analyses confirmed the presence of multiple genotypes (GI.3, GI.6, and GII.17) and intra-genotype variants in some of the frozen raspberry samples. Variants of genotype GI.3 and GI.6 had 100% homology with sequences from patient samples. Similar strains were also reported in previous outbreaks. Confirmation approaches based on sequencing the norovirus capsid genes using Next-Generation Sequencing can be applied at trace level contaminations and could be useful to assess risk and assist in source tracking.

Benchmarking Bioinformatic Tools for Amplicon-Based Sequencing of Norovirus

“…However, there remains room for improvement with respect to the tools and standardized methodologies that are available to manage viral foodborne outbreaks. While much work has been conducted to date to understand the limits of shotgun metagenomics and amplicon-based sequencing for genotypic characterization of norovirus in foodstuffs ( 32 , 33 ), there has been only one bioinformatic study to date ( 18 ). Importantly, this single study did not focus on amplicon-based approaches or foodstuffs.…”

Microbiological hazards associated with the use of water in the post‐harvest handling and processing operations of fresh and frozen fruits, vegetables and herbs (ffFVHs). Part 1 (outbreak data analysis, literature review and stakeholder questionnaire)

“…(2015)Raspberries5724NRNRRaymond et al. (2022)Raspberries13900NRNRSarvikivi et al. (2012)Raspberries4151NRSaupe et al.…”

Development of an amplicon-based high throughput sequencing method for genotypic characterisation of norovirus in oysters

“…A few studies have applied HTS-based methods for the detection and characterisation of norovirus in complex matrices such as food or wastewater (4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16). Notwithstanding these studies, the performance of the various methods, whether it be shotgun metagenomics, capture probe hybridisation, long-read sequencing or amplicon-based HTS, has varied.…”

“…Despite these challenges, several recent studies have demonstrated the capability of various amplicon HTS assays for the genotypic characterisation of norovirus in shellfish (5,17), with similar success observed in berry samples (6).…”

“…ONT combined with adaptive sampling has had limited success in food samples (7), while, to date, long-read ONT sequencing of norovirus amplicons has not been successful (17). Despite these challenges, several recent studies have demonstrated the capability of various amplicon HTS assays for the genotypic characterisation of norovirus in shellfish (5, 17), with similar success observed in berry samples (6). However, these studies focused on application rather than optimisation and did not confirm HTS results with the gold standard Sanger methods.…”