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.
Highlights
Human norovirus (HuNoV) is one of the leading causes of acute gastroenteritis
(MLR2 + qPCR) or next generation sequencing (NGS) (MLR2 + NGS), using RNA extracted from frozen raspberries spiked with a HuNoV GI.5
These 16 positive HuNoV GI.5 subsamples detected by the MLR2 + qPCR analysis were subsequently tested by NGS and 13 were sequenced
Summary
Human norovirus (HuNoV) is one of the leading causes of acute gastroenteritis. HuNoV is transmitted mainly via the fecal–oral route. HuNoV outbreaks (Boqvist et al, 2018; Bozkurt et al, 2020). HuNoV contaminated berries were involved in 46 foodborne outbreaks with over 15,000 cases reported globally between 1983 and 2018 (Bozkurt et al, 2020). Frozen raspberries were involved as a food vehicle for more than 80% of documented HuNoV outbreaks (Boqvist et al, 2018; Bozkurt et al, 2020). Several HuNoV outbreaks associated to frozen raspberries were reported in Canada and in the US (CDC, 2018; Fiset et al, 2018)
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