Application of Next Generation Sequencing on Norovirus‐contaminated oyster samples

Type Article
Date 2022-06
Language English
Author(s) Ollivier Joanna1, Lowther James2, Desdouits MarionORCID1, Schaeffer Julien1, Wacrenier Candice1, Oude Munnink Bas B.3, Besnard Alban1, Mota Batista Frederico2, Stapleton Tina2, Schultz Anna Charlotte4, Aarestrup Frank4, Koopmans Marion3, de Graaf Miranda3, Le Guyader Soizick1
Affiliation(s) 1 : Ifremer, Microbiology laboratory ,France
2 : Centre for Environment, Fisheries and Aquaculture Science ,UK
3 : Erasmus University Medical Center,the Netherlands
4 : Technical University of Denmark, Denmark
Source EFSA Supporting Publications (2397-8325) (Wiley), 2022-06 , Vol. 19 , N. 6 , P. EN-7348E (144p.)
DOI 10.2903/sp.efsa.2022.EN-7348
Note The present document has been produced and adopted by the bodies identified above as author(s). This task has been carried out exclusively by the author(s) in the context of a contract between the European Food Safety Authority and the author(s), awarded following a tender procedure. The present document is published complying with the transparency principle to which the Authority is subject. It may not be considered as an output adopted by the Authority. The European Food Safety Authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors.

Bivalve molluscan shellfish (BMS) contamination with gastroenteritis viruses such as norovirus (NoV) is recognized as a significant public health risk worldwide. These foodborne epidemics are characterized by very low viral concentrations in the implicated foods, and by diverse viruses or multiple NoV strains originating from human sewage, resulting in different strains (co-)infecting the consumers. Next-generation sequencing (NGS) offers promising means to describe the diversity of strains present in BMS or to retrace transmission chains in outbreak settings, but their sensitivity and reproducibility remained to be assessed for this application. In this work, we evaluated the ability of three promising NGS methods to sequence diverse NoV in environmental or food BMS and human stool samples. Using laboratory-prepared samples of known NoV composition, we evaluated the sensitivity, reproducibility, repeatability and selectivity of metabarcoding, capture-based metagenomics and long amplicon sequencing, considering representative NoV strains from genogroup I and II, and the impact of the BMS matrix. The metabarcoding, with separate amplification of polymerase and capsid gene segments followed by Illumina sequencing, was the most sensitive method. It was applied to a selection of 212 BMS samples collected through the European Commission’s NoV baseline survey (BLS), demonstrating a high diversity of NoV sequences found in the BMS which reflect the diversity of NoV strains circulating in the European human population. Besides, a capture-based metagenomics with enrichment of vertebrate viruses was applied on 20 of these BLS samples as well as 20 BMS linked to outbreaks and 10 related human stool samples. In BMS, it yielded NoV sequences compatible with the genomes identified in stool samples, but they were too short to allow definitive confirmation of the infection source. The present report describes NGS methods, including the bioinformatic pipelines, applicable to molecular epidemiology of NoV in BMS, their current limitations and expected outcomes

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Annex A 255 KB Open access
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Annex C 79 KB Open access
Annex D 2 116 KB Open access
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Ollivier Joanna, Lowther James, Desdouits Marion, Schaeffer Julien, Wacrenier Candice, Oude Munnink Bas B., Besnard Alban, Mota Batista Frederico, Stapleton Tina, Schultz Anna Charlotte, Aarestrup Frank, Koopmans Marion, de Graaf Miranda, Le Guyader Soizick (2022). Application of Next Generation Sequencing on Norovirus‐contaminated oyster samples. EFSA Supporting Publications, 19(6), EN-7348E (144p.). Publisher's official version : , Open Access version :