Metagenomic to detect norovirus and other Human enteric viruses in oysters: impact on hexamer selection and targeted capture-based enrichment.

Human virus transmission through food consumption has been identified since many years and preventative measures have been undertaken in many countries. Nonetheless, these remain imperfect, and the international trade increases the risk of dissemination of viral pathogens {Li, 2018 #3560}. The main sources of contamination are infected food handler, zoonotic transmission (essentially for hepatitis E virus) or during the production process by contact with sewage-contaminated waters {Bosch, 2018 #3564}. A direct test, targeting the virus excreted by the food handler or by the animal, may potentially identify the contaminant in the foodstuff. However, when dealing with food contaminated during production virus identification may be more challenging. A variety of human enteric viruses may be excreted in the environment mainly through sewage discharge {Metcalf, 1995 #983}. With the increase of human population and the demands of waters for agriculture or human activities, wastewater re-use will be necessary, increasing the risk for virus pollution of the environment, and subsequent viral transmission through contaminated food {Sano, 2016 #3341}. Thus being able to identify the viral contamination of a food sample will be of primary importance for risk manager and consumer safety.Bivalve molluscan shellfish such as oyster grown in coastal environments, have been identified for many years as a high-risk food. If actual regulations based on bacterial indicators, succeed on lowering outbreaks due to bacterial contamination, shellfish are still one of the food the most frequently identified in viral outbreaks {Yu, 2015 #3297}. Although some reporting bias in outbreaks identification, contribute to increase their image of risky food, different factors such as their mainly raw mode of consumption, and their capacity to accumulate viruses explain the documented implication of shellfish in viral outbreaks on several occasions {McLeod, 2017 #3455}{Venugopal, 2018 #3511}{Bellou, 2013 #3393}{Yu, 2015 #3297}. Most of these outbreaks are due to a norovirus contamination. Belonging to the Caliciviridae family, noroviruses rank as the leading cause of acute gastroenteritis in human, and thus are frequently detected in human sewage {Atmar, 2018 #3590}{Sano, 2016 #3341}. Yet, other human enteric viruses such as hepatitis A virus or rotavirus have also been implicated in shellfish borne outbreaks {Boxman, 2016 #3321}{Kittigul, 2014 #3101}. Thus, following a flooding event close to a shellfish production lagoon, we detected up to seven different virus strains in oyster and clinical samples, including an Aichi strain identified for the first time in Europe in an oyster sample {Le Guyader, 2008 #2317}. Metagenomic sequencing applied to a variety of samples demonstrated its interest in diverse fields {Kallies, 2019 #3691}{Giu, 2019 #3696}{Alberti, 2017 #3544}. Metagenomic refers to the description of all nucleic acid sequences present in a sample allowing the description of bacteria, archea, eukaryotes or viruses. However, despite the abundance of viruses on the earth, the virome is still poorly described {Rosani, 2019 #3695}. Viral metagenomics is generally performed by removing as much host and bacterial nucleic acid as possible followed by nuclease treatment to remove free nucleic acids. This step is of primary importance to identify contamination with human enteric viruses, which have small RNA genomes for most of them. This mean that a cDNA conversion, and enrichment or amplification steps are also needed.