Introduction and objectives

Norovirus contamination of oysters is a public health and economic issue for the oyster industry. This human pathogen originates from human wastewater. Detecting it upstream of oyster-farming sites would make it possible to prevent oyster contamination. The objectives of this study were to use passive sampling on the scale of a watershed to identify input channels, and to analyze the genetic diversity of the contamination using targeted metagenomics.

Materials and methods

Passive samplers using nylon membran were immersed for 48 h at twelve selected sites in the watershed, including two in oyster farming areas, where oysters were also positioned for 18 months. Nucleic acids were extracted from both matrices (membran and oyster) and norovirus were detected by RT-qPCR. Two regions of the genome of genogroups I and II were amplified: the capsid (VP1) and the polymerase (RdRp) following an adapted protocol. Amplicons were sequenced using Illumina MiSeq. An automated, standardized targeted metagenomics analysis workflow was adapted and optimized to reconstruct and identify norovirus sequences.

 Results, discussion and conclusion

Noroviruses were identified in 15% of membranes (50/334) and 41% of oysters (23/56). Sequencing revealed a high degree of genetic diversity. Six genotypes were identified for norovirus of the GI-VP1 genogroup (GI.1, GI.2, GI.3, GI.4, GI.5 and GI.6), nine for noroviruses GII-VP1 (GII.1, GII.2, GII.3, GII.4, GII.6, GII.7, GII.12, GII.13 and GII.17), and three for GI-P-type noroviruses (GI.P1, GI.P2, GI.P4). Genotyping carried out on oysters and membranes showed complementarity between the two matrices, greater genetic diversity in the winter period, and inter-site variability.

For the first time, genotyping using passive samplers was possible. This innovative study demonstrates the benefits of passive sampling for diagnosing the geographical distribution of norovirus contamination in a watershed. However, it needs to be crossed with other data for a more detailed characterization of contamination pathways (knowledge of wastewater networks, river flow, marine currents).