Optimization of next generation sequencing methods for monitoring norovirus in sewage and oysters

Shellfish grown in coastal areas can be contaminated with human viruses through sewage discharge. Norovirus (NoV) is the main agent responsible for non-bacterial gastroenteritis and the primary pathogen of shellfish-associated... [ view full abstract ]

Shellfish grown in coastal areas can be contaminated with human viruses through sewage discharge. Norovirus (NoV) is the main agent responsible for non-bacterial gastroenteritis and the primary pathogen of shellfish-associated outbreaks globally. The distribution and evolutionary patterns of NoV in oysters are not well defined. With the advance of next generation sequencing (NGS), NoV diversity can be described in detail in environmental samples and can provide important phylogenetic information for tracking the sources of NoV contamination.

One of the challenges for deep sequencing environmental samples is to detect human enteric viruses present at very low abundance compared to the overall microbiome. To tackle this challenge, we have optimized concentration steps in the nucleic acid extraction protocol, through the use of PEG (polyethylene glycol) precipitation. Subsequently, a NoV capture was used, followed by Illumina deep sequencing.

The suitability of this new method was tested with a set of sewage samples, bioaccumulated and naturally contaminated oysters. Results indicated that including the PEG precipitation step enriched the total viral nucleic acid extracted. In sewage samples, 0.8 - 5% of reads were NoV-specific. For bioaccumulated oysters, most sequence reads were NoV and, lower abundance (0.2 - 6.4% of total read) were found in naturally contaminated oysters. Complete NoV genomes could be obtained from nearly all samples and interestingly multiple strains of NoV were detected. The method is now being refined, and will provide an important tool for monitoring oyster Norovirus contamination and help in developing interventions to improve shellfish quality.