Development of an amplicon-based high throughput sequencing method for genotypic characterisation of norovirus in oysters

Norovirus is a highly diverse RNA virus often implicated in food-borne outbreaks, particularly shellfish. Shellfish are filter feeders, and when harvested in bays exposed to wastewater overflow or storm overflows, they can harbour various pathogens, including human pathogenic viruses. The application of Sanger or amplicon-based High Throughput Sequencing (HTS) technologies to identify human pathogens in shellfish faces two main challenges i) distinguishing multiple genotypes/variants in a single sample and ii) low concentrations of norovirus RNA. Here we have assessed the performance of a novel norovirus capsid amplicon HTS method. We generated a panel of spiked oysters containing various norovirus concentrations with different genotypic compositions. Several DNA polymerase and Reverse Transcriptase (RT) enzymes were compared, and performance was evaluated based on i) the number of reads passing quality filters per sample, ii) the number of correct genotypes identified, and iii) the sequence identity of outputs compared to Sanger-derived sequences. A combination of the reverse transcriptase LunaScript and the DNA polymerase AmpliTaq Gold provided the best results. The method was then employed, and compared with Sanger sequencing, to characterise norovirus populations in naturally contaminated oysters. Importance While foodborne outbreaks account for approximately 14% of norovirus cases (Verhoef L, Hewitt J, Barclay L, Ahmed S, Lake R, Hall AJ, Lopman B, Kroneman A, Vennema H, Vinjé J, Koopmans M. 2015. 1999-2012. Emerg Infect Dis 21:592–599), we do not have standardised high-throughput sequencing methods for genotypic characterisation in foodstuffs. Here we present an optimised amplicon high- throughput sequencing method for the genotypic characterisation of norovirus in oysters. This method can accurately detect and characterise norovirus at concentrations typically detected in oysters. It will permit the investigation of norovirus genetic diversity in complex matrices and contribute to ongoing surveillance of norovirus in the environment.