Amplicon-Based High-Throughput Sequencing Method for Genotypic Characterization of Norovirus in Oysters

Abstract

Norovirus is a highly diverse RNA virus often implicated in foodborne outbreaks, particularly those associated with shellfish. Shellfish are filter feeders, and when harvested in bays exposed to wastewater overflow or storm overflows, they can harbor 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 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 polymerases and reverse transcriptases (RTs) 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 characterize norovirus populations in naturally contaminated oysters. IMPORTANCE While foodborne outbreaks account for approximately 14% of norovirus cases (L. Verhoef, J. Hewitt, L. Barclay, S. Ahmed, R. Lake, A. J. Hall, B. Lopman, A. Kroneman, H. Vennema, J. Vinjé, and M. Koopmans, Emerg Infect Dis 21:592-599, 2015), we do not have standardized high-throughput sequencing methods for genotypic characterization in foodstuffs. Here, we present an optimized amplicon high-throughput sequencing method for the genotypic characterization of norovirus in oysters. This method can accurately detect and characterize norovirus at concentrations found in oysters grown in production areas impacted by human wastewater discharges. It will permit the investigation of norovirus genetic diversity in complex matrices and contribute to ongoing surveillance of norovirus in the environment.

Keywords: Caliciviridae; DNA polymerase; DNA sequencing; Illumina; calicivirus; environmental microbiology; environmental virology; food safety; reverse transcriptase; virology.

FIG 1

(A) Violin plots with internal box plots of the mean Phred score (Q) obtained in spiked shellfish samples and naturally contaminated oysters. RT and DNA polymerase combinations are ordered from highest mean Phred score to lowest (left to right). (B) Violin plots with internal box plots of the mean expected errors (EE). RT and DNA polymerase combinations are ordered from the highest mean expected error score to the lowest (left to right).

FIG 2

(A) FAMD biplot demonstrating the variance-maximizing distribution patterns of the total mean Phred scores in the map space and their clustering patterns based on DNA polymerase and RT. (B) FAMD biplot for DNA polymerase demonstrates the variance-maximizing distribution patterns of the total mean expected errors in the map space and their clustering patterns based on DNA polymerase and RT.

FIG 3

Regions of norovirus genome used for genotypic characterization and detection by RT-qPCR.