Wastewater-based epidemiology at a global scale for norovirus surveillance: From metagenomics to phenotypical characterization

Ray William Izquierdo Lara, Erasmus MC

Ray W. Izquierdo-Lara1, Putri Ayu Fajar1,2, Nathalie Worp1, Mark Olthof1, Sol Ribó Pons1, Bas B. Oude Munnink1, Claudia M.E. Schapendonk1, Divyae K. Prasad1, Frank M. Aarestrup3, Global Sewage Surveillance Consortium, NoroNet, Marion P.G. Koopmans1, Miranda de Graaf1* 1 Department of Viroscience, Erasmus University Medical Center, Rotterdam, The Netherlands. 2 Pandemic and Disaster Preparedness Center, Zuid-Holland, The Netherlands. 3 Research Group for Genomic Epidemiology, Technical University of Denmark, Kgs, Lyngby, Denmark. * Corresponding author email: m.degraaf@erasmusmc.nl

Food & Environmental Virology-II (Wastewater & others)

Norovirus is the leading cause of gastroenteritis worldwide. Tracking norovirus transmission, evolution and phenotypic changes is essential to develop sustainable strategies to combat this threat. Here, we analyzed the worldwide diversity of noroviruses by using a capture-based metagenomics method on 327 wastewater (WW) samples collected between 2017 and 2019, from 62 cities across 6 continents. This approach resulted in a median of 5094-fold enrichment of Caliciviridae reads compared with an unbiased sequencing approach on the same samples. We identified a total of 34 capsid and 44 polymerase genotypes, predominantly from genogroups GI and GII. We detected changes in genotype distribution over time, along with seasonal variations. Some genotypes/P-types were widespread, while others have a more restricted circulation. Phylogenetic analyses revealed that in several instances, WW-derived sequences were closely related to those reported in outbreaks of the same region. Nonetheless, novel sequence clusters were detected in African samples for genotypes GI.3 and GII.12, which harbored 11 and 2 amino-acid substitutions in the P2-domain compared to their closest related strains, respectively. To assess the phenotypic impact of these mutations, we expressed these and other representative capsids and tested their ability to bind salivary histo-blood group antigens (HBGAs) and their antigenicity against human sera. Although phenotypical differences were detected at the intra-genotype level, the novel clusters showed minimal differences in HBGA binding or antigenic profiles to their closest strains. WW metagenomics can uncover norovirus diversity within the population. However, phenotypic characterization is key to understand the impact of such diversity on virus epidemiology.