Characterization of norovirus GI.3 and GII.12 variants detected in wastewater: proof-of-concept for integrating phenotypic insights in wastewater-based epidemiology
Putri Ayu Fajar, Ray W. Izquierdo-Lara, Nathalie Worp, Claudia M. E. Schapendonk, Sol Ribó Pons, Lonneke Leijten, Gijsbert P. van Nierop, Frank Møller Aarestrup, Marion P. G. Koopmans, Miranda de Graaf
Source abstract
ABSTRACT Norovirus is the leading cause of acute gastroenteritis, responsible for 685 million cases and 200,000 deaths annually. Wastewater-based epidemiology (WBE) enables not only global surveillance of dominant strains but also the detection of cryptic and emerging viral variants. An important question, however, is how to link genomic diversity to clinical impact and phenotypic traits of the strains detected in wastewater. In the Global Urban Wastewater Project, phylogenetically distinct norovirus GI.3 and GII.12 lineages were detected in wastewater from Cameroon. In this study, we characterized their host- and tissue-binding specificity and antigenic makeup in comparison with other GI.3 and GII.12 lineages using a luciferase immunoprecipitation system-based assay, alongside sequence analysis of the VP1 P2 subdomain. We observed intra-genotype variation among GI.3 variants. GI.3 Cameroon/2017 shared phenotypes with its closest reference strain (GI.3 Mexico/2016), and their histo-blood group antigen (HBGA)-binding profiles suggested a narrower susceptible population compared to other GI.3 lineages. Conversely, GII.12 strains showed no significant differences in HBGA binding, antigenic profiles, and tissue attachment. Together, these results reveal distinct patterns of phenotypic diversity between the two genotypes. While GI.3 variants exhibited lineage-specific traits, the GII.12 strains appeared phenotypically conserved across lineages. These findings highlight the feasibility and importance of integrating phenotypic analysis with WBE to assess potential public health risks posed by novel viral variants. IMPORTANCE While detection of norovirus through case-based surveillance is limited to variants causing symptomatic infection, wastewater-based epidemiology (WBE) allows for detection of cryptic and emerging variants. However, validation, such as phenotypic characterization, is needed to assess the clinical importance of a variant detected in WBE. In this study, we investigated the phenotypic traits of genetically distinct GI.3 and GII.12 lineages detected in wastewater from Cameroon. We utilized NanoLuc luciferase immunoprecipitation system (N-LIPS)-based assays to assess binding specificity and antigenicity. Compared to traditional methods involving virus isolation and virus-like particle production, N-LIPS-based assays are considerably less laborious and faster. This allows for rapid characterization of novel variants, making the timely assessment of phenotypic changes of such variants for outbreak or pandemic preparedness feasible. Finally, by integrating insights from phenotypic assays, we showed the possibility of expanding the public health value of wastewater surveillance in assessing the risk of novel viral variants.
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