Miranda de Graaf

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.

The epidemiology of noroviruses, a major cause of gastroenteritis in humans, is shaped by their continuous evolution. Novel genotypes and variants periodically emerge, replacing previously dominant strains. In 2024, a novel GII.17 variant led to an increased number of notified outbreaks, becoming the dominant genotype detected in Europe. To investigate if this surge in GII.17 detection is attributable to changes in the binding and antigenicity of this novel variant, we compared properties of a GII.17 2024 strain with representative GII.17 strains from 2005 (Clade B), 2014 (Clade C), and 2015 (Clade D), and the epidemic GII.4 Sydney 2012 variant. Norovirus virus-like particles (VLPs) were used to assess binding to saliva samples and human intestinal tissues with different histo-blood group antigen (HBGA) profiles. Binding specificity to individual glycans was determined using synthetic HBGAs. Additionally, VP1-NanoLuc (NLuc) fusion proteins were used in binding blocking assays to evaluate the antigenic properties of the variants using 75 sera from healthy adults that were collected during different time periods of GII.17 circulation (the winters of 2009–2010, 2015 and 2024). Compared with other GII.17 variants, the 2024 strain exhibited amino acid substitutions in the P2 domain, which contains both antigenic sites and the HBGA binding site. Binding-blocking assays with human sera confirmed that the 2024 variant is antigenically distinct from the 2005 and 2015 GII.17 strains but closely related to the 2014 variant. All post-2005 GII.17 strains showed a broader HBGA binding profile compared to the GII.17 2005 strain, likely contributing to their more successful spread. These findings show that the recent re-emergence of GII.17 coincided with antigenic drift, enabling it to escape from pre-existing immunity, but not with major changes in HBGA binding specificity. Understanding the factors influencing norovirus emergence is essential for the development of preventive strategies against future norovirus epidemics.

Hepatitis A and E viruses (HAV, HEV) are the main causes of enterically transmitted hepatitis. Many infections remain undiagnosed due to their mild clinical course or asymptomatic presentation, and limited testing. Yet, knowledge of circulating genotype diversity is needed to understand their epidemiology and guide interventions. Wastewater surveillance may complement clinical monitoring by capturing infections missed through routine diagnostics. We applied capture-based metagenomic sequencing to characterize HAV and HEV genetic diversity in wastewater from 62 cities across 38 countries (2017-2019), complemented with longitudinal sampling from five European cities (2020-2021). Rocahepevirus ratti (rat HEV) genotype C1 was detected in 70% of cities, extending its known geographic range by 12 countries. Rat HEV sequences clustered by city and country, though some lineages spanned multiple continents. Paslahepevirus balayani (human HEV), predominantly genotype 3, was most prevalent in European cities. HAV genotype distribution largely reflected regional endemicity, although some subgenotypes were detected in regions where they are rarely reported clinically. Faecal source analysis suggested that rat HEV detected in wastewater originates primarily from rodent contamination rather than human infection. These findings reveal the global distribution of HAV and HEV genotypes and the widespread occurrence of rat HEV, demonstrating the value of wastewater metagenomics for population-level monitoring of both human and zoonotic hepatitis viruses.

Understanding global viral dynamics is critical for public health. Traditional surveillance focuses on individual pathogens and symptomatic cases, which may miss asymptomatic infections or newly emerging viruses, delaying detection and response. Wastewater-based epidemiology has been used to track pathogens through targeted molecular assays, but its reliance on predefined targets limits detection of the full viral spectrum. Here, we analyse longitudinal wastewater samples from 62 cities across six continents (2017–2019) using metagenomics and capture-based sequencing with probes targeting viruses associated with gastrointestinal disease. We detect over 2500 viral species spanning 122 families, many with human, animal, or plant health relevance. The bacteriophage family Microviridae and plant virus family Virgaviridae dominate the metagenomic dataset, while Astroviridae and Picornaviridae prevail in the capture-based sequence dataset. Virus distributions are broadly similar across continents at the family and genus levels, yet distinct city-level fingerprints reveal geographical and temporal variation, enabling spatiotemporal surveillance of viruses such as astroviruses and enteroviruses. Global wastewater-based epidemiology enables early detection of emerging viruses, including Echovirus 30 in Europe and Tomato brown rugose fruit virus. These findings highlight the potential of wastewater sequencing for the early detection of emerging viruses and population-wide virome monitoring across diverse hosts.

Left-ventricular assist devices (LVADs) are increasingly used as a bridge to heart transplantation and destination therapy. These devices, especially the driveline, are susceptible to difficult-to-treat infections, associated with high morbidity and mortality rates. Staphylococcus aureus ( S. aureus ) is a major causative pathogen of LVAD infections. Antibiotic resistance and biofilm formation can complicate the treatment of these infections. A novel in vitro assay was developed to study the antibiotic susceptibility of S. aureus biofilm grown on LVAD drivelines. Besides antibiotic monotherapy, the effect of various antibiotics combined with rifampicin was studied. Additionally, we explored the efficacy of four individual phages and phage-antibiotic combinations as potential treatment strategies. Our data showed a decrease of susceptibility of the S. aureus biofilms to antibiotic monotherapy compared to planktonic S. aureus . With only rifampicin and erythromycin monotherapy resulting in full bacterial clearance. Combining antibiotics with rifampicin showed similar antimicrobial efficacy against S. aureus biofilms as rifampicin monotherapy. While both individual phages and a phage cocktail were effective against planktonic bacteria, phage efficacy was limited against S. aureus in biofilm. Combining phages with antibiotics did not clearly improve treatment efficacy, compared to antibiotic monotherapy. Contrarily, it even increased bacterial growth when phage administration preceded antibiotic treatment. Here, both antibiotic- and phage monotherapy showed reduced efficacy on LVAD-driveline biofilms. Additionally, phages did not show an additive value to antibiotic treatment of LVAD driveline infections. Further studies are needed to elucidate optimal treatment strategies for LVAD-driveline infections. IMPORTANCE: Current treatment strategies for S. aureus LVAD-driveline infections are based on in vitro antibiotic susceptibility of planktonic bacteria. However, LVAD infections are most often biofilm-related, which decreases antibiotic susceptibility significantly, resulting in discrepancies between in vitro antibiotic susceptibility and in vivo treatment success. Here, we have developed a novel in vitro assay to determine antibiotic susceptibility of S. aureus biofilm, grown in conditions relevant to LVAD-driveline infections. Next to antibiotic susceptibility, the susceptibility of this biofilm to bacteriophage mono- and combination treatment with antibiotics was evaluated as an alternative treatment strategy. In the future, this assay can be used to provide a better insight in in vivo antibiotic- and bacteriophage susceptibility of LVAD-driveline biofilms. Thereby improving in vivo treatment strategies for LVAD-driveline infections.

Understanding global viral dynamics is critical for public health1. Traditional infectious disease surveillance primarily focuses on individual pathogens and relies on the identification and reporting of symptomatic cases, which may not capture asymptomatic infections or newly emerging viruses, leading to delayed detection and response.2–4 Wastewater-based epidemiology has been used to track individual pathogens through targeted molecular approaches, but its reliance on predefined targets limits the ability to capture the full spectrum of circulating viruses.5–7 Here, we analyzed longitudinal and biannual wastewater samples from 62 major cities across six continents (2017-2019) using shotgun metagenomics and capture-based sequencing targeting viruses associated with gastrointestinal disease. Over 2,500 viral species spanning 122 families were detected, many with human, animal, or plant health relevance. The Bacteriophage families Microviridae and Virgaviridae dominated the metagenomic dataset, while in the enriched dataset Astroviridae and Picornaviridae were the most prevalent. Virus distributions were broadly similar across continents, but distinct city-level fingerprints emerged, reflecting spatiotemporal variation of viruses like astrovirus, and enterovirus. Global wastewater-based epidemiology enabled the early detection of several emerging viruses, including Echovirus E30 in Europe, and Tomato brown rugose fruit virus before agricultural outbreaks. These findings highlight the potential of wastewater metagenomics for early detection of emerging viruses and population-wide virome monitoring across diverse hosts.

Metagenomic shotgun sequencing (mNGS) can serve as a generic molecular diagnostic tool. An mNGS proficiency test (PT) was performed in six European veterinary and public health laboratories to detect porcine astroviruses in fecal material and the extracted RNA. While different mNGS workflows for the generation of mNGS data were used in the different laboratories, the bioinformatic analysis was standardized using a metagenomic read classifier as well as read mapping to selected astroviral reference genomes to assess the semiquantitative representation of astrovirus species mixtures. All participants successfully identified and classified most of the viral reads to the two dominant species. The normalized read counts obtained by aligning reads to astrovirus reference genomes by Bowtie2 were in line with Kraken read classification counts. Moreover, participants performed well in terms of repeatability when the fecal sample was tested in duplicate. However, the normalized read counts per detected astrovirus species differed substantially between participants, which was related to the different laboratory methods used for data generation. Further modeling of the mNGS data indicated the importance of selecting appropriate reference data for mNGS read classification. As virus- or sample-specific biases may apply, caution is needed when extrapolating this swine feces-based PT for the detection of other RNA viruses or using different sample types. The suitability of experimental design to a given pathogen/sample matrix combination, quality assurance, interpretation, and follow-up investigation remain critical factors for the diagnostic interpretation of mNGS results. IMPORTANCE: Metagenomic shotgun sequencing (mNGS) is a generic molecular diagnostic method, involving laboratory preparation of samples, sequencing, bioinformatic analysis of millions of short sequences, and interpretation of the results. In this paper, we investigated the performance of mNGS on the detection of porcine astroviruses, a model for RNA viruses in a pig fecal material, among six European veterinary and public health laboratories. We showed that different methods for data generation affect mNGS performance among participants and that the selection of reference genomes is crucial for read classification. Follow-up investigation remains a critical factor for the diagnostic interpretation of mNGS results. The paper contributes to potential improvements of mNGS as a diagnostic tool in clinical settings.