Unveiling the global urban virome through wastewater metagenomics

Abstract

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.

Fig. 1. Overview of the 62 wastewater collection sites.

Cities are coloured by continent and categorized by sampling strategy: colour-filled dots represent cities with biannual sampling, while outlined dots represent cities with longitudinal sampling.

Fig. 2. Heatmap of the viral family diversity in the global wastewater samples.

The heatmap is categorized by continent and host association and ordered by RPM value of the GastroCap sequence data. The colour gradient represents the log-transformed relative abundance of reads normalized to genome length. Viral families marked with an orange dot are RNA viruses; all others are DNA viruses. OCE Oceania, SA South America, Metagenomic shotgun metagenomic dataset, and capture GastroCap sequence dataset.

Fig. 3. Pathogenic enteric viruses were widespread and showed distinct spatiotemporal patterns in urban wastewater.

Reads per million viral reads (normalized by genome length) per genus are given for the biannual (a) and longitudinal (b) GastroCap sample set. The heatmap is ordered by reads per million viral reads (normalized by genome length) and categorized by continent (a) and city (b). The colour gradient represents log-transformed relative abundance of reads. SA South America.

Fig. 4. PCA clustering of the urban wastewater virome compositions from 62 cities based on the shotgun metagenomic sequence dataset reveals city-level differences without clear continental clustering.

The virome compostions of the biannual samples (a, b) coloured by continent and longitudinal samples (c, d) coloured by city at the family (a, c) and genus (b, d) levels. Principal components were derived from genome size adjusted read counts subjected to a CLR transformation. Arrows represent viral families or genera, with their direction showing their contribution to the principal components and their length indicating the strength of their contribution to the variance in virome composition.

Fig. 5. PCA clustering of the urban wastewater virome compositions from 62 cities based on the GastroCap sequence dataset highlight city-level clustering, primarily driven by Mamastrovirus.

The virome compostions of the biannual samples (a, b) coloured by continent and longitudinal (c, d) samples coloured by city at the family (a, c) and genus (b, d) levels. Principal components were derived from genome size adjusted read counts subjected to a CLR transformation. Arrows represent viral families or genera, with their direction showing their contribution to the principal components and their length indicating the strength of their contribution to the variance in virome composition.

Fig. 6. Global prevalence and abundance of species within the Tobamovirus genus in urban wastewater.

Biannual (a) and longitudinal data (b) are shown. In each panel, stacked bar plots display the number of reads per million viral reads assigned to the Tobamovirus genus (green) and to specific species (red), followed by the relative abundances of Tobamovirus species. The biannual samples were grouped using hierarchical clustering based on species-level composition. YoMV Youcai mosaic virus, TVCV Turnip VeinClearing virus, TSAMV Tropical soda apple mosaic virus, ToMV Tomato mosaic virus, ToMMV Tomato mottle mosaic virus, TMV Tobacco mosaic virus, TMGMV Tobacco mild green mosaic virus, TBRFV Tomato brown rugose virus, RMV Ribgrass mosaic virus, RheMV Rehmannia mosaic virus, PMMoV Pepper mild mottle virus, PaMMV Paprika mild mottle virus, HLSV Hibiscus latent Singapore virus, HLFPV Hibiscus latent Fort Pierce virus, CMoV Cucumber mottle virus, CGMMV Cucumber green mild mottle virus, BPMV Bell pepper mottle virus, SA South America.

Fig. 7. Global prevalence and abundance of Mamastrovirus genotypes in urban wastewater.

Biannual (a) and longitudinal data (b) are shown. In each panel, stacked bar plots display the number of reads per million viral reads assigned to the Mamastrovirus genus (green) and to specific genotypes (red), followed by the relative abundances of Mamastrovirus genotypes. The biannual samples were grouped using hierarchical clustering based on genotype composition. HAstV Human Astrovirus, RAstV Rat astrovirus, CAstV Canine astrovirus, FAstV Feline astrovirus, ChAstV Cheetah astrovirus, YakAstV Yak astrovirus, Other genotypes detected in fewer than 4 samples.

Fig. 8. Phylogenetic analysis of Human astrovirus type 5 (HAstV-5) from global wastewater and publicly available reference sequences.

Maximum likelihood phylogenetic tree of HAstV-5 based on partial ORF2 gene (capsid) sequences. Sequences obtained from GenBank are indicated in coloured squares, while sequences derived from wastewater with a minimum length of 500 bp are denoted in coloured dots. Colours represent the continent of origin (Europe, Asia, Africa, Oceania, North America, and South America). Bootstrap values > 70 are shown.

Fig. 9. Global prevalence and abundance of Enterovirus genotypes in urban wastewater.

Biannual (a) and longitudinal data (b) are shown. In each panel, stacked bar plots display reads per million viral reads assigned to the Enterovirus genus (green) and to specific genotypes (red), followed by the relative abundances of Enterovirus species and of genotypes. The biannual samples were grouped using hierarchical clustering based on genotype composition. Other = genotypes detected in fewer than 10 samples, including (vaccine-derived) poliovirus type 3. SA South America.

Fig. 10. Phylogenetic analysis of Echovirus 30 (E30) from global wastewater and publicly available reference sequences.

Maximum likelihood phylogenetic tree of E30 based on partial VP1 gene (capsid) sequences. Reference sequences obtained from GenBank are indicated with coloured squares, while sequences derived from wastewater with a minimum length of 500 bp are denoted in coloured dots. Colours represent the continent of origin (Europe, Asia, Africa, Oceania, North America, and South America). Bootstrap values > 70 are shown.