Monitoring SARS-CoV-2 Circulation and Diversity through Community Wastewater Sequencing, the Netherlands and Belgium

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has rapidly become a major global health problem, and public health surveillance is crucial to monitor and prevent virus spread. Wastewater-based epidemiology has been proposed as an addition to disease-based surveillance because virus is shed in the feces of ≈40% of infected persons. We used next-generation sequencing of sewage samples to evaluate the diversity of SARS-CoV-2 at the community level in the Netherlands and Belgium. Phylogenetic analysis revealed the presence of the most prevalent clades (19A, 20A, and 20B) and clustering of sewage samples with clinical samples from the same region. We distinguished multiple clades within a single sewage sample by using low-frequency variant analysis. In addition, several novel mutations in the SARS-CoV-2 genome were detected. Our results illustrate how wastewater can be used to investigate the diversity of SARS-CoV-2 viruses circulating in a community and identify new outbreaks.

Keywords: Belgium; COVID-19; CoV; Illumina; Nanopore; SARS-CoV-2; coronavirus disease; epidemiology; public health; respiratory infections; severe acute respiratory syndrome coronavirus 2; sewage; the Netherlands; viruses; wastewater; zoonoses.

Figure 1

Quantitative reverse transcription PCR C_t of severe acute respiratory syndrome coronavirus 2 RNA in sewage samples as determined by N gene (N1–N3) and E gene assays against the percentage of genome covered (>10×) by nanopore reads, the Netherlands and Belgium. A) N1 gene; B) N2 gene; C) N3 gene; D) E gene. C_t, cycle threshold.

Figure 2

Phylogenetic analysis of severe acute respiratory syndrome coronavirus 2 genome consensus sequences detected in sewage samples, the Netherlands and Belgium. A) The Netherlands subsample dataset; B) global subsample dataset. Lines with dots in green indicate samples sequenced in this study. Clades (19A, 19B, 20A, 20B, and 20C) were assigned by using the Nextclade tool (https://clades.nextstrain.org). For the global subsample tree, samples in orange indicate the Netherlands sequences. Samples in purple indicate Belgium sequences. Scale bars indicate inferred number of nucleotide substitutions per site.

Figure 3

Phylogenetic trees showing 4 low-frequency variants detected in sewage samples in study of severe acute respiratory syndrome coronavirus 2 circulation and diversity through community wastewater sequencing, the Netherlands and Belgium. A) The Netherlands–Belgium subsample; B) global subsample. Patient sequences containing the mutation are shown in magenta. Lines in green indicate sewage samples sequenced in this study. Clades (19A, 19B, 20A, 20B, and 20C) are indicated in colors at the left of the figure. Blue arrows show the consensus sequences (if available) of the sewage samples in which the low-frequency variant was detected. Scale bars indicate the inferred number of nucleotide substitutions per site.