Persistent detection of two genetically and geographically distinct cryptic SARS-CoV-2 lineages in the Netherlands

The causative agent of coronavirus disease 2019 (COVID-19), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is shed by infected individuals into wastewater. The widespread use of wastewater-based surveillance for monitoring SARS-CoV-2 has in several cases led to the detection of 'cryptic' lineages, which denotes unique sets of mutations that are absent or extremely rare in individual-based clinical surveillance. Previously, our group reported a cryptic lineage detected in wastewater over a two-month period. In this study, we report the consistent detection of two cryptic lineages in wastewater from two wastewater treatment plants (WWTPs) over multiple years. These WWTPs were routinely sampled as part of the Dutch national sewage surveillance programme, and their samples exhibited significantly higher viral loads than samples collected from any other WWTP. Consequently, these results were excluded from country-wide aggregated data. Whole-genome targeted sequencing and subsequent lineage abundance estimation identified two pre-Alpha lineages: B.11 and B.1.221(.3). We describe their temporal dynamics and reconstruct the evolution of these distinct lineages during their periods of detection. We demonstrate how these reconstructed lineages improve lineage estimation modelling and highlight mutations common to other cryptic wastewater lineages.