# Wastewater sequencing from a rural community enables identification of widespread adaptive mutations in a SARS-CoV-2 alpha variant

## TL;DR

Wastewater sequencing from a rural area revealed rare mutations in a SARS-CoV-2 Alpha variant that persisted for months.

## Contribution

The study identifies novel adaptive mutations in a SARS-CoV-2 Alpha variant using wastewater sequencing from a rural site.

## Key findings

- Wastewater samples revealed a SARS-CoV-2 Alpha variant with rare mutations over 20–28 months.
- Novel mutations were found in non-structural and structural genes not seen in global clinical samples.
- Wastewater sequencing from small plants can detect rare mutations and reconstruct viral genomes effectively.

## Abstract

Central Michigan University (CMU) participated in a state-wide wastewater monitoring program starting in 2021. One rural site consistently produced higher concentrations of SARS-CoV-2 genome copies. Samples from this site were sequenced retrospectively and exclusively contained a derivative of Alpha variant lineage B.1.1.7 that shed from the same site for 20–28 months. Complete reconstruction of each SARS-CoV-2 open reading frame (ORF) and alignment to an early B.1.1.7 clinical isolate identified novel mutations that were selected in non-structural (nsp1, nsp2, nsp3, nsp4, nsp5/3CLpro, nsp6, RdRp, nsp15, nsp16, ORF3a, ORF6, ORF7a, and ORF7b) and structural genes (Spike, M, and N). These were rare mutations that have not accumulated in clinical samples worldwide. Mutational analysis revealed divergence from the reference Alpha variant lineage sequence over time. We present each of the mutations on available structural models and discuss the potential role of these mutations during a chronic infection. This study further supports that small wastewater treatment plants can enhance resolution of rare events and facilitate reconstruction of viral genomes due to the relative lack of contaminating sequences and identifies mutations that may be associated with chronic infections.

The online version contains supplementary material available at 10.1038/s41598-025-03771-5.