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

Background 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. Competing Interest Statement The authors have declared no competing interest. Funding Statement Michigan Department of Health and Human Services (MDHHS) Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: N/A I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes Data Availability FASTQ files for each sample are available in the NCBI SRA database (Submission ID: SUB13897431; BioProject ID: PRJNA1027333).