Chronic Shedding of a SARS-CoV-2 Alpha Variant Lineage Q.3/Q.4 in Wastewater

Central Michigan University (CMU) participated in a state-wide SARS-CoV-2 wastewater monitoring program since 2021. Wastewater samples were collected from on-campus sites and nine off-campus wastewater treatment plants servicing small metropolitan and rural communities. SARS-CoV-2 genome copies were quantified using droplet digital PCR and results were reported to the health department. One rural, off-campus site consistently produced higher concentrations of SARS-CoV-2 genome copies. Samples from this site were sequenced and initially contained predominately Alpha variant lineage Q.3, which transitioned to lineage Q.4. Alpha variant lineage Q.3/Q.4 was detected at this site beginning in fall 2021 and continued until summer 2023. Mutational analysis of reconstructed genes revealed divergence from the Alpha variant lineage Q.3 clinical sequence over time, including numerous mutations in the surface glycoprotein RBD and NTD. We discuss the possibility that a chronic SARS-CoV-2 infection accumulated adaptive mutations that promoted long-term infection. This study reveals 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.