Analysis of SARS-CoV-2 amino acid mutations in New York City Metropolitan wastewater (2020-2022) reveals multiple traits with human health implications across the genome and environment-specific distinctions

We characterize variant diversity, amino acid mutation frequency, functionality and associations with COVID-19 infections in one of the largest datasets of SARS-CoV-2 genome sequences collected from wastewater in the New York metropolitan area. Variant diversity differed within parts of the New York City sewershed and between wastewater sludge and influent samples. P314L, D614G and T3255I occurred in >95% of wastewater samples. Enhanced infectivity, transmissibility and escape from antibody neutralization were dominant traits in the wastewater. Strikingly, over 60% of the most frequently occurring mutations were found in regions other than the spike (S) protein, and nearly 50% remain uncharacterized for functional impacts warranting further investigation. We demonstrate strong correlations between P314L, D614G, T95I, G50E, G50R, G204R, R203K, G662S, P10S, P13L and mortality rates, percent positive test results, hospitalization rates and % of population fully vaccinated. The results from our study suggest that there are relatively understudied mutations in the spike protein (H655Y, T95I) and understudied mutations occurring in non-spike proteins (N, ORF1b, ORF9b and ORF9c), that are enhancing transmissibility and infectivity among human populations, warranting further investigation.