Phylogenetic analysis of the SARS-CoV-2 spike (S) glycoprotein sequence implicates small mammals and wastewater as potential sources of infection

Understanding host diversity, temporal distribution, and phylogenetic relationships of SARS-CoV-2 is critical for elucidating spillover and reverse zoonotic events.This study aimed to analyze partial surface glycoprotein (S) gene sequences from multiple hosts and sources across various countries and time periods.A total of 208 sequences annotated as SARS-CoV-2 spike (S) glycoprotein were retrieved and analyzed, including seven SARS-CoV-2 sequences retrieved using "spillover," "spillback," or "reverse zoonosis" as search words, and one MERS-CoV sequence as an out-group.Metadata on host, year, and country were examined using descriptive statistics and heatmaps in R. Sequences were aligned with MAFFT, and a maximum-likelihood phylogenetic tree was constructed using the Hasegawa-Kishino-Yano (HKY) substitution model with empirical rate heterogeneity and 1,000 bootstrap replicates.The resulting consensus tree was visualized and rooted on the MERS-CoV out-group using iTOL.The sequences, originating from 16 countries, clustered into six phylogenetic groups.Cluster 1, the largest (101 sequences), exhibited the highest host diversity, including humans, cats, dogs, and deer mouse (Peromyscus maniculatus).Cluster 5 (51 sequences) included wastewater-derived sequences, suggesting environmental sources.Clusters 2, 3, and 6 were strongly supported (bootstrap 93-100) and predominantly human-derived, with cluster 2 also including two white-tailed deer (Odocoileus virginianus) sequences and a single Omicron variant, indicating co-circulation of divergent lineages.Temporal analysis revealed consistent representation from 2021 to 2022, whereas the sequences from 2020 and 2024 were unevenly distributed, reflecting sampling gaps.SARS-CoV-2 exhibits considerable phylogenetic and host diversity, with evidence of environmental sources and cross-species transmission.These findings underscore the importance of continued onehealth surveillance across multiple hosts and geographic regions to monitor spillover events and emerging variants.