Beyond the Spike Glycoprotein: Mutational Signatures in SARS-CoV-2 Structural Proteins

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Background: The continuous emergence of SARS-CoV-2 variants represents a major public health concern. Next-generation sequencing (NGS) enables genomic surveillance, facilitating the detection and monitoring of mutations that impact viral evolution. Methods: In this study, full-length SARS-CoV-2 genomes were analyzed between February 2022 and March 2024 as part of routine genomic surveillance conducted in Verona, Italy. Mutations in the envelope (E), membrane (M), and nucleocapsid (N) structural proteins were investigated. Only substitutions with a total prevalence of greater than 1% in the study dataset were considered. Results: A total of 178 mutations were identified across the three proteins (E: 16; M: 33; N: 129), of which 18 met the inclusion threshold (E: 3; M: 5; N: 10). Mutations were classified according to temporal dynamics as fixed, emerging, or transient. Throughout the study period, fixed mutations were consistently prevalent, emerging mutations appeared later but persisted with an ascending trend, while transient mutations displayed a single frequency peak before disappearing. Several mutations were reported with potential structural or functional relevance based on the existing literature, while others remain of unknown significance. Conclusions: The mutational patterns detected in this study broadly reflect global evolutionary trends of SARS-CoV-2. These findings emphasize the importance of continued genomic surveillance and underline the need for integrated experimental approaches to clarify the biological and epidemiological impact of poorly characterized mutations.

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