Five Years of SARS-CoV-2 Spike Protein Evolution in Goa, India

This is the first spike protein molecular evolution analysis for SARS-CoV-2 in Goa, India, covering all 1,274 GISAID EpiCoV sequences from December 2020 to June 2025 (EPI_SET_260610gh). Using Nextclade v3.21.2, we characterise the complete spike mutation landscape across five pandemic waves, documenting 229 unique spike mutations in 495 good-quality sequences, near-universal D614G fixation (99.8%), furin cleavage site mutations in 65.3% of sequences (P681R and P681H), and a 7.5-fold higher immune escape burden in Omicron vs. Delta sequences. We document 33 convergent spike mutations independently emerging in three or more WHO variant groups, providing direct evidence of positive natural selection at functionally constrained sites. ORF8 was disrupted in 45.2% of sequences through the Delta-associated D119/F120 deletion. Phylogenetic placement of all 1,274 sequences on the global reference tree confirms clade-concordant clustering. This paper constitutes Paper 2 of a planned comparative genomic surveillance series for the West Coast and Western Ghats corridor of India. All data are cross-referenced with Paper 1 of this series (doi:10.5281/zenodo.20632518).