P1 variant and amino acid mutations at Spike gene identified using Sanger protocol

Cabral, G. B.; Ahagon, C. M.; Lopez-Lopes, G. I. S.; Hussein, I. M.; Guimaraes, P. M.; Cilli, A.; Silva, V. O.; Timenetsky, M. d. C. S.; Alves, I. d. J.; Bombonatte, A. G.; dos Santos, F. C. P.; Brigido, L. F. d. M.

SARS-coV-2 variants, along with vaccination, mark the second year of the pandemic. The spike region is a focal point in COVID-19 pathogenesis, with different amino acid changes potentially modulating vaccine response and some being part of variant signatures. NGS is the standard tool to sequence the virus but limitations of different sources hinders expansion of genomic surveillance in many places. To improve surveillance capability we developed a Sanger based sequencing protocol to obtain coverage of most (>95%) spike gene. Eleven nasopharyngeal swabs collections had RNA extracted for real time PCR diagnosis and leftover RNA had up to 3785 bp sequenced at an ABI3500 using dye termination chemistry of nested PCR products of two reactions of one Step RT-PCR. P1 amino acid mutations signatures were present in 18% (2/11), with 82% (9/11) with three or more additional amino acid changes (GISAID CoVsurver list). Most sequences (86%, 7/8) from 2021 have the E484K, whereas the mutation was not present in samples collected in 2020 (0/4, p=0.015). The swiftness that favorable mutations to the virus may prevail and their potential impact in vaccines and other current interventions need broader surveillance and more public health attention.