se of Sanger protocols to identify variants of concern, key mutations and track evolution of SARS-CoV-

Author(s):Show less -

Cabral, Gabriela Bastos ^[1] ; Ahagon, Cintia Mayumi ^[2] ; de Souza Guimaraes, Paula Morena ^[2] ; Silva Lopez-Lopes, Giselle Ibette ^[2] ; Hussein, Igor Mohamed ^[2] ; Cilli, Audrey ^[2] ; Alves, Ivy de Jesus ^[1] ; Coelho Bombonatte, Andrea Gobetti ^[1] ; Sampaio Tavares Timenetsky, Maria do Carmo ^[2] ; da Silva Santos, Jaqueline Helena ^[2] ; de Oliveira Santos, Katia Correa ^[2] ; Pereira dos Santos, Fabiana Cristina ^[2] ; de Macedo Brigido, Luis Fernando ^[2]

Affiliation

^[1] Adolfo Lutz Inst, Santos Reg Ctr, Santos, SP - Brazil

^[2] Adolfo Lutz Inst, Virol Ctr, Sao Paulo, SP - Brazil

• Document type: Journal article
• Source: Journal of Virological Methods; v. 300, FEB 2022.
• Web of Science Citations: 0

Abstract

Vaccination and the emergence of SARS-CoV-2 variants mark the second year of the pandemic. Variants have amino acid mutations at the spike region, a viral protein central in the understanding of COVID-19 pathogenesis and vaccine response. Variants may dominate local epidemics, as Gamma (P.1) in Brazil, emerging in 2020 and prevailing until mid-2021. Different obstacles hinder a wider use of Next-Generation Sequencing for genomic surveillance. We describe Sanger based sequencing protocols: i) Semi-nested RT-PCR covering up to 3.684 kb (>96 %) spike gene; ii) One-Step RT-PCR for key Receptor Binding Domain (RBD) mutations (codons 417-501); iii) One-Step RT-PCR of partial N region to improve genomic capability. Protocols use leftovers of RNA extracted from nasopharyngeal swabs for quantitative RT-PCR diagnosis; with retro-transcribed DNA sequenced at ABI 3500 using dye termination chemistry. Analyses of sequences from 95 individuals (late 2020/early 2021) identified extensive amino acid variation, 57 % with at least one key mutation at the Receptor Binding Domain, with B.1.1.28 lineage most prevalent, followed by Gamma and Zeta variants, with no Delta variant observed. The relatively low cost and simplicity may provide an accessible tool to improve surveillance of SARS-CoV-2 evolution, monitor new variants and vaccinated breakthroughs. (AU)

• FAPESP's process: 16/25212-9 - Therapeutic vaccine based on aDC1 dendritic cells pulsed with inactivated autologous virus for the control of viremia after ATI in HIV infected individuals
• Grantee: Alberto José da Silva Duarte
• Support Opportunities: Research Projects - Thematic Grants
• FAPESP's process: 18/14384-9 - Identification and characterization of acute cases of Human Immunodeficiency Virus (HIV-1) infection
• Grantee: Luís Fernando de Macedo Brígido
• Support Opportunities: Research Grants - Research in Public Policies