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

Autor(es):Mostrar menos -

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]

Afiliação do(s) autor(es)

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

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

• Tipo de documento: Artigo Científico
• Fonte: Journal of Virological Methods; v. 300, FEB 2022.
• Citações Web of Science: 0

Resumo

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)

• Processo FAPESP: 16/25212-9 - Vacina terapêutica baseada em células dendríticas aDC1 e vírus autólogo inativado visando o controle viral de indivíduos infectados pelo HIV-1 em interrupção de terapia antirretroviral
• Beneficiário: Alberto José da Silva Duarte
• Modalidade de apoio: Auxílio à Pesquisa - Temático
• Processo FAPESP: 18/14384-9 - Identificação e caracterização de casos agudos de infecção pelo vírus da imunodeficiência humana (HIV-1)
• Beneficiário: Luís Fernando de Macedo Brígido
• Modalidade de apoio: Auxílio à Pesquisa - Pesquisa em Políticas Públicas