MEM INST OSWALDO CRUZ, RIO DE JANEIRO, FAST TRACK
PAGES: DOI: 10.1590/0074-02760170248 Full paper
Simple Protocol for population (Sanger) sequencing for Zika virus genomic regions

Gabriela Bastos Cabral1, João Leandro de Paula Ferreira1, Renato Pereira de Souza2, Mariana Cunha2, Cristina Figueiredo3, Luís Fernando de Macedo Brígido1+

1NDSS, Retrovirus Laboratory, Virology Center, Adolfo Lutz Institute, São Paulo, Brazil
2NDTV, Virology Center, Adolfo Lutz Institute, São Paulo, Brazil
3NDR, Virology Center, Adolfo Lutz Institute, São Paulo, Brazil

Abstract

The Flaviviridae Zika (ZIKV), first identified in Uganda, initially noticed in Brazil in 2015, was probably introduced in the Americas from Oceania. The pathogenic potential associated to the Brazilian outbreak made the recognition and characterization of this infection a priority. Genetic sequences from ZIKV has allowed to study the molecular signatures of the agent and trace its expansion. Next-generation sequencing (NGS) is increasingly used to understand the genetic diversity of different agents, but sanger sequencing may be an alternative for many applications as it is an easy to use, robust, affordable, rapid, and specific tool to obtain sequences. Objective: The aim of this study is to develop a simple protocol of populational (sanger) genetic sequencing of the envelope and NS5 regions of the Zika virus Materials and methods: Six isolates of Zika virus, obtained from supernatant of infected Vero cells and clinical sample (urine) were evaluated in this study. One of the samples was previously sequenced by NGS. A one-step PCR reaction were performed for the amplification of complete envelope and partial NS5 region of the ZIKV using SuperScript®III One-step RT-PCR system with Taq High Fidelity, followed by a cycle sequence reaction using big dye terminator. Finding: The method was effective for amplification and obtaining the genetic sequences of the 6 samples for envelope region and 5 from NS5 region. Although sequences from NGS can provide additional information, nucleotide alignments and phylogenetic trees topology show that Sanger sequencing may be as informative as NGS for some applications. Conclusion: The present study provides a simple protocol to amplify and sequence segments of the Zika genome. As currently only a few zika sequences are available, this method can facilitate the production of additional sequences in resource limited settings.

+ Corresponding author: This e-mail address is being protected from spambots. You need JavaScript enabled to view it.

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