MEM INST OSWALDO CRUZ, RIO DE JANEIRO, FAST TRACK
PAGES: DOI: 10.1590/0074-02760170248 Full paper

Gabriela Bastos Cabral1, João Leandro de Paula Ferreira1, Renato Pereira de Souza2, Mariana Sequetin Cunha2, Adriana Luchs3, Cristina Adelaide Figueiredo4,†, Luís Fernando de Macedo Brígido1+

1Instituto Adolfo Lutz, Centro de Virologia, Núcleo de Doenças Sanguíneas e Sexuais, Laboratório de Retrovirus, São Paulo, SP, Brasil
2Instituto Adolfo Lutz, Centro de Virologia, Núcleo de Transmissão Vetorial, São Paulo, SP, Brasil
3Instituto Adolfo Lutz, Centro de Virologia, Núcleo de Doenças Entéricas, São Paulo, SP, Brasil
4Instituto Adolfo Lutz, Centro de Virologia, Núcleo de Doenças Respiratórias, São Paulo, SP, Brasil

Abstract

BACKGROUND A number of Zika virus (ZIKV) sequences were obtained using Next-generation sequencing (NGS), a methodology widely applied in genetic diversity studies and virome discovery. However Sanger method is still a robust, affordable, rapid and specific tool to obtain valuable sequences.

OBJECTIVE The aim of this study was to develop a simple and robust Sanger sequencing protocol targeting ZIKV relevant genetic regions, as envelope protein and nonstructural protein 5 (NS5). In addition, phylogenetic analysis of the ZIKV strains obtained using the present protocol and their comparison with previously published NGS sequences were also carried out.

METHODS Six Vero cells isolates from serum and one urine sample were available to develop the procedure. Primer sets were designed in order to conduct a nested RT-PCR and a Sanger sequencing protocols. Bayesian analysis was used to infer phylogenetic relationships.

FINDINGS Seven complete ZIKV envelope protein (1,571 kb) and six partial NS5 (0,798 Kb) were obtained using the protocol, with no amplification of NS5 gene from urine sample. Two NS5 sequences presented ambiguities at positions 495 and 196. Nucleotide analysis of a Sanger sequence and consensus sequence of previously NGS study revealed 100% identity. ZIKV strains described here clustered within the Asian lineage.

MAIN CONCLUSIONS The present study provided a simple and low-cost Sanger protocol to sequence relevant genes of the ZIKV genome. The identity of Sanger generated sequences with published consensus NGS support the use of Sanger method for ZIKV population studies. The regions evaluated were able to provide robust phylogenetic signals and may be used to conduct molecular epidemiological studies and monitor viral evolution.

 

Financial support: Adolfo Lutz Instute, FINEP (grant no. 0219/2016), FAPESP (grant no. 194417/2013).
+ Corresponding author: This e-mail address is being protected from spambots. You need JavaScript enabled to view it.
† In memoriam
Received 28 June 2017
Accepted 23 October 2017

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