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Memórias do Instituto Oswaldo Cruz
Fundação Oswaldo Cruz, Fiocruz
ISSN: 1678-8060 EISSN: 1678-8060
Vol. 113, No. 1, 2018, pp. 38-44
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Bioline Code: oc18011
Full paper language: English
Document type: Research Article
Document available free of charge
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Memórias do Instituto Oswaldo Cruz, Vol. 113, No. 1, 2018, pp. 38-44
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Simple protocol for population (Sanger) sequencing for Zika virus genomic regions
Cabral, Gabriela Bastos; Ferreira, João Leandro de Paula; Souza, Renato Pereira de; Cunha, Mariana Sequetin; Luchs, Adriana; Figueiredo, Cristina Adelaide & Brígido, Luís Fernando de Macedo
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.
Keywords
Zika virus; Sanger sequencing; PCR; phylogeny
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