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Viruses 2018, 10(10), 547; https://doi.org/10.3390/v10100547

An Alanine-to-Valine Substitution in the Residue 175 of Zika Virus NS2A Protein Affects Viral RNA Synthesis and Attenuates the Virus In Vivo

1
Department of Molecular and Cell Biology, Centro Nacional de Biotecnología (CNB-CSIC), Campus Universidad Autónoma de Madrid, 3 Darwin street, 28049 Madrid, Spain
2
Department of Microbiology and Immunology, University of Rochester Medical Center, 601 Elmwood Avenue, Rochester, NY 14642, USA
These authors contributed equally to this work.
*
Authors to whom correspondence should be addressed.
Received: 12 September 2018 / Revised: 2 October 2018 / Accepted: 4 October 2018 / Published: 7 October 2018
(This article belongs to the Special Issue New Advances on Zika Virus Research)
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Abstract

The recent outbreaks of Zika virus (ZIKV), its association with Guillain–Barré syndrome and fetal abnormalities, and the lack of approved vaccines and antivirals, highlight the importance of developing countermeasures to combat ZIKV disease. In this respect, infectious clones constitute excellent tools to accomplish these goals. However, flavivirus infectious clones are often difficult to work with due to the toxicity of some flavivirus sequences in bacteria. To bypass this problem, several alternative approaches have been applied for the generation of ZIKV clones including, among others, in vitro ligation, insertions of introns and using infectious subgenomic amplicons. Here, we report a simple and novel DNA-launched approach based on the use of a bacterial artificial chromosome (BAC) to generate a cDNA clone of Rio Grande do Norte Natal ZIKV strain. The sequence was identified from the brain tissue of an aborted fetus with microcephaly. The BAC clone was fully stable in bacteria and the infectious virus was efficiently recovered in Vero cells through direct delivery of the cDNA clone. The rescued virus yielded high titers in Vero cells and was pathogenic in a validated mouse model (A129 mice) of ZIKV infection. Furthermore, using this infectious clone we have generated a mutant ZIKV containing a single amino acid substitution (A175V) in the NS2A protein that presented reduced viral RNA synthesis in cell cultures, was highly attenuated in vivo and induced fully protection against a lethal challenge with ZIKV wild-type. This BAC approach provides a stable and reliable reverse genetic system for ZIKV that will help to identify viral determinants of virulence and facilitate the development of vaccine and therapeutic strategies. View Full-Text
Keywords: Zika virus; Full-length cDNA infectious clones; Bacterial artificial chromosome; NS2A protein Zika virus; Full-length cDNA infectious clones; Bacterial artificial chromosome; NS2A protein
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Márquez-Jurado, S.; Nogales, A.; Ávila-Pérez, G.; Iborra, F.J.; Martínez-Sobrido, L.; Almazán, F. An Alanine-to-Valine Substitution in the Residue 175 of Zika Virus NS2A Protein Affects Viral RNA Synthesis and Attenuates the Virus In Vivo. Viruses 2018, 10, 547.

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