2024-03-28T23:56:53Zhttp://digital.csic.es/dspace-oai/requestoai:digital.csic.es:10261/1714822022-05-06T10:36:51Zcom_10261_25com_10261_1col_10261_278
An Alanine-to-Valine Substitution in the Residue 175 of Zika Virus NS2A Protein Affects Viral RNA Synthesis and Attenuates the Virus In Vivo
Márquez-Jurado, Silvia
Nogales, Aitor
Ávila-Pérez, Ginés
Iborra, Francisco J.
Martínez-Sobrido, Luis
Almazán, Fernando
Ministerio de Economía y Competitividad (España)
National Institutes of Health (US)
Zika virus
Full-length cDNA infectious clones
Bacterial artificial chromosomes
NS2A protein
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.
This research was funded by the Spanish Ministry of Economy and Competitiveness (MINECO) (grant number BFU2016-79127-R) to F.A. and F.J.I. and the National Institute of Health (NIH) (grant number
1R21AI120500) to L.M.-S. and F.A.
Peer reviewed
2018-10-24T08:17:44Z
2018-10-24T08:17:44Z
2018-10-07
2018-10-24T08:17:44Z
artículo
http://purl.org/coar/resource_type/c_6501
Viruses 10(10): 547 (2018)
1999-4915
http://hdl.handle.net/10261/171482
10.3390/v10100547
http://dx.doi.org/10.13039/501100003329
http://dx.doi.org/10.13039/100000002
30301244
#PLACEHOLDER_PARENT_METADATA_VALUE#
info:eu-repo/grantAgreement/MINECO/Plan Estatal de Investigación Científica y Técnica y de Innovación 2013-2016/BFU2016-79127-R
Publisher's version
https://doi.org/10.3390/v10100547
Sí
open
Multidisciplinary Digital Publishing Institute