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

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.

In vivo rescue of recombinant Zika virus from an infectious cDNA clone and its implications in vaccine development

“…For in vitro rescues, tissue culture supernatants were collected after 3-4 days, when cytopathic effect (CPE) was apparent (60-80%), and stored at −80 °C. Virus stocks were propagated in Vero cells and titrated by immunofocus assay in Vero cells using the pan-flavivirus envelope (E) protein monoclonal antibody (mAb) 4G2 from BEI Resources (NR-50327) 27 . Viral titers were expressed as focus-forming units per ml (FFU/ml) 27 .…”

“…Viral titers were determined by FFU assays in Vero cells 27 . enzyme-linked immunosorbent assay (eLiSA). To assess the levels of virus-specific antibodies present in the sera of vaccinated mice, ELISAs were performed as previously described 27,54,55 . Briefly, 96-well ELISA Figure 9.…”

“…However, there are intrinsic limitations in the in vitro recovery of recombinant viruses using reverse genetic approaches, mostly associated with cell culture-adaptive mutations that might restrict and/or change the phenotype of the virus in vivo 25,26 . In the case of LAVs, these mutations could lead to the reversion to a virulent phenotype 27,28 . Likewise, the current need of first generating recombinant viruses in cultured cells rather than directly in validated animal models of infection delays studies aimed to understand and assess viral infections in vivo.…”

“…Recently, we have described a reverse genetic approach for ZIKV based on the use of a bacterial artificial chromosome (BAC), by assembling the full-length genome of ZIKV Rio Grande do Norte Natal (RGN) 27,29 or Paraiba/2015 strains 30 under the control of the cytomegalovirus (CMV) immediate-early promoter. Importantly, we demonstrated the feasibility of using this infectious clone to generate a virulent rZIKV with similar in vitro and in vivo characteristics to the natural Paraiba/2015 isolate 30 .…”

Rescue of Recombinant Zika Virus from a Bacterial Artificial Chromosome cDNA Clone

“…Herein, we describe the detailed protocol for the engineering of a full-length cDNA clone of the ZIKV strain ZIKV-RGN 13 , using a BAC to overcome the toxicity problem, and the rescue of infectious rZIKV by direct transfection of the BAC cDNA clone into Vero cells 32 , a cell line approved by the Food and Drug Administration (FDA) for the development of human vaccines 33 . In this system, the full-length cDNA copy of the viral genome is assembled in the BAC plasmid pBeloBAC11 34 (Figure 2A), a low-copy-number plasmid (one to two copies per cell) derived from the Escherichia coli F-factor 35 , which minimizes the toxicity of flavivirus sequences during its propagation in bacteria.…”