Abstract
Zika virus (ZIKV) is a mosquito-borne member of the Flaviviridae family that has been known to circulate for decades causing mild febrile illness. The more recent ZIKV outbreaks in the Americas and the Caribbean associated with congenital malformations and Guillain-Barré syndrome in adults have placed public health officials in high alert and highlight the significant impact of ZIKV on human health. New technologies to study the biology of ZIKV and to develop more effective prevention options are highly desired. In this study we demonstrate that direct delivery in mice of an infectious ZIKV cDNA clone allows the rescue of recombinant (r)ZIKV in vivo. A bacterial artificial chromosome containing the sequence of ZIKV strain Paraiba/2015 under the control of the cytomegalovirus promoter was complexed with a commercial transfection reagent and administrated using different routes in type-I interferon receptor deficient A129 mice. Clinical signs and death associated with ZIKV viremia were observed in mice. The rZIKV recovered from these mice remained fully virulent in a second passage in mice. Interestingly, infectious rZIKV was also recovered after intraperitoneal inoculation of the rZIKV cDNA in the absence of transfection reagent. Further expanding these studies, we demonstrate that a single intraperitoneal inoculation of a cDNA clone encoding an attenuated rZIKV was safe, highly immunogenic, and provided full protection against lethal ZIKV challenge. This novel in vivo reverse genetics method is a potentially suitable delivery platform for the study of wild-type and live-attenuated ZIKV devoid of confounding factors typical associated with in vitro systems. Moreover, our results open the possibility of employing similar in vivo reverse genetic approaches for the generation of other viruses and, therefore, change the way we will use reverse genetics in the future.
Highlights
Zika virus (ZIKV), a member of the Flaviviridae family, became a global public concern because of the correlation of Zika virus epidemic with fetal developmental defects, including highly publicized cases of microcephaly[1]
The pBAC-ZIKV cDNA clone was complexed with Lipofectamine 2000 and inoculated in IFNAR−/− A129 mice using different inoculation routes, which are typically used for ZIKV infections
Vero cells (1 × 106 cells, triplicates) were transfected with 5 μg of the pBAC-ZIKV cDNA clone using Lipofectamine 2000 (LPF) (1:3 ratio of cDNA:LPF, referred as pBAC-ZIKV/LPF)
Summary
Zika virus (ZIKV), a member of the Flaviviridae family, became a global public concern because of the correlation of Zika virus epidemic with fetal developmental defects, including highly publicized cases of microcephaly[1]. The establishment of plasmid-based reverse genetic systems for RNA viruses entails the rescue of recombinant viruses from cDNA clones containing the entire viral genome on a plasmid from transfected culture cells[18]. In vivo reverse genetics or recover of virus directly from validated animal models could overcome the concern of virus adaptation to cell cultures and facilitate and simplify the study of the virus and the development of LAVs based on attenuated forms of these viruses. More Importantly, we demonstrate the feasibility of using this in vivo rescue approach with a BAC cDNA infectious clone encoding an attenuated rZIKV (rZIKVatt)[30] that resulted in sterilizing immunity against aggressive ZIKV challenge. In vivo recovery of fully infectious and/or attenuated RNA viruses expands the potential use of reverse genetic systems and open the possibility of developing similar approaches for other viruses, which could be an innovative technology for their study or the future development of LAVs
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