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

Abstract

Zika virus (ZiKV) is a mosquito-borne member of theFlaviviridae 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 liveattenuated 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.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 viral genome is made of a positive sense, single-stranded RNA molecule (~10.8 kb) that contains a single open reading frame flanked by 5′ and 3′ untranslated regions 2-4 . The viral RNA is translated as a single polyprotein that is co-and post-translationally processed by viral and cellular proteases into three major structural proteins (capsid, pre-membrane and envelope) involved in viral entry, fusion and assembly 5 , and seven non-structural (NS) proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5) that are involved in viral RNA replication and transcription, assembly and evasion of the host antiviral responses [6][7][8][9] .Although the majority of ZIKV infections are asymptomatic or associated with a mild febrile illness, infection during pregnancy has been associated with miscarriage and severe congenital malformations, including fetal

Rescue of SARS-CoV-2 from a Single Bacterial Artificial Chromosome

“…Assembly of the SARS-CoV-2 genome in the BAC. We used a BAC-based approach, similar to the one that we previously described for Zika virus (16)(17)(18)(19)(20)(21), to assemble an infectious clone of SARS-CoV-2 based on the USA-WA1/2020 strain ( Fig. 1A).…”

Development and application of reverse genetic technology for the influenza virus

“…The reverse genetics of viruses is often called viral rescue, which is achieved by constructing an infectious clone of the virus and manipulating it in vitro at the DNA level to study the structure and function of the virus [ 40 , 41 ]. Infectious molecular clones include infectious cDNA and infectious in vitro transcripts.…”

Monitoring SARS-CoV-2 infection using a double reporter-expressing virus

“…The copyright holder for this preprint this version posted June 24, 2022. ; https://doi.org/10.1101/2022.06. 23.497376 doi: bioRxiv preprint Reverse genetics systems have permitted the generation of recombinant RNA viruses entirely from cloned cDNA, facilitating studies to better understand multiple aspects of the biology of viruses, including, among others, mechanisms of viral infection, pathogenesis, transmission, and disease (19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29). Another application of reverse genetics is the generation of recombinant viruses containing gene mutations and/or deletions that result in viral attenuation for their implementation as safe, immunogenic, and protective liveattenuated vaccines (LAV) (20,(30)(31)(32)(33)(34)(35).…”