Reverse genetic system, genetically stable reporter viruses and packaged subgenomic replicon based on a Brazilian Zika virus isolate

Abstract

Zika virus (ZIKV, genus Flavivirus) has emerged as a major mosquito-transmitted human pathogen, with recent outbreaks associated with an increased incidence of neurological complications, particularly microcephaly and the Guillain-Barré syndrome. Because the virus has only very recently emerged as an important pathogen, research is being hampered by a lack of reliable molecular tools. Here we report an infectious cDNA (icDNA) clone for ZIKV isolate BeH819015 from Brazil, which was selected as representative of South American ZIKV isolated at early stages of the outbreak. icDNA clones were assembled from synthetic DNA fragments corresponding to the consensus sequence of the BeH819015 isolate. Virus rescued from the icDNA clone had properties identical to a natural ZIKV isolate from South America. Variants of the clone-derived virus, expressing nanoluciferase, enhanced green fluorescent or mCherry marker proteins in both mammalian and insect cells and being genetically stable for multiple in vitro passages, were obtained. A ZIKV subgenomic replicon, lacking a prM- and E glycoprotein encoding region and expressing a Gaussia luciferase marker, was constructed and shown to replicate both in mammalian and insect cells. In the presence of the Semliki Forest virus replicon, expressing ZIKV structural proteins, the ZIKV replicon was packaged into virus-replicon particles. Efficient reverse genetic systems, genetically stable marker viruses and packaged replicons offer significant improvements for biological studies of ZIKV infection and disease, as well as for the development of antiviral approaches.

Antiviral Activity of Benzavir-2 against Emerging Flaviviruses

“…In this recombinant infectious virus, the ZsGreen gene was fused to the sequence encoding foot and mouth disease virus (FMDV) 2A autoprotease and cloned in-between two copies of the sequence encoding ZIKV capsid protein ( Figure 1B). The infectivity of the rZIKV-ZsGreen was similar compared to wild-type ZIKV, as previously reported [21].…”

“…The recombinant ZIKV with ZsGreen-insertion (rZIKV-ZsGreen, Brazilian strain isolate) was a valuable screening tool. The infection was easily quantified and the infectivity of the recombinant virus was very similar to wt ZIKV [21]. We used this recombinant virus for primary assessment of the anti-ZIKV activity of benzavir-2.…”

“…Plasmids containing a full-length infectious clone (icDNA) of the Brazilian ZIKV isolate, containing the ZsGreen reporter (pCCI-SP6-ZIKV-ZsGreen) were kindly provided by Dr. A. Merits (Tartu University, Tartu, Estonia), and the strategy previously described for ZIKV icDNA rescue was used [21]. Briefly, 5 µg of pCCI-SP6-ZIKV-ZsGreen were linearized using AgeI restriction enzyme prior to in vitro transcription.…”

Basic insights into Zika virus infection of neuroglial and brain endothelial cells

“…While MR766 does not represent the natural African lineage due to it being highly adapted, it nevertheless has been used in many other studies, thus allowing a good comparison with those studies. (ii) BeH89015, an Asian genotype, derived from an infectious clone based on the sequence of the Brazilian strain [24]. (iii) PRVABC59, an Asian genotype, is a human isolate from a Puerto Rico patient [25] and was kindly provided by Dr Jill Carr, Flinders University, Australia.…”

ISG15/USP18/STAT2 is a molecular hub regulating autocrine IFN I-mediated control of Dengue and Zika virus replication

“…Pmax TM GFP expression vector was acquired from Lonza (cat numb #D-00061). Expression plasmid for ZIKV NS5 was generated by amplifying the NS5 coding sequence (amino acids 2521-3423 in the polyprotein) from a previously described plasmid-based ZIKV reverse genetic system (Mutso et al, 2017) buffer. Immunoprecipitates were subjected to SDS-PAGE and western blotting, as described above.…”