Simple reverse genetics systems for Asian and African Zika viruses

Abstract

Zika virus (ZIKV), a typical example of a re-emerging pathogen, recently caused large outbreaks in Pacific islands and the Americas, associated with congenital diseases and neurological complications. Deciphering the natural history, ecology and pathophysiology of this mosquito-borne pathogen requires effective reverse genetics tools. In the current study, using the bacterium-free 'Infectious Subgenomic Amplicons' (ISA) method, we generated and made available to the scientific community via the non-profit European Virus Archive collection, two simple and performing reverse genetics systems for ZIKV. One is based on an Asian ZIKV strain belonging to the outbreak lineage (French Polynesia 2013). The second was designed from the sequence of a low-passaged ZIKV African strain (Dakar 1984). Using the ISA procedure, we derived wild-type and a variety of specifically engineered ZIKVs in days (intra- and inter-lineage chimeras). Since they are based on low-passaged ZIKV strains, these engineered viruses provide ideal tools to study the effect of genetic changes observed in different evolutionary time-scales of ZIKV as well as pathophysiology of ZIKV infections.

Figure 1. Schematic representation of the ISA method used to recover infectious ZIKVs.

The entire viral genome, schematically represented in the figure, flanked respectively at the 5′ and 3′ untranslated regions by the pCMV and the HDR/SV40pA, was de novo synthesized in three double-stranded overlapping DNA fragments. Each of these was then amplified by PCR and all subgenomic products were pooled and transfected into permissive cells to generate infectious ZIKVs.

Figure 2. Virus replication kinetics with recombinant and parental PF ZIKV strains.

An moi of 0.01 was used to infect BHK-21 cells with recombinant or parental PF ZIKVs produced in BHK-21 cells. Cells were washed and cell supernatant media were harvested at 24, 48, 72 and 96 hours post-infection and then analyzed using a real-time RT-PCR assay.

Figure 3. Rapid generation of chimeric ZIKVs using the ISA method.

By simply exchanging DNA fragments, inter- and intra-lineage chimeric ZIKVs were produced in days using the ISA method. The MART-II DNA fragment was generated by RT-PCR from the cell culture supernatant medium of the MART ZIKV strain.