Abstract
Zika virus (ZIKV) is transmitted by Aedes mosquitoes and exhibits genetic variation with African and Asian lineages. ZIKV Natal RGN strain, an Asian-lineage virus, has been identified in brain tissues from fetal autopsy cases with microcephaly and is suggested to be a neurotropic variant. However, ZIKV Natal RGN strain has not been isolated; its biological features are not yet illustrated. This study rescued and characterized recombinant, single-round infectious particles (SRIPs) of the ZIKV Natal RGN strain using reverse genetic and synthetic biology techniques. The DNA-launched replicon of ZIKV Natal RGN was constructed and contains the EGFP reporter, lacks prM-E genes, and replicates under CMV promoter control. The peak in the ZIKV Natal RGN SRIP titer reached 6.25 × 106 TCID50/mL in the supernatant of prM-E-expressing packaging cells 72 h post-transfection with a ZIKV Natal RGN replicon. The infectivity of ZIKV Natal RGN SRIPs has been demonstrated to correlate with the green florescence intensity of the EGFP reporter, the SRIP-induced cytopathic effect, and ZIKV’s non-structural protein expression. Moreover, ZIKV Natal RGN SRIPs effectively self-replicated in rhabdomyosarcoma/muscle, glioblastoma/astrocytoma, and retinal pigmented epithelial cells, displaying unique cell susceptibility with differential attachment activity. Therefore, the recombinant ZIKV Natal RGN strain was rescued as SRIPs that could be used to elucidate the biological features of a neurotropic strain regarding cell tropism and pathogenic components, apply for antiviral agent screening, and develop vaccine candidates.
Highlights
Zika virus (ZIKV) was first isolated in monkeys in Uganda in 1947, and detected in Aedes africanus mosquitoes in 1948
ZIKV Natal RGN strain using reverse genetics and synthetic biology techniques based on the published genome sequence (GenBank accession number KU527068)
The synthetic DNA segments were used as templates via PCR to amplify the gene fragments (A–D) that were cloned into the low-copy-number plasmid pBR322 to construct a DNA-launched ZIKV Natal RGN replicon under the control of the CMV promoter (Figure 1B,C)
Summary
Zika virus (ZIKV) was first isolated in monkeys in Uganda in 1947, and detected in Aedes africanus mosquitoes in 1948. ZIKV belongs to a mosquito-borne flavivirus of the family Flavivirus, which spread from Africa to south-eastern Asia through transmission by Aedes mosquitoes, such as A. ZIKV exhibits genetic variation that is classified into African and Asian lineages [1]. The ZIKV Asian-lineage strains spread from south-east Asia toward the Pacific, leading to three large outbreaks on Yap island (Micronesia) in 2007, French Polynesia in 2013–2014, in Brazil in 2015–2016, and across the Americas in 2016 [3].
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