The Rescue and Characterization of Recombinant, Microcephaly-Associated Zika Viruses as Single-Round Infectious Particles

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.

Keywords: Zika virus; cell susceptibility; replicon; reporter; single-round infectious particle.

Figure 1

Construction of the pBR322-based ZIKV Natal RGN replicon and pcDNA3.1-ZIKV prME. Two synthetic DNA segments in the pUC18 plasmid contained the entire ZIKV Natal RGN strain genome: CMVp, EGFP, FMDV-2A, and BGH-pA sequences (A). Four PCR products (Fragments A–D) were cloned into the indicated restriction sites (EcoRI, NotI, ClaI, RsrII, and XhoI) of the pBR322 plasmid and then assembled as the in-frame fusion of the ZIKV Natal RGN replicon with the EGFP reporter under CMV-promoter control (B). Those four PCR products were analyzed using agarose gel electrophoresis (C). The PCR product of ZKIV prM and E genes was cloned into restriction sites (EcoRI and XhoI) of the pcDNA3.1-His-C plasmid (D).

Figure 2

Analysis of the cytopathic effect (CPE) and EGFP reporter expression in prM-E-expressing packaging cells transfected with the ZIKV Natal RGN replicon. CPE and EGFP reporter expressions in mock cells (top), packaging cells (middle), and replicon-transfected packaging cells (bottom) were photographed using light and fluorescence microscopes 24 (A) and 72 (B) h post-incubation. Scale bar, 100 μm.

Figure 3

Analysis of viral proteins and genome synthesis in packaging cells transfected with the ZIKV Natal RGN replicon. ZIKV E, NS1, and NS5 expressions in mock cells (top), packaging cells (middle), and replicon-transfected packaging cells (bottom) were examined using the indicated primary antibodies and Alexa Fluor 546-conjugated secondary antibodies (A). Finally, cell imaging was conducted via immunofluorescence microscopy. In addition, relative copy numbers of sense (top) and antisense (bottom) genomes in the indicated cells were quantitated 72 h post-transfection using real-time PCR, and were then normalized to GAPDH mRNA (B). ***, p value < 0.001 compared with mock cells. Scale bar, 100 μm.

Figure 4

Antigenicity and quantitative analysis of ZIKV Natal RGN single-round infectious particles (SRIPs). The antigenicity, relative viral genome levels, and titer of SRIPs collected from the supernatant of the packaging cells transfected with the ZIKV Natal RGN replicon were analyzed using dot blot (A), real-time RT-PCR (B), and TCID50 (C) assays, respectively.

Figure 5

Infectivity of ZIKV Natal RGN SRIPs in prM-E-expressing packaging cells. The cells were infected with ZIKV Natal RGN SRIPs at MOIs of 3, 0.3, 0.03, and 0.003. The cytopathic effect (top) and the EGFP reporter (middle) in SRIP-infected cells were photographed using light and fluorescence microscopy 72 h post-infection. In addition, infected cells were washed, fixed, and stained by anti-NS1 antibodies and Alexa Fluor 546-conjugated secondary antibodies (bottom). Finally, cell imaging was taken by immunofluorescence microscopy. Scale bars, 100 μm for CPE and EGFP images (top and middle) and 200 μm for the NS1 image.

Figure 6

Cell susceptibility to ZIKV Natal RGN SRIPs. Three cell lines, ARPE-19, TE671, and SF268, were infected with SRIPs at an MOI of 0.15 TCID50/cell. The cytopathic effect (top), EGFP reporter (middle), and ZIKV NS1 protein (bottom) in SRIP-infected cells were photographed using light and fluorescence microscopy 72 h post-infection (A). In addition, relative viral genome levels from SRIPs attached to the surfaces of these cell lines were measured using real-time RT-PCR mRNA (B). **, p value < 0.01; ***, p value < 0.001 compared with mock cells. Scale bar, 100 μm.