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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.

SUBMITTER: Lu CY

PROVIDER: S-EPMC6893733 | biostudies-literature | 2019 Oct

REPOSITORIES: biostudies-literature

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

Lu Chien-Yi CY Lin Chen-Sheng CS Lai Hsueh-Chou HC Yu Ya-Wen YW Liao Chih-Yi CY Su Wen-Chi WC Ko Bo-Han BH Chang Young-Sheng YS Huang Su-Hua SH Lin Cheng-Wen CW

Viruses 20191031 11

Zika virus (ZIKV) is transmitted by <i>Aedes</i> 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 Z ...[more]

PMID: 31683628

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Project description:Birnaviruses are unconventional members of the icosahedral double-stranded (dsRNA) RNA virus group. The main differential birnavirus trait is the lack of the inner icosahedral transcriptional core, a ubiquitous structure conserved in all other icosahedral dsRNA viruses, that shelters the genome from cellular dsRNA sensors and provide the enzymatic machinery to produce and extrude mature messenger RNAs. In contrast, birnaviral particles enclose ribonucleoprotein (RNP) complexes formed by the genome segments, the dsRNA-binding VP3 polypeptide and the virus-encoded RNA polymerase (RdRp). The presence of RNPs suggests that the birnavirus replication program might exhibit significant differences with respect to those of prototypal dsRNA viruses. However, experimental evidences supporting this hypothesis are as yet scarce. Of particular relevance for the understanding of birnavirus replication is to determine whether RNPs act as intracellular capsid-independent transcriptional units. Our study was focused to answer this question using the infectious bursal disease virus (IBDV), the best characterized birnavirus, as model virus. Here, we describe the intracellular assembly of functional IBDV RNPs in the absence of the virus-encoded VP2 capsid polypeptide. Recombinant RNPs are generated upon coexpression of the IBDV VP1 and RdRp polypeptides and transfection of purified virus dsRNA. Presented data show that recombinant RNPs direct the expression of the IBDV polypeptide repertoire and the production of infectious virus in culture cells. Results described in this report constitute the first direct experimental evidence showing that birnaviral RNPs are intracellularly active in the absence of the virus capsid. This finding is consistent with presented data indicating that RNP formation precedes virus assembly in IBDV-infected cells, and supports the recently proposed IBDV replication model entailing the release of RNPs during the initial stages of the infection. Indeed, results presented here also support the previously proposed evolutionary connection between birnaviruses and positive-strand single-stranded RNA viruses.

Dataset Information

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

Publications

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

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