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01.12.2017 | Research article | Ausgabe 1/2017 Open Access

BMC Infectious Diseases 1/2017

Determination of the Cell Permissiveness Spectrum, Mode of RNA Replication, and RNA-Protein Interaction of Zika Virus

Zeitschrift:
BMC Infectious Diseases > Ausgabe 1/2017
Autoren:
Wangheng Hou, Najealicka Armstrong, Lilian Akello Obwolo, Michael Thomas, Xiaowu Pang, Kevin S. Jones, Qiyi Tang
Wichtige Hinweise

Electronic supplementary material

The online version of this article (doi:10.​1186/​s12879-017-2338-4) contains supplementary material, which is available to authorized users.

Abstract

Background

Two lineages of Zika virus (ZIKV) have been classified according to the phylogenetic analysis: African and Asian lineages. It is unclear whether differences exist between the two strains in host cell permissiveness, this information is important for understanding viral pathogenesis and designing anti-viral strategies.

Methods

In the present study, we comparatively studied the permissive spectrum of human cells for both the African (MR766) and Asian strains (PRVABC59) using an RNA in situ hybridization (RISH) to visualize RNA replication, an immunofluorescence technology, and a western blot assay to determine viral protein production, and a real-time RT-PCR to examine viral RNA multiplication level. The experiments were undertaken in the condition of cell culture.

Results

We identified several human cell lines, including fibroblast, epithelial cells, brain cells, stem cells, and blood cells that are susceptible for the infection of both Asian and African strains. We did not find any differences between the MR766 and the PRVABC59 in the permissiveness, infection rate, and replication modes. Inconsistent to a previous report (Hamel et al. JVI 89:8880–8896, 2015), using RISH or real-time RT-PCR, we found that human foreskin fibroblast cells were not permissive for ZIKV infection. Instead, human lung fibroblast cells (MRC-5) were fully permissive for ZIKV infection. Surprisingly, a direct interaction of ZIKV RNA with envelop (E) protein (a structure protein) was demonstrated by an RNA chromatin immunoprecipitation (ChIP) assay. Three binding sites were identified in the ZIKV RNA genome for the interaction with the E protein.

Conclusion

Our results imply that the E protein may be important for viral RNA replication, and provide not only the information of ZIKV permissiveness that guides the usage of human cells for the ZIKV studies, but also the insight into the viral RNA-E protein interaction that may be targeted for intervention by designing small molecule drugs.
Zusatzmaterial
Additional file 1: Figure S1. Immunofluorescent assay and RISH to show ZIKV viral RNA replication and protein production in Vero cells. Vero cells were infected with MR766 (A1-D1, A3-D3) or PRVABC59 (PR) (A2-D2, A4-D4) at an MOI of 0.5 for 24 h. After fixation with 1% formaldehyde, the cells were permeabilized and immunostained for viral protein in red (B1-B4). After refixation with 4% formaldehyde, the cells were hybridized with single strand DNA probe (labeled with biotin) made from ZIKV genomic DNA. The RNA was stained in green (A1-A4). The cell nuclei were shown in blue using DAPI staining (C1-C4). The merged image was shown in D1-D4. In the panels 1 and 2, the pictures were taken using a 40× lens. In the panels 3 and 4, the pictures were taken using an 100× lens. Scale bar: 10 μm. (PSD 5413 kb)
12879_2017_2338_MOESM1_ESM.psd
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