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, 9 (1), 9951

Human Schwann Cells Are Susceptible to Infection With Zika and Yellow Fever Viruses, but Not Dengue Virus

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Human Schwann Cells Are Susceptible to Infection With Zika and Yellow Fever Viruses, but Not Dengue Virus

Gaurav Dhiman et al. Sci Rep.

Abstract

Zika virus (ZIKV) is a re-emerged flavivirus transmitted by Aedes spp mosquitoes that has caused outbreaks of fever and rash on islands in the Pacific and in the Americas. These outbreaks have been associated with neurologic complications that include congenital abnormalities and Guillain-Barré syndrome (GBS). The pathogenesis of ZIKV-associated GBS, a potentially life-threatening peripheral nerve disease, remains unclear. Because Schwann cells (SCs) play a central role in peripheral nerve function and can be the target for damage in GBS, we characterized the interactions of ZIKV isolates from Africa, Asia and Brazil with human SCs in comparison with the related mosquito-transmitted flaviviruses yellow fever virus 17D (YFV) and dengue virus type 2 (DENV2). SCs supported sustained replication of ZIKV and YFV, but not DENV. ZIKV infection induced increased SC expression of IL-6, interferon (IFN)β1, IFN-λ, IFIT-1, TNFα and IL-23A mRNAs as well as IFN-λ receptors and negative regulators of IFN signaling. SCs expressed baseline mRNAs for multiple potential flavivirus receptors and levels did not change after ZIKV infection. SCs did not express detectable levels of cell surface Fcγ receptors. This study demonstrates the susceptibility and biological responses of SCs to ZIKV infection of potential importance for the pathogenesis of ZIKV-associated GBS.

Conflict of interest statement

G.D. and R.A. declare no competing interests. D.E.G. is a member of the Takeda Zika virus vaccine data monitoring committee.

Figures

Figure 1
Figure 1
- Replication of ZIKV strains 1968 Nigeria (IBH 30656), 2014 Thailand (SCV0127/14) and 2015 Brazil (Fortaleza), YFV (17D) and DENV2 (NGC) in immortalized human Schwann cells. (A) hSC cells were infected with strains of ZIKV from Africa (Nigeria), Asia (Thailand) and Brazil (Fortaleza) (MOI = 5). Virus production was measured by plaque formation in Vero cells. Each value represents the average +/− standard deviation from three independent experiments. *P < 0.05 (Fortaleza vs Nigeria/Thailand). (B) hSC cells were infected with DENV2 and YFV (MOI = 5). Virus production was measured by focus formation in Vero cells. Each value represents the average +/− standard deviation from three independent experiments ****P < 0.0001 (DENV vs YFV). (C) Immunofluorescence images of mock, ZIKV Fortaleza, ZIKV Nigeria, ZIKV Thailand, DENV, and YFV infected hSCs at 24-96 h after infection (MOI = 5). Cells were stained with pan flavivirus 4G2 antibody followed by anti mouse Alexafluor594 (red). Nuclei were stained with DAPI (blue). The insets show a higher magnification of infected cells (400X). (D) The number of infected cells (red) as a percentage of total cells remaining in the culture (blue). Each value represents the average +/− standard deviation from three different fields. *P < 0.05 ****P < 0.0001 DENV vs Fortaleza/YFV, ####P < 0.0001 Fortaleza vs Nigeria/Thailand/DENV/YFV (E) hSC viability after infection with three strains of ZIKV, YFV and DENV (MOI = 0.1) as determined by MTT assay. Readings taken at 570 nm were plotted as a percentage of the value for mock-infected cells. Each value represents the average +/− standard deviation from four independent infections. ****P < 0.0001 (DENV vs Fortaleza/Nigeria/Thailand/YFV) (F) hSC viability after infection with three strains of ZIKV, YFV and DENV (MOI = 5) as determined by trypan blue exclusion. Each value represents the average +/− standard deviation from three independent experiments of the numbers of viable cells compared to d0 expressed as a percentage. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (DENV vs Fortaleza/Nigeria/Thailand/YFV), #P < 0.05, ##P < 0.01, ###P < 0.001 (Fortaleza vs Nigeria/YFV).
Figure 2
Figure 2
Expression of immune response protein mRNAs and proteins induced by ZIKV and YFV infection of human Schwann cells. (A) hSC cells were infected with ZIKV Fortaleza and YFV (17D) (MOI = 5) and mRNA expression was measured by qRT-PCR. ZIKV-infected cells (red) and YFV-infected cells (black) were compared to mock-infected cells (blue). CT values were normalized to Gapdh and fold change was calculated relative to uninfected 0-hour (ΔΔCT) data. Each value represents the average +/− SD from three independent experiments *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (mock vs infected). (B,C) hSC cells were infected with ZIKV (Fortaleza and Nigeria) and YFV (17D) (MOI = 5) and supernatant fluids were collected. Levels of IFNβ1 (B) and IFNλ1/IL-29 (C) protein were measured by EIA and optical density was plotted. Data are presented as the mean ± SD for three independent infections. *P < 0.05, ***P < 0.001, ****P < 0.0001(Fortaleza vs YFV/Nigeria).
Figure 3
Figure 3
Expression of Fcγ before and after ZIKV infection of human Schwann cells. (A) hSC cells were infected with ZIKV Fortaleza at an MOI of 5 and Fcγ receptor mRNA was measured by qRT-PCR. CT values were normalized to Gapdh, and fold change was calculated relative to uninfected 0-hour (ΔΔCT) data. Each value represents the mean +/− SD from three independent experiments. (B) hSC cells were stained with Fcγ receptor-specific antibodies and analyzed by flow cytometry. The blue line in the histogram corresponds to specific staining with the receptor antibody while the red line corresponds to staining with the isotype control antibody. Data are representative of 3 independent sets of experiments.
Figure 4
Figure 4
Expression of potential flavivirus receptors. hSC cells were infected with ZIKV (Fortaleza) at an MOI of 5 and levels of cellular receptor mRNA were measured by qRT-PCR. Mock-infected cells (blue) were compared to ZIKV-infected cells (red). CT values were normalized to Gapdh, and fold change was calculated relative to uninfected 0-hour (ΔΔCT) data. Each value represents the average +/− SD from three independent experiments **P < 0.01, ***P < 0.001, ****P < 0.0001 (mock vs infected).

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