Differentiation enhances Zika virus infection of neuronal brain cells

Abstract

Zika virus (ZIKV) is an emerging, mosquito-borne pathogen associated with a widespread 2015-2016 epidemic in the Western Hemisphere and a proven cause of microcephaly and other fetal brain defects in infants born to infected mothers. ZIKV infections have been also linked to other neurological illnesses in infected adults and children, including Guillain-Barré syndrome (GBS), acute flaccid paralysis (AFP) and meningoencephalitis, but the viral pathophysiology behind those conditions remains poorly understood. Here we investigated ZIKV infectivity in neuroblastoma SH-SY5Y cells, both undifferentiated and following differentiation with retinoic acid. We found that multiple ZIKV strains, representing both the prototype African and contemporary Asian epidemic lineages, were able to replicate in SH-SY5Y cells. Differentiation with resultant expression of mature neuron markers increased infectivity in these cells, and the extent of infectivity correlated with degree of differentiation. New viral particles in infected cells were visualized by electron microscopy and found to be primarily situated inside vesicles; overt damage to the Golgi apparatus was also observed. Enhanced ZIKV infectivity in a neural cell line following differentiation may contribute to viral neuropathogenesis in the developing or mature central nervous system.

Figure 1

ZIKV infection of SH-SY5Y neuroblastoma cells. (A) Infection protocol for undifferentiated cells (top) and cells after 2 days of differentiation with retinoic acid (bottom). Syringe icons denote the day of infection with ZIKV. After one hour of incubation, virus was washed twice and collection of time 0 samples was performed. (B) Immunofluorescent staining of SH-SY5Y or Vero cells infected with ZIKV-UG (left) or ZIKV-PR (right) at a multiplicity of infection (MOI) of 1. The 3 columns within each set of 9 panels correspond to staining with a monoclonal antibody against the flaviviral envelope protein (“anti-Env”, staining red, column 1), DAPI stain for cell nuclei (“DAPI”, staining dark blue, column 2), and the merged images (“merged”, column 3). Scale bars represent 50 µm. Abbreviations: IFA, immunofluorescence assay; qRT-PCR, quantitative reverse transcription-polymerase chain reaction; hpi, hours post-infection; DAPI, 4′6-diamidino-2-phenylindole. The images shown are representative images taken from 3 independent experiments, with observation of a minimum of 10 fields under both low (10X) and high (63X) magnification per experiment.

Figure 2

ZIKV infection of SH-SY5Y neuroblastoma cells following longer periods of retinoic acid-induced differentiation. (A) Infection protocol for undifferentiated cells (top) and cells after 2 to 8 days of differentiation with retinoic acid (bottom). Syringe icons denote the day of infection with ZIKV-UG. After one hour of incubation, virus was washed twice and collection of time 0 samples was performed. Infection was monitored after 48 hours by IFA, plaque assay and qRT-PCR. (B) Plot of viral titers as determined by plaque assay versus days of differentiation prior to ZIKV infection. (C) Plot of viral titers as determined by qRT-PCR at different days of differentiation. Gray bars represent control data from 0 hours post infection (hpi), and black bars represent data from 48 hpi. Titers and error bars are calculated from the average and standard deviation of three independent experiments. *p < 0.05 by analysis of variance (ANOVA); **p < 0.05 by one-tailed t-test; ***p = 0.07 by one-tailed t-test. Abbreviations: IFA, immunofluorescence assay; qRT-PCR, quantitative reverse transcription-polymerase chain reaction; hpi, hours post-infection; PFU, plaque-forming units.

Figure 3

Transmission electron microscopy of uninfected and ZIKV-infected differentiated SH-SY5Y cells. (A) Uninfected control. Arrowheads (black) show typical neurotransmitter vesicles. (B–D) Differentiated SH-SY5Y cells at 48 hours post-infection with ZIKV-UG. Arrows (black) point to representative viral particles. G, intact Golgi complex; G*, disrupted Golgi complex; MM, multi-membranous areas. Cells are treated with 2 days of retinoic acid prior to ZIKV infection. The scale bar represents 0.5 µm.

Figure 4

Infection of differentiated SH-SY5Y cells with ZIKV strains representing African and multiple Asian lineages. Viral infection is monitored by immunofluorescent staining of differentiated SH-SY5Y cells at 48 hours post-infection using the anti-Env monoclonal antibody (green). Cells are treated with 2 days of retinoic acid prior to ZIKV infection. The images shown are representative images taken from 3 independent experiments, with observation of a minimum of 10 fields under both low (10X) and high (40X and 63X) magnification per experiment. The scale bar represents 50 µm.