Production, Titration and Imaging of Zika Virus in Mammalian Cells

Abstract

Since the outbreak of Zika virus (ZIKV) in Latin America and the US in 2016, this flavivirus has emerged as a major threat for public health. Indeed, it is now clear that ZIKV is vertically transmitted from the infected mother to the fetus and this may lead to severe neurological development defects including (but not restricted to) neonate microcephaly. Although ZIKV has been identified in the late 1940s, very little was known about its epidemiology, symptoms and molecular biology before its reemergence 60 years later. Recently, tremendous efforts have been made to develop molecular clones and tools as well as cell culture and animal models to better understand ZIKV fundamental biology and pathogenesis and to develop so-far-unavailable antiviral drugs and vaccines. This bio-protocol describes basic experimental procedures to produce ZIKV stocks and to quantify their concentration in infectious virus particles as well as to image and study this pathogen within infected cells using confocal microscopy-based imaging.

Keywords: Double-stranded RNA; Immunofluorescence microscopy; NS4B; Plaque assay; Virus titration; Zika virus.

Figure 1.. Serial dilution plan and plate layout for plaque assays.

Schematic representation of the virus stock 10-fold dilutions and of 24-well plates used for plaque assays. Two samples per plate can be titered. Each dilution is assessed in biological duplicates.

Figure 2.. Typical plaque assay for ZIKV MR766 and H/PF/2013.

Vero E6 cells were infected with 10-fold serial dilutions of ZIKV MR766 or H/PF/2013 stocks (Day 3 harvests). Five days post-infection, cells were fixed and stained with crystal violet. In this specific example, plaques can be counted in the 10^-3 and 10^-4 dilution well of ZIKV MR766 and H/PF/2013 (indicated in red), respectively. According to the formula described above, infectious titers are 1.3 x 10⁵ PFU/ml for MR766 and 1.45 x 10⁴ PFU/ml for H/PF/2013.

Figure 3.. Virus amplification kinetics of ZIKV strains MR 766 and H/PF/2013 in Vero E6 cells.

Vero E6 cells were infected with ZIKV MR766 or H/PF/2013 strains at an MOI of 0.01. Virus supernatants were collected at 3, 4, 5, 6 and 7 days post-infection. Infectious virus titers were determined using plaque assays in Vero E6 cells.

Figure 4.. Imaging of Zika virus infection in Huh7 cells.

Huh7 cells were infected with ZIKV MR766 or H/PF/2013 strains at an MOI of 1 or left uninfected. Forty-eight hours post-infection, cells were fixed, permeabilized and labeled with anti-dsRNA and anti-ZIKV NS4B antibodies. Nuclei were visualized with DAPI staining. Cells were observed with a Zeiss LSM780 confocal microscope. For ZIKV H/PF/2013, an infected cluster is shown. Scale bars: 20 μm.