Detection of Zika Viral dsRNA Replication Intermediates Using Immunocytochemistry

Abstract

Source: Contreras, D., et al. Zika Virus Infectious Cell Culture System and the In Vitro Prophylactic Effect of Interferons. J. Vis. Exp. (2016)

The video demonstrates the detection of Zika viral dsRNA intermediates in infected epithelial cells using immunocytochemistry. Infected cells are fixed and then blocked with a blocking solution. The primary and secondary antibodies are added. The nuclei are stained with a fluorescent dye. The labeled viral dsRNA is then seen under a fluorescence microscope.

Protocol

1. Zika viral genome replication assay

1. Seed naïve Vero cells at 1 x 10⁵ cells per well in 2 ml volumes using a 12-well plate. The following day, prepare viral inocula (Multiplicity of Infection (MOI) of 0.01 and 0.1; 400 µl/well) of low and high titer using serum-free media in triplicate. For mock infection, use serum-free media (400 µl/well) only.
2. Incubate the inoculated plate at 37 °C with 5% CO₂ for 4-6 hr. Spread the inoculum by gently tilting the plate sideways every one hr.
3. At the end of incubation, replace the inoculum with serum-supplemented media (2 ml per well).
4. At the 48 and 96 hr time points, collect the samples for immunocytochemistry (ICC).
5. Perform ICC using the methanol-fixed cells.
   1. Wash the fixed cells three times with 1x phosphate-buffered Saline (PBS) and block with ICC blocking buffer (3% goat serum, 3% Bovine serum albumin (BSA), 0.1% Triton-x 100 in PBS).
   2. Use mouse monoclonal anti-dsRNA antibody J2 (1 µg/ml) at a dilution of 1:100 in blocking buffer and incubate overnight at 4 °C.
   3. Wash the cells with 1x PBS and add a secondary antibody goat anti-mouse IgG-594 (1 µg/ml) at a 1:1,000 dilution in the blocking buffer and incubate for one hour at room temperature.
   4. Wash cells with 1x PBS and stain for nuclei using Hoechst dye and observe the cells using a fluorescent microscope at 100X magnification (Figure 1).

Results

Figure 1: Assays for evaluating Zika virus (ZIKV) replication. (A) The graph shows Zika virus genomic content measured by Reverse Transcription Quantitative Polymerase Chain Reaction (RT-qPCR). Both pan-genotype (ZIKV GEN) and PRVABC59-strain (ZIKV UNI) primers showed equal sensitivity and specificity. As expected, hepatitis C viral (HCV) primers did not amplify any product. (B) The graph presents the Zika viral growth kinetics at the indicated time points in low and high-titer infected cells compared to that of the uninfected mock control. Mean values and standard deviations are given. (C) Immunocytochemistry assay for investigating viral replication. Zika virus-infected cells were specifically stained with dsRNA antibodies. At 48 hpi, infected cells are in a few clusters, whereas at 96 hpi, most of the cells are infected. Hoechst stain was used for the visualization of nuclei (Scale bar = 50 µm). BF: Bright field image.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Dulbecco’s modified Eagle’s medium (DMEM)	Sigma Life Science	D5796
HEPES	Life Technologies	15630080
Glutamax	Life Technologies	35050061
Mouse monoclonal anti-dsRNA antibody J2	English & Scientific Consulting Kft.	10010200
Goat anti-rabbit IgG Alexa Fluor 594	Life Technologies	A11020
Vero Cell Line	ATCC	CCL-81
Zika viral strain PRVABC59	Centers for Disease Control and Prevention (CDC)
Nikon Eclipse Ti Immunofluorescence Microscope with Nikon Intenselight C-HGFI	Nikon	Visit Nikon for Request