Zika virus-infected astrocytoma cells stained with DAPI (blue), antibody to AXL (red), and antibody to flavivirus E glycoprotein (green). Photograph by Amy Rosenfeld.
Our work on Zika virus has utilized two different antibodies to detect Zika virus replication within cells. I described one antibody, directed against double-stranded RNA (dsRNA), in a previous article. A second antibody directed against a Zika virus protein has also been useful.
When we first began working on Zika virus, Carolyn Coyne graciously sent us not only antibody against dsRNA, but also a monoclonal antibody called 4G2. This antibody was produced by immunizing mice with dengue virus type 2. The antibody 4G2 reacts with the E glycoprotein of not only dengue virus type 2 but also other flaviviruses, including Zika virus. The binding site of the antibody is the fusion loop, revealed by structural studies. You can learn more about this antibody and how it binds Zika virus on Virus Watch (embedded below).
We are using antibody 4G2 to show the locations of the Zika virus E glycoprotein in infected cells. It will detect the E glycoprotein either unassembled, or as part of immature or mature virus particles. The results obtained by staining infected cells with this antibody are therefore different from those obtained by staining infected cells with an antibody to dsRNA.
The photograph shown in this article was taken of Zika virus-infected astrocytoma cells—a cell line prepared from a human brain tumor. The infected cells (about 96 hours postinfection) were stained with three different reagents. The first, DAPI (4',6-diamidino-2-phenylindole) is a fluorescent stain that binds to AT-rich regions in DNA and is used to identify the cell nucleus (blue in the image). The infected cells were also stained with an antibody against the cell protein AXL, which is believed to be one of the cell entry receptors for Zika virus (red in the image). Finally, the infected cells were stained with monoclonal antibody 4G2, which detects Zika virus E glycoprotein (green in the image). The image shows distinct foci of Zika virus E glycoprotein several of the infected cells, especially in those cells at the top of the image.
As with many other Zika virus-related reagents, monoclonal antibody 4G2 is currently out of stock at the supplier. Therefore we have purchased the hybridoma cell line that produces the antibody so that we can make our own stocks. I’ll describe more about that process next time.
(Updated 7/21/16 to indicate that the photograph was taken 96 hours post-infection, not 8 hours).
Vincent Racaniello is a virologist at Columbia University and science communicator. He is using Zika Diaries to communicate the personal and behind the scenes experiences of his laboratory as it moves from working on poliovirus (for 35 years) to Zika virus.