Stephanie Carlson and Dr. Bradford Berges, Molecular and Microbiology
While working on this project, we had several unforeseen setbacks. Human herpesvirus 6 (HHV- 6) proved much more difficult to work with than we had supposed. It was tricky to propagate (make it replicate) and hard to quantify. It does not behave like other viruses we have worked with and had a few tricks up its sleeve. We were expecting a dormant virus but were surprised to see a lytic strain. The cells we used to propagate HHV-6 kept dying, so we spent time looking for protocols and reading up on what others have done to maintain the virus. I spent several months culturing the virus and working with it, getting familiar with its behavior. We learned to feed the culture fresh cells weekly in order to maintain viral replication. We did eventually optimize propagation and we were able to harvest virus stock to be used for experiments.
I also began setting up the quantitative PCR (qPCR) protocol that we will use to titer (quantify) the virus. I made a clone of an HHV-6 gene (U65) that is common to both A and B strains and transformed the clone into bacteria. Once the bacteria grew, I re-isolated the plasmid from several bacterial colonies via DNA extraction and prepared serial dilutions in order to run qPCR. The serial dilutions provide decreasing concentration of the DNA to be tested, and the qPCR machine can set up standard curves that are used to figure out how much DNA is in an unknown sample. I have run the prepared dilutions several times, but have not as yet optimized the standard curves. We did try one unknown sample, but it came out relatively high, which is very unlikely for HHV-6, which generally produces an extremely low titer. This low titer presents another issue with this particular virus. We developed several ways and methods to prevent this from becoming a major setback.
Another way we chose to solve the quantification problem was to secure a green fluorescent protein (GFP) positive HHV-6 strain. We have begun propagating that viral strain and will be able to count how many cells fluoresce. From a smaller area, we can calculate how many cells per milliliter are infected, or we can run them through the flow cytometer. We will use the GFP virus to run several other infection experiments, including an ex vivo experiment. In the ex vivo procedure, we will remove cells from a humanized mouse and infect them with HHV-6 (GFP positive). We can run the cells through the flow cytometer to detect fluorescence. Before we run them we can add in antibodies for specific cell markers and determine which cells are infected. We will check for B cells, T cells, dendritic cells and monocytes; these are common cellular targets of HHV-6.
We also had unforeseen issues with our mice. Due to the nearby construction on the new life sciences building, the Widtsoe building often shakes. This disturbed our mice and they did not breed well. We hardly had any new pups born. We were able to set aside several mice specifically for this HHV-6 project, but we have to wait until we are sure they are engrafted, which takes 8-12 weeks after they are born. We saved the CD34 depleted lymphocytes from the cord blood samples we used to humanize those mice. Now we can infect the lymphocytes with HHV-6 and inject them into the mice when we are ready to do the in vivo infections. This will give us a higher titer of the virus than if we simply injected pure virus. Another advantage to using these “matched” lymphocytes is that our mice will not reject them because the cells are their own.
We worked on a pilot grant to the HHV-6 Foundation after a conference call with Dr. Ablashi, the Scientific Director of the Foundation. I ran a flow cytometry experiment to see if humanized mice express CD46, a major target receptor of HHV-6 in humans. It is a relatively ubiquitous marker, expressed on many cell types—especially on white blood cells. We found that humanized mice do express CD46 (see Fig. 1), and unengrafted mice do not, making humanized mice a prime animal model for HHV-6. We submitted our grant to the HHV-6 Foundation and received funding to expand our experiments. With these added tests to confirm HHV-6 infection in humanized mice, we anticipate a published, peer-reviewed paper upon completion of this project.
This coming year, we expect to carry out the in vivo infections (injecting humanized mice with HHV-6 infected cells) and weekly tail bleeds to monitor viral load and infection progress. We also expect to carry out the ex vivo experiment I mentioned previously, which will provide in depth information on infected cell types. We hope to perform RNA in situ hybridization on several lymphoid organs of the mice as well, which will tell us where the virus is harbored in the body. Now that we have worked out the major kinks of this project, we should be able to proceed smoothly. We have viral stocks, ready to be titered and used to infect humanized mice. We also have mice engrafted and set aside. We are quite confident that we will be able to establish humanized mice as animal models for HHV-6 infection. These findings will give us publishable results and will lead to further research on mechanisms of disease and potential vaccine development.