Here, we present the protocols to identify 1) virus-encoded immunomodulators that promote arbovirus replication and 2) eukaryotic host factors that restrict arbovirus replication. These fluorescence- and luminescence-based methods allow researchers to rapidly obtain quantitative readouts of arbovirus replication in simplistic assays with low signal-to-noise ratios.
This method can help answer key questions in the field of viral immunology, such as, which host factors restrict arbovirus replication, and, in turn, which virus-encoded immune evasion proteins overcome these host restriction mechanisms. The main advantage of this technique is that it allows one to use simplistic luminescence and fluorescent space assays to define cellular conditions that allow for arboviruses to replicate in an otherwise abortive infection in lepidopteran insect cells. Because there's minimal background replication of arboviruses in lepidopteran cells, it becomes relatively straightforward for one to detect conditions that allow for arbovirus replication.
To begin, maintain LD652 cells in 10 centimeter tissue culture-treated dishes, and passage the cells when they reach 80%confluency. Pipette media onto the cell monolayer repeatedly to dislodge the cells, then dilute the sample in growth media to an approximate density of 100 thousand cells per milliliter. After this, transfer one milliliter of the diluted culture to each well of a 24-well plate.
Then, allow the cells to settle for at least one hour. 30 minutes prior to the infection, thaw stalks of VSV luciferase and vaccinia virus on ice. Then, dilute VSV luciferase with and without vaccinia virus into SFM, such that an MOI of 10 and 25, respectively, is achieved.
Aspirate mature LD652 media carefully, to avoid disturbing the cell monolayer, and inoculate each well with 0.2 milliliters of virus. Next, add sterile SFM to additional wells to serve as mock-infected negative controls. Incubate the cells in inoculum for two hours at 27 degrees Celsius.
Then, remove the inoculum via aspiration and replace it with one millimeter of LD652 growth media in each well. After this, allow the infection to proceed for 24 to 72 more hours. First, carefully aspirate the supernatant and add one milliliter of DPBS to each well.
Using a syringe plunger, scrape the cells into DPBS. Then, transfer the cells to a micro-centrifuge tube and centrifuge for 20 minutes at 400 times gravity and four degrees Celsius. Meanwhile, dilute five times reporter lysis buffer in sterile water.
After the centrifugation, aspirate the DPBS without disturbing the pellet. Next, re-suspend the pellet in 150 microliters of one times RLB. Then, incubate the sample at negative 80 degrees Celsius, followed by a rapid thaw in a room-temperature water bath.
After this, store the lysates at negative 80 degrees Celsius for further analysis. Thaw the lysates on ice and pipette 20 microliters of lysate into the wells of a 96-well plate. Next, add 100 microliters of luciferase assay region to each well of the plate.
Immediately, measure the light intensity using a lumenometer. Under single-infection conditions, LD652 cells restrict VSV luciferase replication. Thus, only a small luciferase signal is observed.
Co-infection, however, results in significantly higher luciferase signals. Under single-infection conditions, LD652 cells also restrict VSV dsRED replication, thus only a small number of cells exhibit a dsRED signal. Co-infection with vaccinia virus, however, results in most cells displaying dsRED signals by the end of the time course.
Approximately 2%of the cells from the single infections were dsRED positive by 65 hours post-infection. In contrast, approximately 77%of the co-infected cells were dsRED positive at the same time point. When attempting this procedure, it's important to remember to optimize the time points at which luciferase assays or live-cell imaging time points will be taken in order to allow enough time for VSV to replicate and produce a detectable signal.
Following this procedure, other methods such as RNA interference-mediated knockdown of host factors, can be conducted in order to identify potential host antiviral factors restricting arbovirus replication. After its development, this technique paved the way for researchers in the field of viral immunology to define the role of pox virus-encoded A51R proteins in promoting pox virus replication and pathogenesis. Don't forget that working with eukaryotic viruses can be extremely hazardous and it is important to take proper precautions, such as wearing personal protective equipment while performing this procedure.
