Propagation of Homalodisca coagulata virus-01 via Homalodisca vitripennis Cell Culture

The overall goal of the following experiment is to determine if Homalodisca coagulata virus one or HOCV-one can be replicated using Homalodisca vitripennis cell cultures and then extracted out for further downstream uses. This is achieved by successfully cultivating H. vitripennis to introduce purified HOCV-one into for replication. As a second step, the medium and cells containing virus are removed from culture, which are then subjected to virus and RNA extractions.

Next extracted RNA from the infected cultures is subjected to PCR in order to determine if viral RNA is present in the cells. The results show that HOCV one can be replicated in vivo based on live cell counts before and after infection and qRT-PCR analysis.

The main advantage of this technique over existing methods like rearing an infecting live insects is that it's more economical and easily managed with minimal facilities and equipments. The use of this method can help answer key questions in the field of biological control. For instance, can an insect virus or a virus that's specific for a species of insect be used to control a pest on a grand scale?

Though this method can provide insight into the management of this specific insect, it can also be applied to other systems such as the management of red imported fire ants using a virus of the same family as HOCV one. Prior to starting this procedure, cultivate and maintain cultures in 25 square centimeter tissue culture flasks using H2G plus leaf hopper medium, a modified WH-two honeybee media. Homogenize whole bodies of virus positive H. vitripennis in phosphate buffer with 0.02% DETCA by vortexing for approximately ten second intervals until there are no more large clumps of tissue present. Extract virus via super-speed centrifugation at 22, 000 g for 16 hours at four degrees Celsius.

After discarding the supernatant, collect the pellet and dissolve it with five milliliters of 10 millimolar phosphate buffer containing 0.4% sodium deoxycholic acid, and 4% polyethylene glycol hexa-desal ether. To mix the pellet well remove the pellet from the side of the tube and crush it if necessary. Following centrifugation at 300 g for 15 minutes. Remove the supernatant, then pass the solution through a 0.45 micrometer filter and collect the filtrate in a large collection tube.

Transfer the filtrate to a dialysis membrane with a molecular weight cutoff of 3.5 kilodaltons using small amounts of 10 millimolar phosphate buffer containing no DETCA if needed. Place the dialysis membrane in a large beaker filled with double distilled water at four degrees Celsius. Change the water every hour for five to six hours until a white precipitate forms in the membrane. At this point, seed cells in 48 Well culture plates.

Once the cell growth is 80%confluent, inoculate each row of cells with the serially diluted virus. Then add 10 microliters of double distilled water to each well as a volume control. In order to establish a starting baseline cell concentration for comparison with experimental cell counts, dissociate the first well of each row prior to the initial inoculation of any wells in the cultures with virus. Then count the cells.

Next, remove all medium from the column that was imaged at each 24 hour time point and store it at negative 20 degrees Celsius for RNA extraction and viral quantification. After extracting virus from the culture cells following the previously described protocol extract RNA from medium samples collected during each one week virus trial using a guanidinium thiocyanate phenol chloroform extraction designed for liquid samples per the manufacturer's protocol. For RT PCR establish viral standards by running traditional PCR using the HOCV RT PCR primer-one primer pair with virus isolate from whole body H. vitripennis

Following this, perform a tenfold serial dilution series of the purified sample ranging from 57 nanograms per microliter to 57 attograms per microliter. Determine detection limits of the dilution series by qRT-PCR using a qRT PCR kit with reliable quantification of low abundance transcripts.

After extracting RNA from the experimental samples as described previously, quantify using spectrophotometry. Next perform qRT-PCR on all samples in duplicates as 25 microliter reactions using a one step qRT-PCR kit with the ability to sense low copy numbers as follows. Make the master mix so that each reaction mixture contains 12.5 microliters of one x master mix, 1.0 microliters of 0.3 micromolar forward primer, 1.0 microliters of 0.3 micromolar reverse primer, 0.25 microliters of reverse transcriptase and variable amounts of template based on standardization values. Then bring the total reaction volume to 25 microliters with RNAs free water. Include five standard concentrations in each PCR run with five times 10 to the negative, 10, five times 10 to the negative eight, five times 10 to the negative six, five times 10 to the negative four and five times 10 to the negative two copies. Set the threshold for each run to just below a fluorescence of 10 x to the negative 2.5 to reduce noise during early acquisition at the beginning of each run.

Once Homalodisca vitripennis cells have been grown in a 12 well plate containing glass cover slips inoculate one column every 24 hours for four days, once a monolayer is achieved with the extracted virus. When finished, fix the cells with cold 4%paraformaldehyde at four degrees Celsius for 30 minutes. Then add 500 microliters of 1X PBS to the cells and wash them for 10 minutes at room temperature on a rocker at low speed. After washing the cells three times, add 500 microliters of 0.1%TritonX-100 to permeabilize the cells for 10 minutes.

Once the cells have been washed with 1X PBS, add 500 microliters of a 5%bovine serum albumin solution to block the cells at room temperature. After two hours, remove the solution from the cells. Then add 250 microliters of a diluted stock rhodamine-red conjugated Phalloidin solution to each well to stain for f-actin. Cover the plate in aluminum foil to prevent the dye from bleaching and incubate cells at four degrees Celsius overnight. Following incubation, remove the rhodamine solution and replace it with 250 microliters of DAPI diluted in 1X PBS containing 5%BSA to stain the nuclei of the cells. After incubating the cells at room temperature for one hour, wash them three times with 1X PBS as previously described.

Finally, gently remove the cover slips from the wells and mount to microscope slides using a mounting media with an Antifade reagent.

Cell attachment and fibroblast development were observed within 48 hours of passage in small and large culture flasks. When newly seeded flasks were disturbed, there was a decline in cell attachment leading to slower growing cultures and sometimes no attachment or growth.

Cells were approximately 80%confluent within one week and formed a monolayer in 10 to 14 days. Infected cultures showed signs of declining cell shape integrity and what appeared to be large holes were present on the cell surfaces at 48 hours post infection. Cells shrunk and detached from the culture surface approximately 72 to 96 hours after being infected with non-diluted HOCV-one. Mean live cell counts were plotted to depict differences in live cell abundance between viral loads over time. The higher viral treatment groups indicate a much more marked decline in culture health with a major drop in live cells between 48 to 72 hours. While the lower viral groups slowly declined until dropping off around 144 hours. Survival probability of cells inoculated with higher viral treatments was lower as tested by Kaplan Meyer curves, correlating to the mean live cell count analyses. While not significant, the data suggests that cells exposed to virus are 88%more likely to exhibit lower survival rates over time. The decline of nuclei present as well as the misshapen appearance of f-actin in the cells exposed to HOCV-one indicate a major impact on culture.

Health cells exposed to the higher one to 10 viral load showed greater distress. While attempting this procedure, it is important to remember to complete each step in the protocol in a timely manner, as some steps are time and temperature sensitive. Following this procedure, other methods like ELISA can be used to answer key questions, for instance, how much virus is present in a cell culture after varying amounts of virus are inoculated into that cell culture? After watching this video, you should have a good understanding of how to use an insect cell line to replicate an RNA virus, extract that virus out and then quantify it using qRT-PCR.