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

1. Cell Culture

NOTE: Homalodisca vitripennis cell lines established by the Dr. Wayne Hunter laboratory at the USDA Agricultural Research Service (Ft. Pierce, FL USA) were used to initiate a lab stock composed of mixed cell stages including initial fibroblasts and monolayers.

1. Perform the following procedures in a sterile laboratory environment maintained at a temperature range of 20-24 °C with 25 cm² culture flasks.
2. Cultivate and maintain cultures in 25 cm² tissue culture flasks using H2G+ Leafhopper medium, a modified WH2 honeybee media¹⁰ (Table 1).
3. Incubate culture flasks at 24 °C with ~53% humidity.
4. Use an inverted microscope at a total magnification (TM) of 100X to monitor culture growth, e.g., check for any abnormal cellular growth, monitor for any potential contaminants, and check growth progress across the culture surface.
5. Perform a complete medium change (~4 ml culture medium per flask) every 7 – 10 days without disturbing the culture surface.
6. Pass cultures when the culture surface is approximately 80% covered by cell growth (confluent) using 0.25% trypsin containing ethylenediaminetetraacetic acid (EDTA) to dissociate cells. Add ~2 – 3 ml of trypsin to each culture flask and expose culture(s) to the enzyme for short periods of time (5 – 10 min) to achieve complete cell dissociation.
7. Add an equal amount of fresh medium to the culture flask(s) (~2 – 3 ml) to stop enzyme activity and transfer the whole solution to a conical tube for centrifugation.
8. Pellet cells by centrifugation at 4 °C for 6 min at 350 x g.
9. Remove the supernatant without disturbing the cell pellet and add ~8 ml of fresh medium to each tube. Gently homogenize cells using a pipette.
10. Split cultures at a 1:2 ratio for 25 cm² flasks (~4 ml of cell solution per flask) and leave freshly passed cultures undisturbed for 48 hr to allow cells in suspension to attach securely to the surface of the flask.
    NOTE: Cultivation of cells in 48-well sterile tissue culture plates with a growth surface of 1 cm² is also possible and they can be maintained in the same manner as culture flasks with a reduction in volume of culture medium to ~250 μl.

2. Whole Virus Extraction

1. Homogenize whole bodies of virus positive H. vitripennis in phosphate buffer, pH ~7.2, with 0.02% sodium diethyldithiocarbamate trihydrate (DETCA) by vortexing for ~10 sec intervals until there are no more large clumps of tissue present. Extract virus via superspeed centrifugation at 124,500 x g for 4 hr at 4 °C or 22,000 x g for 16 hr at 4 °C. If a precipitate forms at the top, remove it with a sterile cotton swab¹¹.
2. Discard the supernatant.
3. Collect the pellet and dissolve with 5 ml of 10 mM phosphate buffer (no DETCA), pH ~7.2, containing 0.4% Na-deoxycholic acid and 4% polyethylene glycol hexadecyl ether (Brij 52).
   1. To mix the pellet well, remove the pellet from the side of the tube and crush up until in solution if necessary.
   2. Add more 10 mM phosphate buffer in ~5 ml increments to help the pellet go into solution if needed and combine into 2 tubes¹¹.
4. Centrifuge the solution at 300 x g for 15 min¹¹.
5. Remove the supernatant and pass the solution through a 0.45 μm filter, and collect the filtrate in large collection tube¹¹.
6. Transfer filtrate to a dialysis membrane with a molecular weight cut off (MWCO) of 3.5 kD, using small amounts of 10 mM phosphate buffer (pH 7) containing no DETCA if needed¹¹.
7. Place the dialysis membrane in a large beaker filled with ddH₂O at 4 °C. Change the ddH₂O every hour for 5 – 6 hr, until a white precipitate forms in the membrane¹¹.
8. Collect the purified virus from within the membrane and subject the 100% virus solution to a 10-fold dilution series by adding 10 μl of solution to 90 μl of ddH₂O. Subsequently add 10 μl of the dilution to another 90 μl of ddH₂O until reaching a dilution of 1:100,000.
9. Store virus solution at -80 °C.

3. Viral Replication

1. Seed cells in 48-well culture plates and grow all rows until cell growth is 80% confluent (approximately 72 hr post-pass if cells are growing at an average rate).
2. Inoculate each row of cells with the serial diluted virus once cell growth reaches confluency, i.e., one row of a 1:10 dilution, one row of a 1:100 dilution, etc., except for the top row. Use the top row as a control and add 10 μl of ddH₂O to each well as a volume control.
   1. In order to establish a starting baseline cell concentration for comparison with experimental cell counts, dissociate and count the first well of each row prior to the initial inoculation of any wells in the cultures with virus.
3. Monitor culture plates every 24 hr after viral inoculation for any color change in the medium indicating a pH change and for any changes in cell morphology.
4. Image one column of the test plate at each time point, using an inverted microscope at 100X TM.
5. Remove all medium from the column that was imaged at each 24 hr time point and store it short-term at -20 °C for RNA extraction and viral quantification or long term at -80 °C.
6. Dissociate cells from the wells after removing the medium as previously described in steps 1.6 – 1.9, using only ~250 μl of 0.25% trypsin EDTA and fresh medium to stop enzyme activity and ~250 μl of fresh medium to re-suspend the cell pellet.
7. Add 10 μl of 0.4% trypan blue stain to each tube containing cells to perform cell counts after cell dissociation for each 24 hr period over one week. Allow stain to sit for 10 min before performing cell counts.
   1. Perform cell counts within 1 hr of exposure to stain or viable cells will begin to uptake stain as well as non-viable cells.
8. Slowly add 10 μl of the stained cell solution to each side of a standard hemocytometer using a micropipette. Allow the solution to be taken up by capillary action to avoid air bubbles.
9. Count the number of viable (non-stained) cells on both sides of the hemocytometer (the equivalent of 4 counts of the 16 squares in the hemocytometer). Perform cell counts for each well of the column that was removed.
10. Average the cell counts from each column and use the following formula to obtain the total cell count number: (average number of cells / 4) x dilution factor = number of cells x 10⁴ ml. This is the cell density or number of viable cells in the culture.

4. Virus Extraction from Cell Culture

1. Remove treated H. vitripennis cells from culture flasks as previously described in steps 1.6 – 1.8, using only ~250 μl of 0.25% trypsin EDTA and fresh medium to stop enzyme activity.
2. Discard the supernatant.
3. Extract virus from culture cells following the protocol previously described in steps 2.3 – 2.8.

5. RNA Extraction

1. 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.
2. Store extracted samples at -80 °C.

6. RT-PCR

1. Establish viral standards for RT-PCR by running traditional PCR using the primer pair HoCV RT-PCR primer 1 (forward 5′-GCTCCCCGGCTTTGCTGGTT-3′, reverse 5′-ACGACGGATCTGCGTGCCAA-3′) with virus isolate from whole body H. vitripennis.
2. Subject PCR product to gel electrophoresis for 60 min at 120 V in a 2% agarose gel containing 0.1% ethidium bromide.
3. Excise the bands from the gel and purify using a gel extraction kit as per the manufacturer’s protocol.
4. Pool all excised and gel purified product and further purify by basic ethanol precipitation. Elute the precipitation product in approximately 30 μl of Tris EDTA (TE) to increase the overall sample concentration.
5. Quantify the level of cDNA in pooled samples via spectrophotometry using 260/230 wavelength settings for nucleic acid detection.
6. Perform a ten-fold serial dilution series of the purified sample ranging from 57 ng/μl to 57 ag/μl (10^-18). Perform the serial dilution similar to the one described in 2.8 with adjustments made for the difference in concentration requirements.
7. Determine detection limits of the dilution series by performing qRT-PCR using a qRT-PCR kit with reliable quantification of low-abundance transcripts.
   NOTE: Viral concentrations lower than 5 x 10^-3 copies are not detectable.
8. Extract RNA from experimental samples as described previously in step 5.1 and quantify using spectrophotometry.
9. Normalize all the extracted samples to 5 ng/μl using nuclease free water.
10. Perform qRT-PCR on all samples, in duplicates, as 25 μl reactions using a one-step qRT-PCR kit with the ability to sense low copy numbers as follows: 50 °C hold for 10 min; 95 °C hold for 5 min; 30 cycles of 95 °C for 10 sec, 60 °C for 30 sec; melt from 50 – 99 °C for 5 sec on each step.
    1. Make the master mix so that each reaction mixture contains 12.5 μl of 1x master mix, 1.0 μl (0.3 μM) of forward primer, 1.0 μl (0.3 μM) of reverse primer, 0.25 μl of reverse transcriptase and variable amounts of template based on standardization values.
    2. Bring the total reaction volume to 25 μl with RNase free water.
    3. Include five standard concentrations in each PCR run with the following copy numbers: 5 x 10^-10, 5 x 10^-8, 5 x 10^-6, 5 x 10^-4, and 5 x 10^-2 copies. Set the threshold for each run to just below a fluorescence of 10x^-2.5 to reduce noise during early acquisition at the beginning of each run.

7. Confocal Microscopy

1. Grow Homalodisca vitripennis cells in a twelve well plate containing glass coverslips measuring 18 mm in diameter in each well.
2. Inoculate one column on the plate every 24 hr for a period of four days, once a monolayer is achieved. Ensure that each column contains a control well, a low viral dilution (1:10) well and a high viral dilution (1:100,000) well. Use virus solutions obtained from step 2.8.
3. Remove all media on the fifth day and wash cells twice with 1x PBS (pH 7.4).
4. Fix the cells with cold 4% paraformaldehyde at 4 °C for 30 min.
5. Add 500 μl of 1x Phosphate buffered saline (PBS) to the cells and wash them for 10 min at RT on a rocker at low speed. Wash the cells three times.
6. Add 500 μl of 0.1% Triton X-100 to the cells to permeabilize them. Let sit for 10 min at RT.
7. Wash cells again as previously described in step 7.5.
8. Add 500 μl of a 5% bovine serum albumin (BSA) solution to block cells at RT. Let sit for 2 hr and then remove.
9. Dilute stock Rhodamine red-conjugated phalloidin (RCP) 1:250 in 1x PBS containing 5% BSA and add 250 μl of the dilution to each well to stain for F-actin.
10. Cover plate in aluminum foil to prevent the dye from bleaching and incubate cells at 4 °C O/N.
11. Remove the RCP after 12 hr of incubation and replace it with 250 μl of 4′,6-diamidino-2-phenylindole (DAPI) (250 μg/ml) diluted in 1x PBS containing 5% BSA, to stain the nuclei of the cells.
12. Incubate the cells containing DAPI at RT for 1 hr.
13. Wash cells three times with 1x PBS as previously described in step 7.5.
14. Gently remove the coverslips from the wells and mount to microscope slides using a mounting media with an anti-fade reagent.
15. Allow the slides to dry in lightproof boxes until viewed under the confocal microscope. View prepared slides as soon as possible as dyes can fade rapidly.
16. Image stained cells using a confocal system equipped with a microscope containing a 63X (oil) plan-apochromate lens.
    1. Set the laser wavelengths to 543 ± 10 nm excitation and 575 ± 10 nm emission for Rhoadmine red-conjucated phalloidin, and 369 ± 10 nm excitation and 450 ± 30 nm emission for DAPI.
    2. Use identical gain and off-set settings for the detector to obtain all images.
    3. Process images using appropriate software for image processing and sorting and import desired images into an image managing software.