The use of recombinant viruses is a powerful technology both to decipher the viral replication mechanisms and to provide development platforms for vaccines. Likewise, generation of good quality viral stock is crucial for every viral study from the most fundamental research to the applied studies. Rescue of recombinant viruses, optimal harvesting, freezing, and titration of RSV stocks are critical methods for all virological studies.
They might be considered traditional but remain delicate and require specific expertise. The day before transfection, suspend BSR-T7/5 cells in complete medium at 0.5 million cells per milliliter concentration. Distribute two milliliters of the cell suspension per well in a six-well plate.
Incubate the plate at 37 degrees Celsius in 5%carbon dioxide until they reach 80-90%confluency the next day. To rescue each virus, first mix thawed reverse genetics vectors in a tube, then proceed to transfection, following the transfection reagent manufacturer's protocol. Next add 250 microliters of the reduced serum medium to the mixed vectors.
In another tube, dilute 10 microliters of this transfection reagent in 250 microliters of the reduced serum medium. Gently vortex both tubes and wait for five minutes. Mix the contents of both tubes and wait for 20 minutes at room temperature.
Meanwhile, rinse the BSR-T7/5 cells with one milliliter of the reduced serum medium and distribute 1.5 milliliter of minimum essential media antibiotics free supplemented with 10%fetal calf serum per well. If necessary, incubate at 37 degrees Celsius in 5%carbon dioxide environment. After the 20-minute incubation, add 500 microliters of the transfection mix into each well.
Incubate the cells at 37 degrees Celsius in 5%carbon dioxide environment for three days. To monitor the rescue efficiency, observe at GFP fluorescence under an inverted fluorescence microscope at 20X magnification once per day. On the third day of transfection, scratch cells in each well of the transfected BSR-T7/5 six-well plate.
Transfer cells and supernatant of each well into a sterile two-milliliter microcentrifuge tube. To release the rescued virus from the cell membranes, vortex each tube vigorously for at least 30 seconds. For the first amplification of the rescued viruses, first remove the culture medium from the HEp-2 plate seeded the day before, then quickly add 500 microliters per well of the fresh passage-zero viral suspension.
Place the HEp-2 plate at 37 degrees Celsius on a seesaw rocker for soft agitation for two hours. To produce the first passage of the rescued viruses, discard 500 microliters of the inoculum and add two milliliters of MEM with 2%FCS. Incubate the plate at 37 degrees Celsius in 5%carbon dioxide for three days.
To monitor the infection under an inverted fluorescence microscope at 20X magnification, observe GFP fluorescence of the HEp-2 cells infected with the passage-zero suspension once per day. Under a brightfield microscope, observe the appearance of small syncytia and cell detachment, which reflects the RSV cytopathic effect. To amplify the rescued viruses, first dilute the viral suspension of FCS-free MEM to obtain a three-milliliter suspension at 50, 000 PFU per milliliter, then remove the medium from the HEp-2 flask.
Quickly add the three-milliliter viral suspension and incubate the flask at 37 degrees Celsius on a seesaw rocker for soft agitation for two hours. Next remove and discard the inoculum and add 15 milliliters of MEM with 2%FCS. Incubate at 37 degrees Celsius in 5%carbon dioxide for two to four days.
To estimate the right time to harvest the viruses, check the cell morphology and GFP fluorescence under an inverted fluorescence microscope at 20X magnification. Note that this is usually when 50%to 80%of the HEp-2 cell layer is detached due to the RSV cytopathic effect that occurs between 48 and 72 hours post infection. Next scrape all the cells using a cell scraper.
Collect both the cells and the supernatant together and transfer them to a 50-milliliter centrifuge tube. Add 1/10 of the volume of the 10X RSV conservation solution. Vortex the tubes vigorously for five seconds and centrifuge for five minutes at 200 times g to clarify the suspension.
Transfer the supernatant to a 50-milliliter tube. Vortex briefly and aliquot the suspension in cryogenic tubes labeled with alcohol-resistant tags. Immerse the tube in pre-cooled, minus 80 degrees-Celsius alcohol for at least one hour and store them at minus 80 degrees Celsius.
To perform a plaque titration assay, first make 2X minimum essential medium by diluting commercial 10X MEM with sterile water and adding L-glutamine, 1, 000 units per milliliter penicillin, and one milligram per milliliter streptomycin. Shake the dilution vigorously and store it at four degrees Celsius, then add 900 microliters of the FCS-free MEM to six tubes. Thaw the virus aliquots and vortex them vigorously for five seconds.
To make serial dilutions, first add 100 microliters of the virus to 900 microliters of the medium in the first tube. Put the cap on the tube and mix its contents by vortexing for a few seconds, then add 100 microliters of the first dilution to 900 microliters of the medium in the second tube. Put the cap on the tube and vortex.
Repeat this procedure until the sixth tube. Write the virus name and the full dilutions on the HEp-2 12-well plates. Add a mark to match the plate and its cover because they may be separated during staining.
Remove the medium from the plates and distribute 400 microliters of one dilution per well. Incubate the plates at 37 degrees Celsius for two hours for virus adsorption. During the virus adsorption, prepare the microcrystalline cellulose overlay by first adjusting the pH of the 2X MEM to around 7.2 with a sterile sodium bicarbonate solution at 7.5%following the color indicator.
To obtain 100 milliliters of the overlay, add 10 milliliters of 2X MEM, 10 milliliters of 2.4%monocrystalline cellulose suspension, and 80 milliliters of the MEM supplemented with 2%FCS and mix vigorously. At the end of the two-hour incubation, add two to three milliliters of the overlay to each well of the 12-well plates without removing the inoculum. Incubate the plate at 37 degrees Celsius in 5%carbon dioxide for six days.
To stain the cells with crystal violet solution, first gently shake the plates to take off the microcrystalline cellulose overlay. Remove the supernatants and wash the cells twice with 1X PBS, then add one to two milliliters of the crystal violet solution and wait 10 to 15 minutes. Remove the solution, which can be reused for subsequent plate-staining.
Finally calculate the virus titers as a fraction of the number of visible plaques in the wells of the dry plates and the inoculum volume and the dilution. Performing a plaque assay on the negative control, transfected with only the expression plasmids of N, P, L, and M2-1 revealed no plaque at the lowest dilution. The titers obtained from the transfected cells were expected to be above 100 PFU per milliliter if the rescue was efficient.
The titers increased over the passages to reach one million to 10 million PFU per milliliter at passage one or two. Thanks to these fluorescent viruses, inhibition of RSV expression by siRNA targeting the protein N and the cellular protein IMPDH can be easily observed and measured. In contrast, a strong GFP signal on control cells transfected with non-targeting siRNA or cells transfected with siRNA against the cellular protein GAPDH was observed and measured.
Likewise these viruses enabled easy observation of the inhibition of RSV multiplication by an antiviral drug compound. Tracking the fluorescent protein M2-1 in HEp-2-infected cells showed IBs and IB-associated granules as very dynamic structures. IBs were observed as mobile and spherical structures able to fuse and to form larger spherical inclusions.
IB-associated granules underwent continuous assembly-disassembly cycles with the formation of small IB-associated granules that grew, fused into large IB-associated granules, and then disappeared. The protocol of plaque titration of RSV using microcrystalline cellulose overlay may be easily adapted to other cells and/or other viruses. It will require adjusting the concentration of the microcrystalline cellulose.
