Using the assays presented here, the difference in phenotype among rotavirus strains can be evaluated, where unique strains are isolated. Specifically, the phenotypic change of disinfectant-resistant strains of rotavirus is investigated. To achieve clear quantitative outputs using the plaque assay, it is critical to select an appropriate combination of rotavirus strain and serum, in which the rotavirus is well adapted to the serum.
Demonstrating the procedure will be Syun-suke Kadoya, a doctoral student from my laboratory. Remove a CryoTube containing MA104 cell lines from the liquid nitrogen container. Place the CryoTube in a water bath at 37 degrees Celsius to thaw the cells.
Add one milliliter of the cell suspension to 20 milliliters of the serum-containing medium in a T75 flask. Incubate the flask in an incubator at 37 degrees Celsius and 5%CO2 for two to three days. Once the cell monolayer reaches 80%confluency, remove the supernatant and wash the cells twice with five milliliters of 1X Dulbecco's PBS.
Add four milliliters of 0.05%trypsin-EDTA to the flask, and incubate at 37 degrees Celsius for five minutes to detach the cells from the flask. Transfer the cell suspension to a 15 milliliter tube, and centrifuge at 190 g for five minutes. Discard the supernatant and resuspend the pelleted cells in one milliliter of serum-containing medium.
Then dilute the resuspended cells 100-fold with the medium. Add three milliliters of the diluted cell suspension to each well of a six-well plate for the plaque assay. Add one milliliter of the diluted cell suspension to each well of a 24-well plate for the cell-binding assay.
Incubate the plates in an incubator at 37 degrees Celsius and 5%CO2 undersaturated vapor for two or three days. Transfer a tube containing one milliliter of the virus suspension in serum-free medium from storage at minus 80 degrees Celsius to a 37 degrees Celsius water bath to thaw. Add one microgram per microliter of trypsin from porcine pancreas to one milliliter of the virus suspension and then vortex.
Incubate the virus suspension at 37 degrees Celsius and 5%CO2 undersaturated vapor for 30 minutes. Dilute the activated virus suspension with serum-free medium to adjust the multiplicity of infection, or MOI, to 0.1 pfu per cell. Add one milliliter of the diluted virus suspension to MA104 cells in a T75 flask three days after cell plating.
Incubate the cells with virus at 37 degrees Celsius for one hour, gently shaking the flask every 15 minutes. Then add 30 milliliters of a serum-free medium containing four micrograms per milliliter of trypsin from porcine pancreas to the flask. Incubate the flask at 37 degrees Celsius and 5%CO2 undersaturated vapor.
Collect one milliliter of the supernatant in the flask at zero, six, 12, 18, 24, and 36 hours post infection, and replace the supernatant in the 1.5 milliliter types using a pipette. Conduct a freeze-thaw cycle within 15 minutes at minus 80 degrees Celsius followed by a 15-minute melt in a water bath at 37 degrees Celsius. Then centrifuge the tubes at 12, 600 g for 10 minutes at four degrees Celsius.
Collect the supernatant. Filter the supernatant with a distilled 0.2 micron filter to remove the cell fraction. Store the supernatant at minus 80 degrees Celsius until applying it to the plaque assay for measuring the virus titer.
When ready to perform the plaque assay, place the tubes containing the collected supernatant in a water bath at 37 degrees Celsius. Add trypsin to one milliliter of 10-fold diluted sample, and incubate at 37 degrees Celsius for 30 minutes. During this 30-minute incubation, carefully wash the MA104 cells in a six-well plate with two milliliters of 1X PBS after removing the serum-containing medium.
Serially dilute the incubated samples with serum-free medium and inoculate one milliliter of the diluted sample into each well. Incubate the plate for 90 minutes at 37 degrees Celsius and 5%CO2 undersaturated vapor, gently shaking the plate every 15 minutes. After incubation, remove the inoculum from the six-well plate.
Add four micrograms per milliliter of trypsin to the prepared medium. Gently but immediately add three milliliters of the medium mixed with agarose gel to each well. Allow the agarose gel to solidify at room temperature for more than 10 minutes.
Then incubate the plate for two days at 37 degrees Celsius and 5%CO2 undersaturated vapor. Next, add one milliliter of 0.015%Neutral Red solution diluted with 1X PBS to each well, and incubate with the same conditions. Remove the dye after three hours and continue to incubate for one day.
The next day, count the number of plaques in each well and calculate the pfu per milliliter. Carefully check the cell confluence before the plaque assay to assure the plaque numbers. For the cell-binding assay, add one microgram per microliter of trypsin from a porcine pancreas to one milliliter of the virus suspension, and then vortex.
Dilute the virus suspension with serum-free medium to adjust the MOI to one pfu per cell. Wash the MA104 cells twice in a 24-well plate with one milliliter of tris-buffered saline. Now inoculate the cells with 100 microliters of diluted virus suspension into each well of the 24-well plate.
Incubate the plate at four degrees Celsius for 90 minutes with gentle shaking every 15 minutes. Remove the virus inoculum, and wash the cells twice with one milliliter of tris-buffered saline. To extract the double-stranded RNA of the rotavirus, add 140 microliters of 1X PBS and 560 microliters of the RNA extraction buffer to each well.
Mix adequately with a pipette until the haze of cells in the buffer is not seen. After recovering the double-stranded RNA, according to the manufacturer's protocol, place the 1.5 milliliter tubes, containing the double-stranded RNA extract, on a heat block at 95 degrees Celsius for five minutes to denature the RNA. Then immediately place the tubes on ice and incubate for more than two minutes.
Synthesize the cDNA by using a reverse transcription kit. Add four microliters of denatured viral RNA solution to a PCR tube containing 16 microliters of reaction mixture, and mix it carefully with a pipette so as not to generate bubbles. After spinning down the tubes, perform the reverse transcription with a thermocycler.
For quantitative PCR, use a quencher-inserting probe targeting the 963 to 1, 049 region of the NSP3 genome segment of rotavirus. Serially dilute the standard plasmid with PCR-grade water, and proceed to make the master mix for quantitative PCR. Add 20 microliters of the master mix to the wells of a 96-well PCR plate.
Then mix five microliters of cDNA samples or five microliters of the standard plasmid by pipetting 10 times. Perform quantitative PCR according to the conditions in the text protocol. Finally, calculate the rotavirus genome bound to the MA104 cell surface as described in the text protocol.
Shown here is a growth curve of rotavirus. By applying the least square method to a modified Gompertz model, the specific growth rate is estimated to be 0.197, and the lag period is estimated to be 6.61 hours. The relative virus titer at the stationary phase to the initial titer is 3.15.
Of the six tested rotavirus clones, the estimated values of the specific growth rate ranged from 0.19 to 0.27. These estimated values are reliable because the coefficient of determination values in the model fitting is more than 0.98. The result of the cell-binding assay is displayed as binding efficiency, which is the ratio of bound viral particles to those present in the inoculum.
Our RV virions binding to cell surfaces are about 10, 000 copies per milliliter with a binding efficiency of around 1%when using a 24-well plate for the cell-binding assay. Since cultivated cells are sensitive to physical stresses, take care to pour the buffer and agar gently but rapidly. Essentially, a new system for the viruses has been introduced.
Using this system, we can evaluate the real cell-binding ability and specific growth rate with instruments. Our protocol is advantageous in measuring the phenotypic trait. By checking different of newly-emergent strains, you can help to evaluate its new vaccines for the viruses.
The viruses are designated as BSL-2 pathogens, so you will need to prepare BSL-2 experimental rooms to perform this procedure.
