This protocol is a useful approach for isolating mosquito-associated viruses and can help in further research into the distribution of pathogens related to mosquitoes and the control of mosquito-borne diseases. Using thin lines to isolate viruses in Biosafety level 2 laboratory is safer and more efficient. Unlike other virus isolation procedures, such as cycling red inoculation with varying fluid, this technique does not necessitate animal experimentation or ethical assessment.
The contamination from samples and the cells drawing during the incubation can lead to the failure of the experiment. Hence, it is necessary to add 2%Penicillin-Streptomycin-Amphotericin into the medium to prevent contamination. Begin with the addition of three-millimeter ceramic beads, 1.5 milliliters of RPMI medium supplemented with 2%Penicillin-Streptomycin-Amphotericin B Solution in a two-milliliter sterile tube placed on ice and then transfer 50 mosquitoes in it.
Next, homogenize the mosquitoes with a low temperature tissue homogenizer by grinding for 30 seconds at 70 hertz and 4 degrees Celsius for 3 cycles. Centrifuge the homogenate at 15, 000 G for 30 minutes at 4 degrees Celsius and transfer the supernatants to the new tubes. To remove the mosquito debris completely, centrifuge the supernatant again at 15, 000 G for 10 minutes at 4 degrees Celsius.
Aliquot the 200 microliters of supernatant P0 into a two-milliliter screw cap storage tube, and store it at minus 80 degrees Celsius. For the virus amplification, use Aedes albopictus RNAi-deficient mosquito cell line C6/36 and a vertebrate cell line, baby hamster kidney, or BHK-21 from a 75-centimeter square flask and seed them in 24 well plates separately. Place the seeded 24 well plate overnight at 28 or 37 degrees Celsius.
The next day, observe the cells under the microscope to confirm 80 to 90%confluency in each well. Prepare 500 milliliters of the cell culture maintenance medium supplemented with antibiotics. Remove the cell culture medium from the 24 well plates and add 100 microliters of the cell culture maintenance medium to each well.
Then inoculate the cells with 100 microliters of the supernatant of the mosquito homogenate or P0.Incubate the plates at 28 or 37 degrees Celsius for 60 minutes and gently shake the plates after every 15 minutes to prevent the drying of the cells. Once done, remove the supernatant and rinse each well with 600 microliters of cell culture maintenance medium to remove the debris completely. Next, add 800 microliters of cell culture maintenance medium to each well before placing the plates in a 5%carbon dioxide humidified incubator at 28 or 37 degrees Celsius for 7 days.
Daily monitor the state of the cells of each well under the microscope. On the seventh day, collect the supernatant of cells known as P1 and store it at minus 80 degrees Celsius. Repeat the procedure to collect P2 and P3 viral supernatants.
Due to the cytopathogenic effect, or CPE, cells from some of the wells die and detach from the surface. To greatly amplify the viruses Collect 300 to 400 microliters of the supernatant from wells showing the CPE effect and transfer it to a new six-well plate having 80 to 90%cell confluency of culture cells. Finally, collect and transfer 500 microliters of the supernatant from the wells of the six-well plate to two-milliliter screw cap storage tubes before storing them at minus 80 degrees Celsius.
The inoculation of C6/36 cells with the mosquito homogenates P0 exhibited a wide intercellular space in exfoliated cells at 120 hours post-infection period compared to the uninoculated or control cells. Incubation of BHK21 cells with the P3 viral supernatants demonstrated a visible CPE at 48 hours post-infection, displaying the death of the cell and detachment from the surface in contrast to the control cells. Commercially available universal primers or Flaviviruses, Alphaviruses and Bunyaviruses were used to generate the PCR products for the viral supernatant.
A PCR product for the Bunyavirus, Ebinur Lake virus, was set as a positive control. The PCR amplification bans in lanes belonging to Bunyaviruses and the positive control highlighted the possibility of the presence of Bunyavirus in the supernatants. To improve the success rate of virus isolation, keep a low temperature during sample transportation, isolation, and grinding.
Add antibiotics when homogenizing the mosquitoes and the culture itself. And handle the culture rapidly during incubation and the removing the culture medium. In addition to this procedure, the grinding solution can be injected into a cycling rat or chicken embryo.
However, the affirmation procedure will raise ethical concerns and increase the expense of the experiment.
