The overall goal of this procedure is to screen field collected mosquitoes for viral infection by Vero cell culture assay. First, the mosquitoes collected in the field are sorted according to species. Mosquitoes are then homogenized using a mixer mill.
Once the mosquito homogenates have been made, Vero cells are cultured in preparation for viral testing, each Vero cell culture is then inoculated with a single mosquito homogenate placed on a shaker for five minutes, and then incubated at 37 degrees Celsius to allow viruses to propagate. Ultimately, the cultures are examined visually for evidence of viral induced cytopathic effects. The main advantage of this technique over existing methods such as R-T-P-C-R, is that allows one to screen mosquitoes for a wide diversity of viral agents that grow in cell culture.
This method can help answer key questions in the medical entomology field, such as the roles different mosquito species play in supporting virus transmission. Use a biosafety level two facility for the following procedure. Place the collection bag containing live mosquitoes at minus 20 degrees Celsius for 15 minutes to anesthetize them.
Once the insects are anesthetized, transfer the bag to an insulated container containing dry ice and expose the mosquitoes to carbon dioxide for one to three minutes to ensure complete knockdown. Next place mosquitoes on a pre chilled pan on a bed of dry ice. Using fine tip forceps, identify the female insects and transfer them to plastic portion cups.
Females can be distinguished from males by examining the mouth parts and antennae. Seal the cups and place them on wet ice. Using a stereo dissecting microscope mounted on an electronic chill table and a taxonomic key.
Identify the species of each mosquito pool mosquitoes of the same species into labeled two milliliter snap cap vials containing a copper ball. Up to 50 individuals can be pooled in a single tube as they become filled. Place vials in labeled bags on a styrofoam container on dry ice.
Once all the mosquitoes have been identified, they can be stored at minus 80 degrees Celsius until homogenization. Use a biosafety level three facility for this procedure and work in a class two biosafety cabinet. Add about one milliliter of PBSG per tube and place tubes containing mosquitoes inside a pre chilled mixer mill cassette.
Secure the loaded cassette into a mixer mill and homogenize the mosquito pools for four minutes at 25 cycles per second. Once the mosquitoes are homogenized, transfer the tubes into an aerosol tight refrigerated micro fuge and centrifuge. The mosquito homogenous at 7, 000 RPM for six minutes.
Following centrifugation tubes may be stored at minus 80 degrees Celsius until virus testing. Decant the culture medium from a T 1 75 flask of confluent farrow cells into a waste beaker containing bleach. Add five milliliters of PBS to the flask and wash cells evenly before decanting the PBS into the waste beaker.
After washing the cells, add 2.5 milliliters of tripsin EDTA and incubate the flask at 37 degrees Celsius and 5%CO2 for five minutes to detach the cells. Once the cells are detached, add five milliliters of MEM and pipette the cells up and down several times to break up any clumps. Then add another 15 milliliters of MEM.
Place 40 T 25 flasks on two racks inside the biosafety cabinet and remove the flask caps, placing them directly in front of each flask using a sterile pipette aliquot, four milliliters of MEM into each flask. Then add 0.5 milliliters of suspended vari cells and cap the flasks. Stack the flasks on a tray and swirl them gently to make sure that the medium uniformly covers the bottom of the flasks.
Incubate the cultures at 37 degrees Celsius and 5%CO2 overnight or until the cells become 80 to 90%confluent. Remove 20 small flasks from the incubator and label them with the corresponding accession number from each mosquito pool. Decant most of the medium from each flask into the waist speaker leaving just enough medium to coat the cell monolayer.
Transfer 100 microliters of supernatant from each mosquito homogenate into the correspondingly labeled flask and seal the flasks tightly. Once the supernatants have been transferred, place the flasks on a shaker set at 35 to 40 RRP M for five minutes. Bring the flasks back into the biosafety cabinet and add four milliliters of growth medium to each.
Recap the flasks and incubate them at 37 degrees Celsius and 5%CO2 for three to seven days. Once the cultures are ready to be screened, first visually inspect the media. Cloudy media indicates either viral induced cytopathic effects or contamination with other microbes, or both examine the cultures, exhibiting cloudy media under an inverted light microscope.
Viruses typically cause cells to become deformed and detach from the culture flasks. If contamination with bacteria, yeast, or fungi is detected, or if the culture contains heavy mosquito debris, retest the original mosquito pool after filtering it through a 0.22 micron syringe. Filter aseptically dispense one to two milliliters of media from virus positive cultures not contaminated with other microorganisms into labeled cryo tubes.
Store the virus containing samples at minus 80 degrees Celsius until identification, using the appropriate diagnostic assays using this assay. Nine different types of viruses from four taxonomic families were recovered from mosquitoes collected in Connecticut during a continuous statewide surveillance period from 1997 to 2010. Several of these viruses, including Eastern equine encephalitis virus, and West Nile Virus are known to cause human disease.
After watching this video, you should have a good understanding of how to sort process and screen mosquitoes for viral infection by viral cell culture assay.
