A look into the lab: mosquito testing

The Vermont Department of Health reports its first positive tests for West Nile Virus in three batches of mosquitoes captured in Springfield, Vermont on July 22nd.
http://digital.vpr.net/post/look-lab-mosquito-testing

A Look Into The Lab: Mosquito Testing By JANE LINDHOLM • 9 HOURS AGO The risk of mosquito-borne illness increases during the summer months until there is a hard frost in the fall. The risk of mosquito-borne illness increases during the summer months until there is a hard frost in the fall. CENTERS FOR DISEASE CONTROL AND PREVENTION Listen Listening...10:56 Every year in June, the Vermont Department of Health starts collecting mosquitoes at various locations around the state. The vials of mosquitoes then travel to Burlington where they're tested for Eastern Equine Encephalitis and West Nile Virus. Vermont Edition met with microbiologist Christine Matusevich in a lab at the Health Department. The room looked a lot like an old high school chemistry lab, but with some newer-looking machines crowding the counters. She explained the process of collecting and testing the mosquitos for diseases they aim to monitor across the state. This season's testing in early June, and as of July 18, 1005 specimens have been collected and tested for disease statewide. Thus far, there have been no positive tests for EEE or West Nile Virus. "We see positives starting early July," says Matusevich. "We haven't see any yet, but I have my own personal theories on that. We've had these cold, cold winters and I think that has had an effect on the amount that we see." Mosquito testing and monitoring began in 2013. Unseasonably warm weather that year resulted in a lot of positive tests, which were not matched in the following two years. The Process Entomologists in the field are given vials that contain two BB bullets, like the kind found in pellet guns, to collect mosquito specimens. The mosquitoes are trapped, sorted by species and put into geographical pools based on where they were collected. The BBs are essential to the testing process. When the vials are shaken, the BBs break up the mosquitoes into small pieces, releasing the RNA to come out from the West Nile and EEE viruses. A liquid medium is put into the vial to suspend the pieces and help release the RNA before the specimens are put into a spinning centrifuge. Microbiologist Christine Matusevich shows the vials of collected mosquitoes during the RNA extraction process. The results from these tests will indicate whether or not EEE or West Nile Virus are present in the state. Microbiologist Christine Matusevich shows the vials of collected mosquitoes during the RNA extraction process. The results from these tests will indicate whether or not EEE or West Nile Virus are present in the state. CREDIT JANE LINDHOLM / VPR "It'll help get all of the debris to the bottom so we can extract some of this the liquid for the testing that we do," says Matusevich. The extracted liquid takes on a dark, mahogany coloring at this stage. This isn't blood from hosts that the mosquitos are carrying, however. The coloration is caused by the mosquito's own innards. The viral RNA is placed into the Mag Max, a machine that processes the specimen and further draws out RNA. The plates that go into Mag Max have 96 wells, which hold the mosquito liquid, magnetic beads and alcohol. After loading these wells with liquid, the "fingers" of Mag Max transfer the beads from plate to plate. These beads gather the RNA, and leave a clear liquid filled with viral RNA. A final instrument is used to process the liquid. "At this point it's like cooking. We're adding different amounts of ingredients that allows the instrument to tell us whether or not we have the virus present in these mosquito pools," says Matusevich. "[This machine] heats up to 95 degrees and then cools down to about sixty degrees and it cycles like that over and over and over for about 45 cycles," says Matusevich. The cycling during this two-hour step is key, as the viral specimen present in the liquid doubles with each cycle. "At the start, if there's that one copy, by the end we'll have about one or two billion copies." Positive Results Getting a positive result doesn't necessarily mean there is immediate reason for concern. "These are actually preliminary results," says Matusevich. "So we have to repeat the test with different primer segment, so segments of the virus, and once we do that once it confirms positive then we contact epidemiology and also the Department of Agriculture, because we're all partnering in this." All of the samples are regionally grouped, so a positive test can be pinned to a specific area. In the past, disease testing was limited to specific parts of the state based on prior records of the disease. Now, mosquito pools have been sampled from all 14 counties in Vermont, and 11 different mosquito species have been collected and tested for EEE. Matusevich expects to see positive tests for EEE or West Nile Disease starting in August. Update, 10:30am July 30th 2015: The Vermont Department of Health reports its first positive tests for West Nile Virus in three batches of mosquitoes captured in Springfield, Vermont on July 22nd. No positive tests for EEE have been detected as of this date.