Midge Vectoring of EHDV-2 Strains

Cohorts of ≤50 midges were fed EHDV-2 infected blood using a Hemotek membrane feeding system (Hemotek Ltd., Blackburn, UK) following published protocols [33 (link)]. Parafilm was used as the membrane for blood feeding using defibrinated bovine blood (Hemostat laboratories, Dixon, CA, USA) in three treatments: Can-Alberta strain, Florida strain, and control (blood without the addition of virus). Blood was collected before and after each blood feeding trial to measure viral titer and to ensure titers were similar between the two infected groups. The calculated titer of virus in the two virus fed treatments were each 5.5 log₁₀ plaque forming unit equivalents (PFUe)/mL. Midges were allowed to blood feed for one hour after which blood-fed females were sorted from the males and unfed females using a CO₂ pad (LabScientific Carbon Dioxide Staging Platform, Cat #BGSU-12, Highlands, NJ, USA) and a stereoscopic light microscope (Nikon SMZ 745). Blood-engorged females were sorted into cups of ≤25 individuals, separated by treatment. After blood-feeding, midges were provided 10% sucrose solution ad libitum on cotton pads and maintained at 25 ± 1 °C, 60–80% RH, and 12:12 (L:D) cycle until processing.