Ae. albopictus mosquitoes (kindly provided by Illia Rochlin, Suffolk County Health Department, Yaphank, NY, USA) were originally collected in Suffolk County in 2014 and subsequently colonized in the NYSDOH Arbovirus Laboratory. F5–F7 female mosquitoes from New York were used for experimental feedings.
Ae. aegypti mosquitoes used for preliminary experiments were collected by C. Mangudo in Salta, Argentina, in 2014 and initially colonized by V. Micieli and L.D. Kramer at the Centro de Estudios de Parasitología y Vectores (La Plata, Argentina) before being shipped to the NYSDOH Arbovirus Laboratory for maintenance. F4–F5 females from Argentina were used for experimental feedings.
Ae. aegypti mosquitoes (kindly provided by G.D. Ebel, Colorado State University, Fort Collins, CO, USA) were originally collected in Poza Rica, Mexico. F7–F8 females from Mexico were used for experimental feedings. For preliminary blood feeding experiments,
Ae. aegypti mosquitoes from Argentina were fed Zika virus PR stock virus diluted 1:1, 1:5, or 1:20 in defibrinated sheep blood (Colorado Serum Co., Denver, CO, USA) with 2.5% sucrose. For feedings with freshly propagated virus, supernatant from infected C6/36 cultures was harvested at 96 h after infection (multiplicity of infection ≈1.0) and diluted 1:1 with blood-sucrose mixture without freezing. Female mosquitoes, 4–7 days of age, were deprived of sucrose for 18–24 h and offered blood meal mixtures by use of a Hemotek membrane feeding system (Discovery Workshops, Acrington, UK) with a porcine sausage casing membrane. For all subsequent experiments assessing dose-dependent vector competence, similarly prepared fresh C6/36 cultures of Zika virus HND and Zika virus CAM were used to feed
Ae. aegypti mosquitoes from Mexico and
Ae.albopictus mosquitoes from New York. In addition to undiluted supernatant, 1:20, 1:400, and 1:8,000 dilutions were made in C6/36 maintenance media before being mixed with blood.
For all blood feeding experiments, mosquitoes were sedated with CO
2 after 1 h of feeding, and fully engorged mosquitoes were transferred to 0.6-L cartons and maintained at 27°C for experimental testing. Infection, dissemination, and transmission rates were determined as previously described (
24 (
link)) on day 14 or 21 after feeding. After the mosquitoes were sedated, the legs were removed from 12–30 mosquitoes and placed in 1 mL mosquito diluent (20% heat-inactivated fetal bovine serum in Dulbecco phosphate-buffered saline plus 50 μg/mL penicillin/streptomycin, 50 μg/mL gentamicin, and 2 μg/mL Fungizone [Sigma Aldrich, St. Louis, MO, USA]). For 30 minutes, mosquitoes were allowed to expectorate into capillary tubes containing ≈20 μL fetal bovine serum plus 50% sucrose (1:1), at which time the mixture was ejected into 250 μL mosquito diluent. Mosquito bodies were then placed in individual tubes with mosquito diluent. All samples were held at −80°C until tested. To test for infection, dissemination, and transmission, we processed and screened bodies, legs, and salivary secretions, respectively, by Zika virus–specific quantitative reverse transcription PCR (
25 (
link)). Zika virus body titers were calculated from standard curves based on infectious particle standards created from matched virus stocks. Data were analyzed by using GraphPad Prism version 4.0. Rates were compared by using Fisher exact tests, and dose dependence was evaluated and compared by using linear regression analyses.
Ciota A.T., Bialosuknia S.M., Zink S.D., Brecher M., Ehrbar D.J., Morrissette M.N, & Kramer L.D. (2017). Effects of Zika Virus Strain and Aedes Mosquito Species on Vector Competence. Emerging Infectious Diseases, 23(7), 1110-1117.