A colony of unknown generations of An. quadrimaculatus (Orlando strain) was obtained from BEI (MRA-139) and were maintained at 27°C under standard rearing [22, 23]. Aedes albopictus colony (Spring Valley, New York, kindly provided by Laura Harrington, Cornell University) was established in 2019 from field-collected eggs. Aedes albopictus were hatched in distilled water, reared and maintained similarly to Anopheles as described .
Experimental Infections Of Mosquitoes With Jamestown Canyon Virus
Five JCV strains originally isolated from mosquito pools in Connecticut (kindly provided by Philip Armstrong, CAES), and previously found to be genetically distinct, were freshly propagated in Vero (African Green Monkey kidney) cell cultures in separated plates maintained at 37°C, 5% CO2. These included 3836-05 (lineage A, 2005; S, M, L accession #s EF681835, EF687050, EF687103), 11497-03 (lineage A, 2003; S, M, L accession #s EF81845, EF87121, EF687059), 1441-01 (lineage B, 2001; S, M, L accession #s EF681814, EF687110, EF687027), 1425-02 (lineage B, 2002; S, M, L accession #s EF681813, EF687091, EF687039), and 4148-03 (lineage B, 2003; S, M, L accession #s EF681827, EF687114, EF687051). At 48 hours following infection (multiplicity of infection ≈ 1.0), the supernatant of each strain was harvested and diluted 1:1 with defibrinated sheep blood plus a final concentration of 2.5% sucrose. Female An. quadrimaculatus mosquitoes (3–5 days old) from unknown generations deprived of sugar for 1–2 hours, and F7 female Ae. albopictus (5–7 days old) were deprived of sugar for 24 hours were offered a JCV-blood suspension for 45 minutes via a Hemotek membrane feeding system (Discovery Workshops, Acrington, UK) with porcine sausage casing membrane at 37°C . Following feeding, females were anesthetized with CO2 and fully engorged mosquitoes were transferred to 0.6L cardboard containers and maintained with 10% sucrose at 27°C until harvested for testing . A 1 ml aliquot of each pre-feeding blood meal was frozen at -80°C to determine JCV blood meal titers.
Evaluation Of Mosquito Vector Competence For Jamestown Canyon Virus
Infection, dissemination, and transmission were determined on days 7 and 14 post infectious blood (dpi) meal as previously described [22, 23]. Blood meals, mosquito bodies, legs, and salivary secretions were assayed for infection by plaque assay on Vero cells [23, 24]. Briefly, Vero cells were seeded in six-well plates at a density of 6.0 × 105 cells per well and were incubated for 3–4 days at 37°C in 5% CO2, to produce a confluent monolayer. The cell monolayers were inoculated with 0.1 ml of 10-fold serial dilutions of the blood meals (diluted in BA-1) in duplicate or with undiluted mosquito bodies, legs, and salivary secretions from each homogenized mosquito sample. Viral adsorption was allowed to proceed for 1 h at 37°C with rocking of the plates every 15 min. A 3 ml overlay of MEM, 5% FBS, and 0.6% Oxoid agar supplemented with 0.2X of Penicillin/Streptomycin per ml, 0.5 µg of fungizone (Amphotericin B) per ml and 20 ug of Gentamicin per ml, was added at the conclusion of adsorption. The infected monolayers were incubated at 37°C in 5% CO2. After 2 days of infection a second overlay, similar to the first but with the addition of 1.5% neutral red (Sigma–Aldrich Co., St. Louis, MO), was added to the wells, and the plates were incubated at 37°C in 5% CO2 overnight. For the blood meal, the plaques were counted, and the viral titer was calculated and expressed as plaque forming units (PFU) per ml. For mosquito samples, presence or absence of plaques was checked.
Dissemination rate is the proportion of mosquitoes with infected legs among the mosquitoes with infected bodies. Transmission rate is the proportion of mosquitoes with infectious saliva collected by capillary transmission method among mosquitoes with disseminated infection [22, 23].
A Fisher’s exact test was used to compare infection rates, dissemination rates, and transmission rates within mosquito species and between both time points and virus strains. All statistical analyses were carried out at a significance level of p < 0.05. OpenEpi, Version 3, open-source calculator–TwobyTwo (http://www.openepi.com/TwobyTwo/TwobyTwo.htm) was used for all statistical analysis.