Cells, viruses and mosquitoes
C6-36 (Ae. albopictus mosquito) cells were purchased from the American Type Culture Collection (ATCC) and cultured in 10% v/v heat-inactivated foetal calf serum (FCS, Life Technologies, Carlsbad, CA, USA)/RPMI 1640 medium (Sigma-Aldrich, St. Louis, MO, USA). The QML16 strain of DENV2 was isolated from a dengue fever patient in Australia and strain QML22 was isolated from a dengue fever patient returning to Australia from Borneo [8]. The virus strains were passaged three times in C6-36 cells and the cell culture supernatant was harvested, aliquoted and stored at -80 °C for further use. One vial of the viral stocks was thawed to determine virus titre (CCID50/ml) using a cell culture enzyme-linked immunosorbent assay (CCELISA) method. As required, remaining vials were removed from the -80 °C freezer immediately thawed, diluted and mixed with blood to prepare artificial viremic blood meals as described previously [12].
Colonies of Ae. aegypti were established from collections in Townsville and Innisfail in north-east Australia and maintained within the Australian Defence Force Malaria and Infectious Disease Institute and QIMR Berghofer Medical Research Institute insectaries, respectively. Both mosquito colonies were established before the release of Wolbachia in northern Australia. Larvae were reared at a density of 200 larvae in 3 liters of water, prepared by reverse osmosis, in plastic trays (48 × 40 × 7 cm) and fed ground TetraMin tropical fish food flakes (Tetra, Melle, Germany) at a rate of 0.25–1.00 mg/larva/day as development progressed. Pupae were transferred to cages (30 × 30 × 30 cm) for adult emergence. Adults were provided with 10% w/v sucrose solution on cotton wool pledgets which were removed 24 h prior to feeding.
Membrane feeding
Approximately one hundred 4–5 day-old mosquitoes were placed into 750 ml containers with gauze covering the opening. Stocks of DENV2 QML16 and QML22 were thawed and immediately mixed with defibrinated sheep blood to contain 108 CCID50/ml. The mosquitoes, in containers, were allowed to feed for 1 h on the blood/virus mixtures through bovine caecum membrane using an artificial feeding apparatus maintained at 37 °C, as previously described [21]. After feeding, mosquitoes were anaesthetized using CO2, placed on a Petri dish on ice and fully engorged females were separated from unfed or partially fed mosquitoes. The engorged mosquitoes were placed into the gauze covered containers, provided with cotton balls soaked with 10% sugar solution, and maintained within an environmental chamber (PHCbi, PA, USA) set at 28 °C, 75% relative humidity and 12:12 h day:night light schedule with 30 min dawn:dusk periods.
In vitro transmission assays
At 7, 10 and 14 days post-infection (dpi), female mosquitoes were anesthetized using CO2 and placed in Petri dishes on ice. Legs and wings were removed, and their virus content used to determine the dissemination rate as described previously [22]. In vitro transmission assays were performed as previously described [23, 24]. For each mosquito, the proboscis was placed in a capillary tube containing 20 µl of a 1:1 solution of 50% sucrose and FCS. After 30 min, the contents were expelled into 0.25 ml MD (MD, 2% v/v FCS in RPMI 1640, 50 µg/ml penicillin/streptomycin, 50 µg/ml gentamycin, 2.5 µg/ml Amphotericin B, 10 mM HEPES) (Life Technologies). Mosquitoes were observed for abdominal contractions during the 30-min salivation period to confirm they had salivated. Those that did not appear to have salivated were discarded.
Determination virus titre
Legs, wings and bodies from individual mosquitoes were placed into separate 2 ml screw cap vials with 1ml MD with 4–5 zirconium silica beads. The samples were homogenized by shaking the tubes for 90 s in a chilled block using a MiniBeadbeater-96 sample homogenizer (Biospec Products, Bartlesville, OK, USA) followed by centrifugation at 17,000× g for 10 min at 4 °C. Supernatants were transferred to sterile tubes.
Virus stocks and virus in mosquito samples were titrated using a modification of the CCELISA procedure of Broom et al. [25]. Briefly, virus stocks and samples were 10-fold serially diluted and inoculated onto monolayers of C6/36 cells grown in RPMI 1640 supplemented with L-glutamine, 5% heat-inactivated FCS, 1% penicillin/streptomycin (Life Technologies) and maintained at 30 °C, 5% CO2. After 7 days of incubation, cells were fixed in acetone:methanol (1:1) for 1 h at 4 °C. Plates were air-dried and antigen was detected using a cocktail of anti-flavivirus monoclonal antibody hybridoma supernatants (4G2 [26]:6B-6C1:3H5 [27], at a ratio of 1:1:1), followed by horseradish peroxidase (HRP-) conjugated goat anti-mouse polyclonal antibody (DAKO, Carpinteria, CA, USA) (1:2000 in PBS-Tween). Antibodies bound to the cell monolayers were detected by the addition of 3,3',5,5'-tetramethylbenzidine (TMB) Liquid Substrate System for Membranes (Sigma-Aldrich). The CCID50 was determined from titration endpoints as described elsewhere [28] and expressed as the C6/36 CCID50/ml.
The infection rate was defined as the proportion of mosquitoes with bodies containing DENV divided by the total number of engorged mosquitoes. Dissemination and transmission rates were defined as the proportions of infected mosquitoes with legs/wings containing DENV and salivary secretions containing DENV divided by the total number of engorged mosquitoes. The Mann-Whitney U-test, t-test and Chi-square tests were employed to compare virus titres in tissues and proportions of infected tissues.
Mosquito immunohistochemistry
Histological analysis of DENV infection within mosquitoes employed indirect immunofluorescence assays (IFA) as described previously [23]. Briefly, mosquitoes with legs and wings removed were fixed in 4% v/v paraformaldehyde/0.5% v/v Triton X-100 for 12 h, dehydrated in xylol followed by a graded ethanol series, embedded in paraffin and 3–4 µM sections fixed to slides. Sections were incubated in Diva antibody retrieval solution (Biocare Medical, Concord, CA, USA) at 125 °C for 5 min in a Biocare Medical Decloaking Chamber. Sections were cooled for 20 min and washed twice in 0.025% v/v Tween 20/PBS pH 7.2 for one minute each wash. Non-specific antibody binding was inhibited by incubating the sections in 2% w/v bovine serum albumin (Sigma-Aldrich)/ Biocare Medical Background Sniper for 30 min. Excess Sniper/BSA was removed from the sections before they were incubated with anti-flavivirus monoclonal antibody, 4G2, for 2 h at room temperature. Sections were washed three times with PBST and Alexa Fluor 488 donkey anti-mouse antibody diluted 1:300 in PBST applied for 30 min. Sections were washed three times with PBST before being counterstained with DAPI for 10 min, and washed 4 times with PBST before being mounted.