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 Technology, USA)/RPMI 1640 medium (Sigma, USA). The DENV-2 strain QML16 was isolated from a dengue fever patient in Australia and QML22 was isolated from a dengue fever patient returning to Australia from Borneo [5]. The virus strains were passaged three times in C6-36 cells to a titer around of 108 CCID50/ml and the cell culture supernatant was stored at -80 °C for further use.
Ae. aegypti colonies were established from collections in Townsville and Innisfail, north Queensland (QLD), Australia, and maintained within the Australian Defence Force Malaria and Infectious Disease Institute and QIMR Berghofer Medical Research Institute insectaries, respectively. Larvae were reared at a density of 200 larvae in 3 L of reverse osmosis water in plastic trays (48 × 40 × 7 cm) and fed ground TetraMin tropical fish food flakes (Tetra, Melle, Germany) at a rate of 0.25–1.0 mg/larva/day as development progressed. Pupae were transferred to cages (30 × 30 × 30 cm) for adult emergence. Adults were provided with 10% sugar solution on cotton wool pledgets which was withheld two day prior to virus feeding. Prior to feeding, mosquitoes (4 day-old) were deprived of sucrose solution for 24 h.
Membrane feeding
Approximately one hundred 3–5 day old mosquitoes were placed into 750 ml containers with gauze covering the opening. DENV-2 strains QML16 and QML22 strains were mixed with defibrinated sheep blood to a titre of 108 CCID50/ml in C6/36 cells. The mosquitoes in containers were allowed 1 hr to feed on the blood/virus mixtures through bovine ceacum 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 the unfed 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 (Panasonic) set at 28 °C, 75% relative humidity and 12:12 h day:night ligiht schedule with 30 min dawn:dusk periods.
In vitro transmission assays
At 7, 10 and 14 dpi, female mosquitoes were anesthetized using CO2 and ice; legs and wings were removed. In vitro transmission assays were performed as previously described [22, 23]. For each mosquito, the proboscis was placed in a capillary tube containing 20 µl of a 1:1 solution of 50% sucrose and FBS. After 30 min, the contents were expelled into 0.25 ml MD (MD, 2% FBS in RPMI 1640, 50 µg/ml penicillin/streptomycin, 50 µg/ml gentamycin, 2.5 µg/ml Amphotericin B and 10 mM HEPES).
Virus titre determination
Legs and wings samples and body samples from individual mosquitoes were placed into 2 ml screw cap vial with 1 ml MD with 4–5 zirconium silica beads. The samples were homogenized by shaking tubes for 1 min 30 s in a chilled block using a MiniBeadbeater-96 sample homogenizer (Biospec Products, Bartlesville, OK, USA) followed by centrifugation (twice at 17,000 × g, 10 min, 4 °C)
Virus stocks and virus in mosquito samples were titrated using a Cell Culture Enzyme-linked Immunosorbant Assay (CCELISA) modified on the method of Broom et al [24]. Briefly, virus stocks and samples were ten-fold serially diluted and inoculated onto a monolayer of C6/36 cells grown in RPMI 1640 supplemented with L-glutamine, 5% heat inactivated FBS, 1% penicillin/streptomycin (Gibco Life Technologies, USA) and maintained at 30˚C, 5% CO2. After 7 d incubation, cells were fixed in acetone: methanol (1:1) for 1 hr at 4˚C. Plates were air-dried and antigen was detected using a cocktail of anti-dengue monoclonal antibody hybridoma supernatants (4G2[25], 6B-6C1:3H5[26], at a ratio of 1:1:1) as the primary antisera, Horseradish peroxidase (HRP-) conjugated goat anti-mouse polyclonal antibody (DAKO, Carpinteria, CA, USA) (1:2000 in PBS-Tween) as the secondary antisera and 3,3’,5,5’-Tetramethylbenzidine (TMB) Liquid Substrate System for Membranes (Sigma-Aldrich, St. Louis, MO, USA) as the substrate. Wells with cell monolayers that stained blue were scored as positive for infection. The CCID50 was determined from titration endpoints as described everywhere [27] and expressed as the CCID50/ml in C6/36 cells.
Infection rate was defined as the proportion of mosquitoes with DENV positive bodies/total number of engorged mosquitoes. Dissemination and transmission rates were defined as the proportions of infected mosquitoes with positive legs/wings and salivary secretions/ the total number of engorged mosquitoes. We compared the virus titers and proportions using Mann-Whitney U-test and Chi square test.
Mosquito immunohistochemistry
Histological analysis of DENV infection within mosquitoes using indirect immunofluorescence assay (IFA), microscopy was performed based on the methods of described in our previous publication [22]. Briefly, mosquitoes with legs and wings removed were fixed in 4% paraformaldehyde/0.5% Triton X for 12 hr, dehydrated in xylol then a graded ethanol series, embedded in paraffin and 3–4 µM sections affixed to slides using standard histological procedures. 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 in PBS + 0.025% Tween 20 (PBST) twice for one minute each wash. Non-specific antibody binding was inhibited by incubating the sections in Biocare Medical Background Sniper + 2% BSA for 30 min. Excess Sniper/BSA was removed from the sections and the sections were incubated with undiluted 4G2 hybridoma supernatant for 2 hr at room temp Sections were washed three times with PBST. Alexa Fluor donkey anti-mouse AF488 diluted 1:300 in PBS was applied for 30 minutes. Sections were washed three times with PBST before being counterstained with DAPI for 10 min, washed several times with PBS before being mounted.