Vector Competence Analyses on Aedes aegypti Mosquitoes using Zika Virus

All procedures performed in these protocols were performed in full compliance with protocols approved by the Institutional Biosafety Committee and the Institutional Animal Care and Use Committee of the University of Texas Medical Branch at Galveston.

1. Amplify ZIKV in Vero cells

1. Grow Vero cells (CCL-81 or VeroE6) in Dulbecco’s modification of Eagle’s minimal essential medium (DMEM) supplemented with 10% v/v heat-inactivated fetal bovine serum (FBS), and 1% (v/v) penicillin-streptomycin (100 U/mL and 100 μg/mL, respectively) in a humidified 37 °C incubator with 5% CO₂ to between 80−90% confluency in a 150 cm² tissue culture flask.
2. In a biosafety cabinet (BSC), remove the medium and dispose of in either 10% bleach or a working dilution of dual quaternary ammonium (Table of Materials). Immediately inoculate the monolayer with 1 mL of viral stock, aiming for 0.1−1 infectious viral particle per cell. Agitate the flask immediately such that the inoculum contacts the monolayer in its entirety.
3. Top up the medium to a volume of 5 mL using DMEM supplemented with 2% v/v heat-inactivated FBS, and 1% (v/v) penicillin-streptomycin. Then move the flask into the humidified 37 °C incubator with 5% CO₂ for 60 min.
4. Remove the flask from the incubator and bring it into a BSC. Add additional medium to a total volume of 15 mL using DMEM supplemented with 2% v/v heat-inactivated FBS, and 1% (v/v) penicillin-streptomycin. Move the flask into a humidified 37 °C incubator with 5% CO₂.
5. Examine the flask daily under phase contrast microscopy for evidence of cytopathic effects (CPE). Proceed to the viral harvest (step 1.7) when only approximately 40−50% of the cells remain in the monolayer, generally 3−5 days postinfection depending on the strain of ZIKV being utilized.
6. Aspirate the supernatant and place in a 50 mL conical vial. Clarify the supernatant of cellular debris by centrifugation (3,500 x g for 20 min).
   NOTE: If multiple flasks were infected identically, supernatants from multiple flasks can be combined into 50 mL conical tubes.
7. Remove the supernatant from the 50 mL conical tube to a fresh one, taking care not to disrupt the pellet. Supplement the supernatant with heat-inactivated FBS to a final concentration of 30% (v/v). Aliquot this mixture into individual screw cap tubes and freeze at -80 °C until use.

2. Preparation of artificial bloodmeals

1. On the day mosquitoes are to be exposed to infectious bloodmeals, turn on the power source (Table of Materials) in an arthropod containment facility such that the feeding units (Table of Materials) are preheated by the time the mosquitoes are prepared for exposure.
2. Prepare artificial bloodmeals using one of the methods described below.
   1. Method 1: Combine freshly harvested (within a week) citrated or heparinized human blood purchased commercially 1:1 v/v with viral stock (prepared as described in section 1).
      NOTE: This method is contingent on the absence of any antibodies to the virus/virus family being present in the blood.
   2. If absence of antibodies cannot be confirmed or the blood source is known to have prior flavivirus exposure, wash and manually pack erythrocytes.
      1. In a BSC, add 30 mL of whole human blood to a 50 mL conical tube and top up to 50 mL with phosphate buffered saline (PBS).
      2. Centrifuge at 3,500 x g for 20 min.
      3. Aspirate the supernatant either by gently pouring into a tray pan containing either 10% bleach or working dilution of dual quaternary ammonium, taking care to not discard the erythrocyte pellet.
      4. Add 10 mL of PBS and gently tap the bottom of the conical tube against the bottom of the BSC such that the erythrocyte pellet has been reconstituted. Bring the volume of the suspension up to 50 mL with PBS. Mix by gentle inversion.
      5. Repeat steps 2.2.2.1−2.2.2.4 a total of 4−6x. Confirm that the supernatant is clear or only slightly pink and no longer opaque. Remove all the supernatant using a serological pipette. Resuspend the erythrocyte pellet in 1−2 mL of PBS.
      6. Assemble the bloodmeal: 350 μL of packed erythrocytes, 100 μL of 10% sucrose, 200 μL of heat-inactivated FBS, 900 μM recombinant ATP, and 2 mL of appropriately diluted virus stock using DMEM supplemented with 2% v/v heat-inactivated FBS, and 1% (v/v) penicillin-streptomycin.
3. Overlay a standard 3 mL reservoir unit (Table of Materials) with the skin of an uninfected mouse (other options include paraffin film, collagenous membranes, or sausage casing).
4. Place the covered reservoir on white paper towels. Add ~2 mL of infectious bloodmeal to the reservoir 1 mL at a time. Inspect the towel underneath the feeder for any evidence of leakage. If leaks are present, recover the bloodmeal from the feeders and discard the covers. Seal the feeders with plugs. Once again confirm that no leaks are present.

3. Backtitration of bloodmeals/plaque assay

1. Using the remaining volume of the prepared bloodmeal, perform a 10x serial dilution series (i.e., 6 dilutions, ranging from diluted 10x to 1,000,000x) using DMEM supplemented with 2% v/v heat-inactivated FBS, and 1% (v/v) penicillin-streptomycin.
2. Aliquot 100 µL of the dilutions into the wells of 24 or 12 well plates from the most dilute to most concentrated.
3. Incubate for 1 h in a 37 °C, 5% CO₂ incubator.
4. At the end of the 1 h incubation period, bring the plates back into the BSC and add 1 mL or 2 mL of methylcellulose overlay to the 24 well or 12 well plates, correspondingly.
5. Place overlayed plates back in a 37 °C, 5% CO₂ incubator and incubate for 3−7 days (virus strain-dependent).
6. Following incubation, remove the plates from the incubator and bring into the BSC. Discard the methylcellulose overlay into a tray pan containing 10% bleach or dual quaternary ammonium disinfectant.
7. Wash each well 2x with PBS, discarding the wash into a tray pan containing 10% bleach or dual quaternary ammonium.
8. Add ~1 mL of methanol:acetone (1:1 v:v) and allow the cells to fix onto plate for at least 30 min in the BSC at room temperature (RT). Discard methanol:acetone according to institutional policy on organic waste.
9. Visualize ZIKV using one of two methods described below.
   1. Following the removal of methanol:acetone, stain immediately with a crystal violet solution (0.25% w/v in 30% methanol) for 5 min. Rinse 2x in tap water and leave to dry, then directly visualize by eye for evidence of plaques or destruction of monolayer.
   2. Alternatively, perform focus forming assay.
      1. Allow the plates to air dry until no organic fixative remains.
         NOTE: This should take ~2−3 h outside of the BSC but can be accelerated by air-drying in a BSC or chemical fume hood.
      2. Wash each well 3x for 15 min each in nonsterile PBS (Mg²⁺ and Ca²⁺ free) on an orbital plate rocker. Remove PBS and add 1 mL of blocking solution (PBS + 3% FBS) to each well and rock for 15 min at RT.
      3. Add 100 μL per well of α-ZIKV or α-flavivirus primary antibody (e.g., flavivirus group hybridoma D1-4G2-4-15 [4G2]) at a 1:2,000 dilution in blocking solution. Incubate with rocking for a minimum of 4 h (preferably overnight, not exceeding 18 h) at RT.
      4. Remove the primary antibody and wash 3x for 15 min each with PBS (Mg²⁺ and Ca²⁺ free) on an orbital plate rocker.
      5. Add 100 µL per well of the secondary antibody (goat α mouse HRP-labeled) diluted 1:2,000 in blocking buffer. Incubate with rocking for 1 h at RT.
      6. Wash 3x for 15 min each with PBS (Mg²⁺ and Ca²⁺ free) on an orbital plate rocker.
      7. Aliquot 100 µL of substrate development reagent (Table of Materials) per well. Rock plates at RT for 15 min.
      8. Halt the reaction upon development of foci/plaques by removing the substrate and rinsing the plates 2x with tap water. Pour off tap water and allow the plates to air dry before quantifying.
      9. Count viral foci to determine the number of FFU present in the given sample.

4. Administration of bloodmeals

1. Use Ae. aegypti mosquitoes 2−4 days post eclosion. Sort female mosquitoes into 0.5 L cardboard cartons with screened lids and deprive them of sugar (generally 36−48 h prior to the infectious bloodmeal). Provide ad libitum water via water-saturated cotton balls.
2. Remove the water-saturated cotton balls on the morning that the mosquitoes will be exposed to the infectious bloodmeal.
3. Attach reservoirs containing artificial bloodmeals to feeding unit leads within a clear plastic glove box.
4. Within the glovebox, place a 0.5 L cardboard carton with a screened lid, containing 50−100 starved Ae. aegypti mosquitoes, underneath the feeding unit attached to the reservoir.
   NOTE: Appropriately starved mosquitoes will generally feed within 20 min. Feeding can be prolonged as needed to increase sample size in slower populations, although this should not extend beyond 60 min, because (ZIKV) viral titer can decrease within the feeder after ~60 min.
5. Upon completion of feeding, remove the reservoir and immerse in freshly made 10% bleach.
6. Cold-anesthetize mosquitoes by incubation for 30 s at -20 °C or for 5 min in a refrigerator.
7. Within the glovebox, pour the mosquitoes into a Petri dish on ice. Count and sort the engorged females from unengorged mosquitoes. Dispose of the unengorged mosquitoes by immersion in a 50 mL tube conical tube filled with 70% ethanol. While the mosquitoes are still anesthetized, pour them back into the 0.5 L cardboard carton and quickly cover with the screen and lid. Trim excess screen mesh off of the carton and secure the mesh with tape.
8. Add a cotton ball saturated with sterile filtered 10% aqueous sucrose to the screen of each carton. Place all mosquito cartons in a large plastic secondary container with a damp sponge to maintain humidity.
9. Place secondary container containing cartons of mosquitoes into an incubator with a temperature of 27 ± 1 °C (or as appropriate to simulate conditions in region of interest) with 80% ± 10% relative humidity and a 16:8 light:dark cycle). Maintain the mosquitoes with ad libitum access to 10% sucrose until completion of the experiments.

5. Sample acquisition and processing

1. On specified days postfeeding, aspirate a predetermined number of mosquitoes from the appropriate cartons using a mechanical aspirator within a glovebox. Cap the collection tube with a cotton round after the requisite number of mosquitoes is acquired.
2. Cold-anesthetize mosquitoes by incubation for 30 seconds at -20 °C or for 5 minutes at 4 °C.
3. Within the glovebox, pour the mosquitoes into a Petri dish on ice. Using two pairs of forceps, remove all six of each mosquito’s legs and place in a prelabeled 2 mL round bottom microcentrifuge tube containing a sterilized stainless steel ball bearing (7/32”) and 500 μL of mosquito collection media (MCM) composed of DMEM, 2% FBS, 1% penicillin-streptomycin, and 2.5 μg/mL amphotericin.
4. Gently place the mosquito onto a drop of mineral oil to restrain it, taking care to not allow any contact between the oil and the mosquito’s head and proboscis.
5. Insert the mosquito proboscis into a 10 μL pipette tip filled with 10 μL of heat-inactivated FBS.
   NOTE: Alternatively, the pipette can be filled with sucrose, blood, or mineral oil. The oil allows for direct visualization of saliva bubbles via light microscopy.
6. Allow the mosquito to salivate for 30 min. Eject the micropipette tip containing FBS + saliva into a microcentrifuge tube containing 100 μL of MCM, then place the carcass into a separate 2 mL round bottom microcentrifuge tube containing a sterilized steel ball bearing and 500 μL of MCM. Ensure that the tubes used for the bodies, legs, and saliva are labeled so that it is clear all three samples originated from the same mosquito.
7. While the mosquito(s) are salivating, perform steps 5.3−5.5 on the remaining mosquitoes.
8. Transport the tubes containing the bodies and legs to a bead milling tissue homogenization device contained within a BSC. Triturate all body and leg samples at 26 Hz for 5 min to liberate viral particles into the supernatant. Clarify all samples by centrifugation at 200 x g for 5 min to pellet cellular debris.
   NOTE: At this point, samples can be frozen at -80 °C, or assayed immediately.

6. Detection of ZIKV by infectious assay

1. In a BSC, prepare 24 well tissue culture plates with Vero cells (10⁵ cells per well) 24 h prior to the onset of the infectious assay. Label each well with the identity of a single mosquito/sample.
2. If samples were frozen at -80 °C, allow to thaw.
3. Remove the media from the Vero cell plates prior to inoculation with samples one plate at a time.
4. For samples containing bodies or legs, carefully aliquot 100 μL of clarified supernatant into each well, taking care not to disturb the mosquito cell debris from the pellet.
   NOTE: Saliva samples can be diluted 1:1 (v/v) with MCM prior to inoculation onto cells to conserve samples for later titration, if necessary.
5. Move the plates into a 37 °C, 5% CO₂ incubator and incubate for 1 h.
6. Return the plates to the BSC and add ~1 mL of methylcellulose overlay to each well. Place the overlayed plates back in a 37 °C, 5% CO₂ incubator and incubate for 3−7 days (virus/strain-dependent).
7. Perform fixation and visualization as described in steps 3.6−3.9.
8. Regarding scoring via a focus forming assay, quantify the positive wells by examination under a light microscope. Detection of intracytoplasmic staining of cells in a well indicates the presence of virus. Samples are scored solely as focus-positive or -negative.