Experiment Summary

Aedes aegypti mosquitoes are the main vectors of many medically relevant arthropod-borne (arbo) viruses, including Zika (ZIKV), dengue (DENV), and yellow fever (YFV). Vector competence studies with Ae. aegypti often involve challenging mosquitoes with an artificial bloodmeal containing virus and later quantifying viral titer or infectious plaque-forming units (PFU) in various mosquito tissues at relevant time points post-infection. However, Ae. aegypti mosquitoes are known to exhibit midgut infection and escape barriers (MIB and MEB, respectively), which influence the prevalence and titer of a disseminated infection and can introduce unwanted variability into studies analyzing tissues such as the salivary glands. To surmount this challenge, we describe herein a protocol for the intrathoracic inoculation of ZIKV in Ae. aegypti. This method bypasses the midgut, which leads to a more rapid and higher proportion of disseminated infections in comparison to oral challenge, and mosquitoes become infected with a consistent dose of virus. Our protocol is advantageous for studies that need a large sample size of infected mosquitoes, need to bypass the midgut, or are analyzing salivary gland infection or escape barriers.

Materials and Reagents

1. 1.5 ml polypropylene Eppendorf tubes
2. Pipette tips
3. 3M Permanent double-sided tape ½"
4. Plastic Petri dishes
5. Glass capillaries, 3.5"
6. Glass Petri dishes, 100 × 15 mm
7. 64 oz. white double poly-coated paper food cup with pen-sized hole punched out covered with rubber stopper
8. Rubber stopper (for mosquito enclosement)
9. 30 G × ½" hypodermic needles
10. 1 ml syringe
11. PCR tubes
12. Kimwipes
13. 1 oz. translucent plastic souffle cups
14. 2 oz. translucent polystyrene souffle cups
15. 3-4 days old Aedes aegypti female mosquitoes
16. Vero cell
17. Sugar source, such as sugar cubes or raisins
18. Ice
19. Ice bucket
20. Mineral oil
21. Kendall/Covidien Curity all-purpose sponges, non-sterile, non-woven, 4-Ply, 3" × 3" (7.6 × 7.6 cm) (Model 9023)
22. White chiffon fabric 8" × 8"
23. Dulbecco's Modified Eagle Medium
24. Frozen ZIKV stock at a concentration of at least 2 × 10⁶-8 × 10⁶ PFU/ml
25. 20% Bleach (Clorox)
26. 70% Ethanol

Equipment

1. Pipettes
2. Forceps
3. Leica GZ4 StereoZoom microscope
4. Dual Gooseneck Microscope Illuminator
5. Sutter Instrument Co. Vertical Micropipette Puller
6. Nanoject II Auto-Nanoliter Injector

Procedure

A. Prepare intrathoracic inoculation needles and virus injection mix

1. Prepare micropipette needles for injections

a) Line the diameter of a plastic Petri dish with Scotch double-sided tape. This will be used to store the prepared micropipette needles so that their delicate tips will not be damaged.

b) Place a single 3.5" glass capillary into the Sutter Instrument Co. Vertical Micropipette Puller.

c) Set the micropipette puller to heat 999, Pull 003.

d) Pull needle.

e) Place the needle on top of the double-sided tape in the previously prepared Petri dish for storage until use.

2. Prepare virus injection mix

a) Thaw frozen ZIKV stock on ice.

b) Dilute virus with DMEM 7% FBS in a 200 μl PCR tube. The concentration should be ~2 × 106-8 × 106 PFU/ml with a target injection concentration of 100-500 PFU in 69 nl (the maximum volume for a single injection with the Nanoject II model instrument). See Recipes for relevant calculations.

c) Keep virus injection mix on ice.

3. Load micropipette with mineral oil

a) Using forceps, gently break the tip of a pulled needle so that it is wide enough to load injection mix (Figure 1) but not too blunt that it would harm the mosquito. Ideally, the tip size should be 10-30 microns in size. The tip of the needle should be visually inspected under the microscope to ensure it is not overly blunt. Overly blunted needles may cause mosquito mortality after injection.

Fig. 1 Micropipette tip preparation for injection.

b) Fill a 1.5 ml Eppendorf tube with mineral oil.

c) Using a 30 G × ½" gauge and syringe, backfill the needle by injecting oil into it through the non-pointed side. The Nanoject II will not operate properly without backfilling the needle before attaching it to the injector.

d) Set the injection volume and speed on the Nanoject II Auto-Nanoliter Injector control panel (Figure 2A) according to manufacturer's instructions (Figure 2B). The volume and speed are controlled by the positions of the dip switches on the side of the control box. Volumes range from 2.3-69 nl, while speeds are either 23 (slow) or 46 (fast) nl/s. Once the volume has been selected, each time the INJECT button is pressed, the selected volume will be dispensed.

Fig. 2 Nanoject II Auto-Nanoliter Injector control panel settings.

e) Carefully place a pulled needle through the metal tip of the Nanoject following operating instructions until it stops into place. Give the needle a slight pull to confirm it is securely mounted.

f) Once the needle is secured, press and hold the EMPTY button until the wire plunger is ejected out ~½" from the end of the needle. Oil that was backfilled will be expelled.

g) Place the tip of the micropipette into the 1.5 ml Eppendorf tube filled with mineral oil. Be careful not to touch the tip of the needle to the bottom of the tube. Press the FILL button to allow the needle to fill with oil.

4. Load needle with virus injection mix

a) Place the tip of the needle into the virus injection solution.

Approximately 50 μl is the minimum volume for virus preparation in a 200 μl PCR tube. A fully loaded micropipette needle will contain approximately 2.5 μl of virus injection solution. If the injection volume in the PCR tube is too small, the user risks sucking air into the micropipette needle that could generate air bubbles, which could cause inaccurate injection volumes.

b) Press and hold the FILL button to allow the plunger to retract, drawing in the virus solution. Do not allow air bubbles to form in the needle. These bubbles can cause inaccurate injection volumes. There should be an obvious boundary between the virus injection mix and the mineral oil. The mineral oil layer also prevents the virus from contaminating any equipment.

c) Load the needle with sufficient virus injection mix to comfortably fill the needle (about halfway up). The amount may depend on one's injection speed: allowing virus to sit in the needle too long may decrease infectivity as the virus is no longer on ice. The Nanoject II will beep twice when the needle is fully loaded.

d) Once the needle is loaded, be careful not to damage the tip by touching it to any hard surface. This may blunt the tip. If tip becomes blunted, repeat Steps A3-A4.

e) To ensure that the virus injection mix flows freely from the needle, perform a few test injections onto a Kimwipe (thereafter spray Kimwipe with 70% ethanol solution to eliminate the virus).

B. Perform ZIKV intrathoracic inoculation on Ae. Aegypti

1. Prepare mosquitoes for intrathoracic inoculation by anesthetizing them at 4°C

a) Place a glass Petri dish with a lid on ice.

ⅰ. Arrange dual gooseneck microscope illuminator so that the light is centered on the Petri dish.

ⅱ. Adjust stereomicroscope so that the eyepiece is in focus on the Petri dish.

b) Place a small carton of Ae. aegypti in a 4°C fridge. Do not anesthetize too many at once, depending on one's injection speed. Leaving mosquitoes on ice for too long (> 30 m) may cause significant mortality.

c) Once mosquitoes are anesthetized (~5 min), pour them into the inside of the glass Petri placed on ice.

d) Using forceps, arrange a single mosquito on its side so that its target injection point is visible.

ⅰ.Using the microscope, visually inspect the mosquito thorax for the injection point. Upon careful examination, there is a small "V" shaped region of soft tissue that is unprotected from the mosquito cuticle. This is the target injection point. For reference, in Figure 3, a mosquito has been injected with 69 nl purple NuPAGE LDS Sample buffer, and the target injection point is indicated.

Figure 3. Ae. aegypti target injection site.

2. Perform ZIKV intrathoracic inoculation

a) Using your dominant hand, gently place the tip of the loaded needle into the mosquito's target injection point (Figure 4).

Figure 4. Intrathoracic inoculation procedure.

b) Inject virus injection mix into mosquito target injection point.

Once the needle tip is inserted into target injection point, press INJECT. The mosquito will slowly rise up a little as the injection mix is flowing. Multiple injections (up to 3 injections [= 207 nl total vol.]) can be made by simply pressing INJECT again. Pressing INJECT again before the first injection is complete will not produce a second injection. A beep sound indicates an injection.

c) Place mosquito back into a paper carton.

ⅰ. Place paper carton that acts as a mosquito cage, with a small hole punched out blocked by a rubber stopper, on its side (Figure 5A).

ⅱ. Remove rubber stopper from carton, and very gently wipe the mosquito off the needle at the edge of the carton's hole using an upward motion (Figure 5B). Replace rubber stopper.

Figure 5. Return infected mosquito into proper containment.

5. Leave carton of infected mosquitoes with sugar and water in proper containment

a) Place sugar source, such as a damp sugar cube or a raisin, on top of the mosquito carton.

b) Place water source on top of carton. We use a small water-filled plastic cup that is covered with 2 layers of gauze secured around the top by rubber bands.

c) Place infected mosquito carton in proper containment.