Experimental Viral Infection in Adult Mosquitoes by Oral Feeding and Microinjection

1. Preparation of viruses and mosquitoes

1. Preparation of viruses
   NOTE: All processes were carried out in a biosafety level 2 (BSL-2) laboratory. The level of biosafety contaiment used should be determined by the pathogen's risk assessment and regulations specific to nations and regions. The process must be performed in a biosafety cabinet.
   1. Inoculate 1 x 10⁶ C6/36 cells into a T75 culture flask. Fill the flask with 10 mL of Roswell Park Memorial Institute (RPMI) 1640 medium containing 10% heat-inactivated fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S).
      NOTE: Leibovitz's L-15 medium (L15) or Dulbecco's modified Eagle's medium (DMEM) can also be used to maintain C6/36 cells.
   2. Maintain the cells in 5% CO₂ at 28 °C overnight and remove the cell supernatant the next day when the cells are 80%-90% confluent.
   3. Add 1 mL of virus dilution (Multiplicity of Infection (MOI) = 0.01) into the flask and incubate the cells for 1 h in 5% CO₂ at 28 °C. Ebinur Lake virus (EBIV)¹⁵, a mosquito-borne orthobunyavirus, was used as a model virus for this protocol.
   4. Remove the supernatant and wash the cells 3x with 5 mL of PBS. Fill the flask with 15 mL of new RPMI medium with 2% FBS. Maintain the flask in 5% CO₂ at 28 °C.
   5. Collect the supernatant containing virus when the required viral titer is reached (after nearly 5 days, depending on the virus used). For EBIV, this titer value was 10⁷ plaque forming units (PFU)/mL. Sub-package them into individual 2 mL screw cap storage tubes and store the tubes containing 0.5-1 mL of virus at -80 °C.
2. Determination of viral titer
   NOTE: Determine viral titer through the plaque assays¹⁶. BHK-21 was used to determine viral titer of EBIV, due to the obvious cytopathic effect (CPE) observed in infected cells.
   1. Inoculate 1 x 10⁵ BHK-21 cells into each well of 24-well plates. Fill each well with 1 mL of DMEM medium containing 10% FBS and 1% P/S. Maintain the cells in 5% CO₂ at 37 °C overnight.
   2. Make six (10^-6) successive 10-fold dilutions of virus-containing supernatant with fresh DMEM medium containing 10% FBS and 1% P/S.
   3. Add 100 µL of virus diluent into each well of 24-well plates containing the monolayer BHK-21. Discard the virus diluent after incubation for 1 h in 5% CO₂ at 37 °C. Add 500 µL of DMEM containing 1.5% methylcellulose to each well.
   4. Culture cells in 5% CO₂ at 37 °C for 48 h (the apparent CPE was found after 48 h for EBIV). Fix the cells overnight with 750 µL of 3.7% formaldehyde.
   5. Stain them with 200 µL of 2% crystal violet for 15 min. Count the number of plaques to calculate the viral titer.
3. Mosquito breeding in the lab
   NOTE: Rear mosquitoes in an arthropod containment level 1 (ACL-1) laboratory.
   1. For the preparation of the dechlorinated water, fill a 20 L bucket with tap water and allow it to stand for 7-8 days without light. Do not cover the lid.
   2. Keep eggs and larvae of Ae. aegypti in a mosquito room with the following conditions: 28 ± 1 °C with a light/dark cycle of 12 h and a relative humidity of 75% ± 5%.
   3. Maintain nearly 1,000 larvae in a shallow dish (22 cm x 15 cm x 6 cm) containing dechlorinated water after the eggs hatch. Feed them with nearly 1/8 teaspoon of mice feedstuff powder. Refresh the food daily and change the water every 2 days.
   4. Once the larvae pupate after 5-7 days, use a one-off dropper to transfer pupae to a plastic cup (7 oz) filled with dechlorinated water.
   5. Put five cups containing pupae (about 200 per cup) into one mesh cage (30 cm x 30 cm x 30 cm) and keep the cages in the insect incubator with the same conditions as in step 1.3.2.
   6. Put a tiny sponge containing an 8% (m/w) glucose solution on each mesh cage to feed adult mosquitoes. After adult emergence, take out the cups without pupae. Keep the mesh cages with about 1,000 adult mosquitoes per cage in the same insect incubator.

2. Preparation for oral infection

1. Prepare female mosquitoes for oral infection. Use the mosquito absorbing machine to collect 5-8-day-old mosquitoes. Anesthetize them at -20 °C for 1 min and check to see whether the mosquitoes are dizzy. If not, anesthetize them for another minute at -20 °C.
2. Pour them quickly into the plastic cups (24 oz) at about 200 mosquitoes (female and male) per cup. Cover each cup quickly with a well-cut mosquito net, followed by a lid with a hole (approximately 6 cm in diameter) in the middle.
3. Starve the mosquitoes for 24 h before the oral-infection and prepare infectious blood meal as follows.
4. In a biosafety cabinet under BSL-2 conditions, take out the virus stock from the -80 °C freezer and thaw them on ice. Dilute the virus stock to a concentration of 2 x 10⁶ PFU/mL with RPMI 1640 medium containing 10% FBS and 1% P/S. Mix the virus dilution with an equal volume of defibrated horse blood.
   NOTE: The processes were carried out in a biosafety cabinet in an ACL-2 laboratory. 

3. Perform oral infection

NOTE: All steps were performed in an ACL-2 laboratory. The process must be performed in the glove box to prevent mosquitoes from escaping.

1. Perform artificial feeding by using the artificial mosquito feeding system for 1 h.
   1. Cut the collagen membranes to the appropriate size (approximately 5 cm in diameter), secure them to the reservoirs with o-rings, then add the virus-blood mixture (approximately 3 mL) to the reservoirs. Use plastic plugs to seal reservoirs and prevent leaks.
      NOTE: These steps were performed in a biosafety cabinet.
   2. Put the plastic cups (24 oz) containing mosquitoes into the glove box. Screw the sealed reservoirs onto the FU1 Feeder and put one reservoir on one cup, then turn on the power unit. Feed the mosquitoes for 1 h
2. Anesthetize mosquitoes by CO₂ for several seconds until they faint. In brief, place the carbon dioxide spray gun in the middle of the net mesh through the hole on the cup containing mosquitoes, open the value, and allow huge amounts of carbon dioxide to be spewed out to anesthetize mosquitoes.
3. Pour them into a Petri dish placed on ice and cover the lid quickly. Pick out the engorged female mosquitoes with forceps. To identify, males have feathery antennae and females have plain antennae. Transfer them to new plastic cups at 50 female mosquitoes per cup.
   NOTE: The engorged female mosquitoes were picked to maintain consistency as feeding amount affects the viral content.
4. Wrap the cup with a cut mosquito net mesh and then cover it with a lid with a hole in the middle. Cut the sponge into a small piece and put it on the cup.
5. Add 8% glucose solution onto the sponge using a plastic disposable dropper. Keep the cups containing female mosquitoes in the incubator at 27 °C at 80% humidity for 10 days. Replace a new glucose-saturated sponge every 72 h.

4. Preparation for intrathoracic inoculation

1. Prepare the microinjection needles as follows. Use a PUL-1000 to make microinjection needles (Figure 1A) with the following parameters in the needle puller program: Heat index = 450, force (g) = 110, distance (mm) = 1.00, and delay (s) = 0.
2. Cut the tip of a pulled needle with tweezers under the dissecting microscope at 16x magnification (Figure 1B). Do not make the tip too big, otherwise it can harm the mosquitoes.
3. Prepare female mosquitoes as in step 2.1.
   NOTE: The following processes were performed in an ACL-2 laboratory. 
4. Prepare the infectious virus dilution as follows:
   1. Take out the virus stock from the -80 °C freezer and thaw them on ice. Dilute virus to a 100 nL virus dilution containing 100-500 PFU virus with RPMI 1640 medium containing 10% FBS and 1% P/S. Keep the virus dilution on ice.
5. Backfill the needle with mineral oil through a disposable sterile syringe. Attach the needle into the injector following the instruction on the machine.
6. Insert the needle into a tube containing the infectious virus dilution. Do not break the tip of the needle. Press the FILL button to fill the needle with virus solution. The final volume of the virus solution in the needle is approximately 4.0 µL. Once the needle is filled with the virus, be careful not to touch and damage it.
   NOTE: The steps were performed in a biosafety cabinet.

5. Perform viral injection under a dissecting microscope

NOTE: All steps were performed in an ACL-2 laboratory. 

1. Anesthetize mosquitoes at -20 °C for 1 min. Pour them into a Petri dish placed on ice and cover the lid quickly.
2. Pick out nearly 50 female mosquitoes with tweezers and put them on the ice plate. Place the ice plate under the injector and insert the needle into the thoracic cavity of mosquito under the dissecting microscope (Figure 1C).
3. Set the injection volume to 100 nL and rate to 50 nL/s. Press the INJECT button to let the virus solution flow into the mosquito. If successful injection occurs, the abdomen will slightly bulge.
4. Put the infected mosquito into a new plastic cup (24 oz) placed on ice. When the number of the infected mosquitoes is enough, wrap the cup with a cut mosquito net mesh and then cover it with the lid.
5. Keep the cup containing infectious mosquitoes in the incubator at 27 °C at 80% humidity for 10 days. Feed the mosquitoes as in step 3.5.

6. Processing of the infected female mosquitoes

NOTE: Three different tissues/organs were dissected from each female mosquito: the gut for calculating the virus infection rate (VIR), the head for calculating the virus dissemination rate (VDR), and saliva for calculating the virus transmission rate (VTR). VIR was defined as the number of mosquitoes with virus-positive guts. VDR was defined as the number of mosquitoes with virus-positive heads divided by the number of mosquitoes with virus-positive guts by 100. VTR was defined as the number of mosquitoes with virus-positive saliva divided by the number of mosquitoes with virus-positive guts by 100.

1. Collection of saliva from the infected female mosquitoes
   NOTE: The processes must be performed in an ACL-2 laboratory. 
   1. Anesthetize the infected female mosquitoes by carbon dioxide anesthetization as in step 3.2. Pour them into a Petri dish placed on ice and close the lid quickly.
   2. Place 10 µL pipette tips filled with immersion oil side by side on the rubber mud.
   3. Pick out female mosquitoes with tweezers and put them on an ice plate. Remove their legs and wings with tweezers. Place the mouthpart of one mosquito on each pipette tip.
   4. Collect the saliva as secreted into the oil by the mosquitoes for the next 45-60 min at room temperature.
   5. Place the pipette tips in 1.5 mL tubes containing 200 µL of virus diluent medium (RPMI 1640 medium containing 10% FBS and 1% P/S). Centrifuge them at 5,000 x g for 5 min at 4 °C to expel saliva into tubes. Store these tubes at -80 °C for subsequent determination of transmission rates.
2. Dissect the infected female mosquitoes
   NOTE: The processes must be performed in an ACL-2 laboratory. 
   1. Use tweezers to cut the heads of the female mosquitoes after saliva collection. Place each head into an individual tube containing 200 µL of RPMI 1640 medium.
   2. Grab the thorax with a tweezer, then the penultimate segment of abdomen with another tweezer and pull it out. The gut will be pulled out. Clean the pulled-out gut of any stray tissue and organs.
   3. Wash the gut with PBS and place each gut into an individual tube containing 200 µL of RPMI 1640 medium. Store these tubes at -80 °C for subsequent determination of the virus infection rate and dissemination rate.

7. Data analysis

1. RNA extraction
   1. Grind the tissues into pieces with a low temperature tissue homogenizer grinding machine set to the following parameters: operating frequency = 60 Hz, operation time = 15 s, pause time = 10 s, cycles = 2, and setting temperature = 4.0 °C.
   2. Centrifuge the samples at 12,000 x g for 10 min at 4 °C. Extract total RNA of each sample by automated nucleic acid extraction system following instrument instructions.
2. Performing qRT-PCR
   1. Use the one-step RT-PCR kit to quantify the viral RNA copies using a quantitative PCR instrument¹⁷. Use the following primers for the detection of EBIV S segment F: ATGGCATCACCTGGGAAAG and R: TTCCAATGGCAAGTGGATAGAA. Set up the reaction process as: stage 1: 42 °C for 5 min, 95 °C for 10 s; stage 2: 40 cycles of 95 °C for 5 s and 60 °C for 20 s.
   2. Determine the lowest virus dose for the infection in the cells through the serial 10-fold dilutions inoculated on cells. Use the BHK-21 cells to determine the lowest virus dose of EBIV, using dilutions till 10^-7 as described in step 1.2.
   3. Calculate the Ct value of the lowest virus dose by qRT-PCR. Use the cutoff value for EBIV-positive by qRT-PCR as < 35. Use the following equation to calculate the copies of EBIV in each sample:
      y = -4.0312x + 3.384 [x = log (the copies of EBIV), y = Ct value, R² = 0.9905]
   4. Calculate the infection rate, dissemination rate and transmission rate as follow:
      Infection rate (%) = 100 x (the number of virus-positive mosquito guts / the number of total mosquitoes)
      Dissemination rate (%) = 100 x (the number of virus-positive mosquito heads / the number of virus-positive mosquito guts)
      ​Transmission rate (%) = 100 x (the number of virus-positive mosquito saliva / the number of virus-positive mosquito guts)
   5. Analyze the differences in continuous variables and differences in mosquito infection rates, dissemination rates, and transmission rates by using the non-parametric Kruskal-Wallis analysis for multiple comparisons and Fisher's exact test where appropriate.