Experimental Viral Infection in Adult Mosquitoes by Oral Feeding and Microinjection

Summary

This methodology, which included oral feeding and intrathoracic injection infection, could effectively assess the influence of midgut and/or salivary gland barriers on arbovirus infection.

Abstract

Mosquito-borne viruses (MBVs), which are infectious pathogens to vertebrates, are spread by many mosquito species, posing a severe threat to public health. Once ingested, the viruses must overcome the mosquito midgut barrier to reach the hemolymph, from where they might potentially spread to the salivary glands. When a mosquito bites, these viruses are spread to new vertebrate hosts. Similarly, the mosquito may pick up different viruses. In general, only a tiny portion of viruses may enter the salivary glands via the gut. The transmission efficiency of these viruses to the glands is be affected by the two physical barriers found in different mosquito species: midgut barriers and salivary glands barriers. This protocol presents a method for virus detection in salivary glands of Aedes aegypti's following oral feeding and intrathoracic injection infection. Furthermore, determining whether the guts and/or salivary glands hinder viral spread can aid in the risk assessments of MBVs transmitted by Aedes aegypti.

Introduction

Mosquito-borne viruses (MBVs), a heterogeneous group of RNA viruses, can persist in mosquito vectors and subsequently spread to vertebrate hosts¹. The clinically important MBVs are majorly distributed in four virus families, namely Flaviviridae, Togaviridae, Reoviridae, and Peribunyavividae^2,3. In recent decades, these viruses have been reported all across the globe, causing public health issues. As one of the most well-known MBVs, Dengue virus (DENV) has become the most prevalent emerging or re-emerging arbovirus in over 100 countries during the last 20 years⁴. Since the discovery of Zika virus (ZIKV) inland, almost all tropical and subtropical countries and territories of the continent have reported human ZIKV infections⁵. In order to assess the risk of virus transmission, numerous studies in recent years have focused on mosquito vector competence for these viruses^6,7. As a result, it is critical to effectively prevent and control vector-borne diseases.

Aedes aegypti (Ae. aegypti), one of the most easily reared mosquitoes in the laboratory, is an important vector of DENV, ZIKV, Chikungunya virus (CHIKV), and yellow fever virus (YFV)⁸. For a long time, Ae. aegypti was solely found in the African continent and in Southeast Asia, but in recent years it has colonized nearly all continents⁹. Furthermore, the global abundance of Ae. aegypti has been continuously growing, with an estimated 20% increase by the end of the century¹⁰. From 2004 to 2009 in China, there was an evident increase in Ae. aegypti vector competence for DENV due to higher day-to-day temperatures¹¹. The status of Ae. aegypti as the pathogenic vector has significantly risen in China. Consequently, to address these challenges, it is necessary to investigate the vector competence of Ae. aegypti's to transmit viruses.

As a haematophagous arthropod, the female mosquito pierces the skin of a vertebrate host and feeds on the blood. Mosquitoes do occasionally acquire viruses from virus-infected hosts and then transfer the viruses to a new host. As such, to determine vector competence, mosquitoes are fed an artificial bloodmeal containing arboviruses through a feeding system in the laboratory setting¹². Individual mosquitoes are separated into heads, bodies, and saliva secretions several days after infection. To measure virus infection, dissemination, and transmission rates, virus titers have been detected by quantitative reverse-transcription PCR (qRT-PCR) or plaque assay. However, not all mosquitoes develop midgut infections and the capacity to transfer a virus to the next host following blood feeding. It is linked with mosquitoes' physiological barriers, which prevent pathogens from penetrating the body and play a vital role in their innate immunity¹³. The midgut barriers, particularly the midgut infection barrier (MIB) and midgut escape barrier (MEB), influence whether the virus could infect the vector systemically and the efficiency with which it spreads. It obstructs the analysis of other tissues' infection, such as salivary glands which also exhibit salivary gland infection and escape barriers^13,14. To better characterize the infection of midguts and salivary glands in the vector, a detailed protocol for oral feeding and intrathoracic inoculation of arbovirus in Ae. aegypti is presented herein. This protocol might be applied to additional arbovirus infections in a variety of mosquito vectors, such as DENV and ZIKV infection in Aedes spp., and could prove to be a practicable procedure.

Protocol

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.

Representative Results

To examine EBIV distribution in the infected mosquitoes via artificial blood feeding (the viral final titer was 6.4 x 10⁶ PFU/mL) and intrathoracic injection (the viral dose was 340 PFU), viral RNAs in saliva, heads, and guts of the mosquitoes at 10 days post infection (dpi) were determined.

For Ae. aegypti, virus titer of EBIV in the guts, heads, and saliva of the intrathoracically inoculated female mosquitoes were much higher than that in the oral-infected female mosquitoes (Figure 2A-C). Infection rate and dissemination rate for the intrathoracically inoculated female mosquitoes was 100%, but for the oral-infected female mosquitoes was 70% and 38.1%, respectively (Figure 2D, E). Transmission rate for the intrathoracically inoculated female mosquitoes reached up to 90%, but for the oral-infected female mosquitoes was only 4.8%. It indicated that the gut of Ae. aegypti had a strong inhibitory effect for virus transmission.

Figure 1: Schematic of micropipette tip and intrathoracic injection. (A) Pulled micropipette tip before breaking the tip. (B) Properly prepared micropipette tip suitable for intrathoracic inoculation. (C) Schematic of intrathoracic injection for the mosquitoes. Please click here to view a larger version of this figure.

Figure 2: Vector competence of adult mosquitoes through oral feeding and intrathoracic inoculation. The infected female mosquitoes were incubated at 28 °C for 10 days. p value ≤ 0.05 was considered statistically significant. The non-parametric Kruskal-Wallis analysis was used for multiple comparisons and Fisher's exact test where appropriate. This figure has been modified from study by Xia et al.¹⁸. Please click here to view a larger version of this figure.

Discussion

The goal of this method was to provide a comprehensive risk assessment of one mosquito-borne virus by evaluating vector competence through oral feeding and intrathoracic inoculation.

In the oral-feeding experiment, engorged-mosquitoes need to be picked out and transferred to a new container, posing a severe risk to the operators. The reason for this is because any mosquito, including uninfected mosquitoes, might be a source of infection¹⁹. Consequently, mosquitoes must be anesthetized first, as specified in the protocol, and then the follow-up steps should be performed. It is important to note that selecting out engorged mosquitoes and transferring them should be performed in a sealed glove box. Keep the glove box closed during the process unless there are no freely moving mosquitoes in the box.

In the infection experiment, it is better for two individuals to work together, and after the two people confirm that there are no escaping mosquitoes at the same time, they can open the glove box to prevent infected mosquitoes from escaping. If an escape takes place, those mosquitoes must be killed or recaptured. It is not recommended to kill infected mosquitoes with bare hands. Operators' infection risk can be reduced by using vacuum aspirators or other appropriate tools (e.g., forceps, paintbrushes, gloved hands). After the experiment, disinfect the inside of the glove box and the surface of the workbench with a conventional disinfectant.

The majority of research about mosquito vector competence for mosquito-borne viruses has solely focused on oral infection^20,21. For oral infection, viruses must overcome several tissue barriers associated with the midgut and salivary glands to be released into saliva¹⁴. The effect of midgut gland and salivary gland on mosquito vectorial capacity was not demonstrated in this experiment. When oral feeding and intrathoracic injection are combined, the different tissue barriers may be thoroughly evaluated. According to the results of this procedure, the gut of Ae. aegypti plays a dominant role in the vector competence for the virus and the salivary gland barrier may not be present (Figure 2). For this, our protocol is beneficial to confirm the presence of midgut or salivary gland barriers to arboviruses infection.

According to the methodology, only viral RNA was identified in the samples. Other investigations, such as seeing the virion with a transmission electron microscope, confirming the infection using plaque assays, or detecting viral protein with an immunofluorescence assay, are required to confirm the infectious viruses in the various samples. This methodology may be modified and used to inoculate additional viruses of interest into a wide range of mosquito vectors.

Materials

Name	Company	Catalog Number	Comments
Aedes aegypti			Rockefeller strain
Automated nucleic acid extraction system	NanoMagBio	S-48
BHK-21 cells	National Virus Resource Center, Wuhan Institute of Virology
Buckets
C6/36 cells	National Virus Resource Center, Wuhan Institute of Virology
Carbon dioxide spray gun	wuhan Yihong	YHDFPCO2
Centrifugal machine	Himac	CF16RN
CFX96 Touch Real-Time PCR Detection System	Bio-Rad	CFX96 Touch
Ebinur Lake virus			Cu20-XJ isolation
Formaldehyde	Wuhan Baiqiandu	B0003
Glove box
Glucose	Hushi	10010518
Immersion oil	Cargille	16908-1
Insect incubator	Memmert	HPP750T7
Low Temperature Tissue Homogenizer Grinding Machine	Servicebio	KZ-III-F
Magnetic Virus Genome Extraction Kit	NanoMagBio	NMG0966-16
mesh cages (30 x 30 x 30 cm)	Huayu	HY-35
methylcellulose	Calbiochem	17851
mice feedstuff powder	BESSN	BS018
Microelectrode Puller	WPI	PUL-1000	PUL-1000 is a microprocessor controlled horizontal puller for making glass micropipettes or microelectrodes used in intracellular recording, patch clamp studies, microperfusion or microinjection.
Mosquito net meshes
Nanoject III Programmable Nanoliter Injector	Drummond	3-000-207
One Step TB Green PrimeScript PLUS RT-PCR Kit	Takara	RR096A
PBS, pH 7.4	Gibco	C10010500BT
Penicillin/streptomycin	Gibco	151140-122
Petri dishes
Plastic cupes (7 oz)	Hubei Duoanduo
Plastic cups (24 oz)	Anhui shangji	PET32-Tub-1
Plastic disposable droppers	Biosharp	BS-XG-O3L-NS
Refrigerator (-80 °C)	sanyo	MDF-U54V
Replacement Glass Capillaries	Drummond	3-000-203-G/X
RPMI medium 1640	Gibco	C11875500BT
Screw cap storage tubes (2 mL )	biofil	FCT010005
Shallow dishes
Sponge
Sterile defibrillated horse blood	Wuhan Purity Biotechnology	CDHXB413
T75 culture flask	Corning	430829
The artificial mosquito feeding system	Hemotek	Hemotek PS6
The dissecting microscope	ZEISS	stemi508
The ice plates
The mosquito absorbing machine	Ningbo Bangning
The pipette tips	Axygen	TF
Trypsin-EDTA (0.25%)	Gibco	25200056
Tweezers	Dumont	0203-5-PO