Vector Competence Analyses on Aedes aegypti Mosquitoes using Zika Virus

An aprox mediated by arbovirus, as countermeasures are largely limited to abatement of mosquitoes. So vector competence analyses are critical prevention strategies because they determine what populations and species of mosquitoes can serve as vectors for a given arbovirus, and therefore should be targeted. So one of the most important components in our toolbox for assessing the role of mosquitoes, and other vectors in transmitting infectious diseases, is the vector competence study.

These are experiments where we can very precisely control, not only the species of mosquito, or tick, for example, but also geographic strains, as well as very specific strains of a different virus or other infectious organism to get very precise and well-controlled data on the risk of different species for transmitting different agents in very specific parts of the world. Working with infected mosquitoes requires dedicated facilities, training, experience, and careful planning to minimize the risks to the experimenter, as well as the risks of an escaped, infected mosquito. It’s therefore quite important for new individuals in these facilities to take the time to work with uninfected blood mosquitoes to familiarize themselves with the techniques and workflow.

On the day of exposure, turn on the power source in an Arthropod containment facility to preheat the feeding units. Combine freshly harvested citrated or heparinized human blood with a viral stock at a one to one volume ratio, and overlay a standard three milliliter reservoir unit with the skin of a naive uninfected mouse. Place the covered reservoir on white paper towels, and add about two milliliters of infectious blood to the reservoir one milliliter at a time.

Then, inspect the paper towels to confirm that there is no leakage. 24 hours prior to performing the infectious assay, prepare 24-well cell culture plates with Vero cells, and label each well with the identity of the mosquito and sample. On the morning that the mosquitoes will be exposed to the infectious blood meal, remove the water saturated cotton balls, and attach reservoirs with the artificial blood meals to the feeding unit leads with a clear plastic containment glove box.

Place the cardboard carton with the starved mosquitoes into the glove box underneath the feeding unit. Upon completion of feeding, remove the reservoir, and immerse it in freshly prepared 10%bleach. Within the glove box, pour cold anesthetized mosquitoes into a Petri dish on ice, and sort the engorged from the un-engorged mosquitoes.

Pour the engorged mosquitoes back into the cardboard carton, quickly covering it with a screen and lid. Trim the excess screen mesh off of the carton, and secure the lid with tape. Add a cotton ball saturated with sterile filtered 10%sucrose to the screen, and place the carton in a large plastic secondary container with a damp sponge to maintain humidity.

Incubate the secondary container at 27 degrees celsius, allowing mosquitoes ad libitum access to the sucrose until completion of experiments. Use a mechanical aspirator to acquire a predetermined number of incubated mosquitoes from the carton, capping the collection tube with a cotton round when finished. Working within the glove box, pour cold anesthetized mosquitoes into a Petri dish on ice.

Remove all six of each mosquitoes legs, and place them in a pre-labeled two milliliter micro centrifuge tube with a sterilized stainless steel ball bearing and 500 microliters of mosquito collection media, or MCM. Gently place the mosquito onto a drop of mineral oil, taking care that the oil does not contact the mosquito’s head or proboscis. Insert the proboscis into a micro pipette tip filled with 10 microliters of heat inactivated FBS, and allow the mosquito to salivate for 30 minutes.

Eject the pipette tip with the saliva into a micro centrifuge tube with 100 microliters of MCM, and place the carcass into a separate two millimeter tube with the sterilized steel ball bearing and 500 microliters of MCM. Transport the tubes to a bead milling tissue homogenization device within a bio safety cabinet, and triturate all body and leg samples at 26 hertz for five minutes to liberate viral particles into the supernatant. Clarify all samples by centrifugation at 200 times G for five minutes to pelite cellular debris.

Immediately before inoculation with samples, remove the media from the previously prepare Vero cells. Carefully aliquot 100 microliters of clarified supernatants from the bodies or legs into each well, taking care not to disturb the mosquito cell debris from the pelite. Then, incubate the plates for one hour.

After the incubation, add one milliliter of methylcellulose overlay to each well, and put the plates back in the incubator for three to seven days. Remove overlay plates from the incubator, and bring them to a bio safety cabinet. Discard the methylcellulose overlay into a tray pan containing 10%bleach, and wash each well twice with PBS, discarding each wash into the tray pan.

Remove the PBS, and add one milliliter of methanol-acetone per well, and allow the cells to fix onto the plate for 30 minutes. Then, discard the fixative, and leave the plates to dry. Wash each well three times with non-sterile PBS on an orbital plate rocker for 15 minutes per wash, then add one milliliter of blocking solution to each well, and rock the plate for another 15 minutes.

Add 100 microliters of anti Zika Virus, or anti flavivirus primary antibody to each well, and incubate the plate with rocking overnight. On the next day, remove the primary antibody, and repeat the washes with PBS. After the last wash, add 100 microliters of secondary antibodies, and incubate the plate while rocking for one hour.

Repeat the washes with PBS, then add 100 microliters of substrate development reagent per well, and rock the plate for 15 minutes. When the foci or plaques develop, halt the reaction by rinsing the plate with tap water. Pour off the tap water, and allow the plates to air dry, then quantify the viral foci to determine the number of focus forming units present in the given sample.

Three mosquito populations were exposed to an outbreak strain of Zika Virus over a range of blood meal titers. Over the following two weeks, subsets of mosquitoes were processed to determine infection, dissemination, and potential transmission rate. At the lower blood meal titers, the mosquitoes from Salvador, Brazil were infected after 4, 10, and 14 days of extrinsic incubation, with no evidence of disseminated infection observed in the assayed legs.

At the highest titer, the virus was detected in the legs of the mosquitoes, but not in the saliva. The mosquitoes from the Dominican Republic proved to be the most susceptible to infection, and the most transmission competent at all tested blood meal titers. Mosquitoes fed all three doses demonstrated dissemination by seven days, and transmission competence at 14 days post infection.

Mosquitoes from the Rio Grande Valley in Texas, exposed to lower blood meal titers, were infected as early as four days post infection, while disseminated infections were found after 14 days. In the cohort exposed to the highest titer, infection was observed beginning from the second day post infection, and reached peaks at 4, 10, and 14 days. Disseminated infections were observed at seven days, and only a single mosquito was transmission capable at 10 days.

When attempting this protocol, it’s important to work safely, as opposed to quickly. So always handle infected or potentially infected mosquitoes within the confines of a glove box or a bio safety cabinet, until they’ve had their legs or wings removed. The basic vector competence workflow can be adapted to produce infected mosquitoes for downstream analyses, such as transmission studies, as well as to examine the interactions between mosquitoes, the virus, and the microbiome.

Vector competence has been a really important tool for understanding the role of mosquitoes in transmitting viruses during epidemics of viral disease, and this has been true for many decades. But it’s also a method that’s evolved over the years to include new information, as we learn it, about the interactions between these viruses and the mosquito vectors. Just by way of two examples, the genetic population of the virus, the complexity of the population within the mosquito turns out the be very important for its ability to transmit, and we can now include that in vector competence studies with new methods for sequencing the viral population.

We also now know that the population of bacteria and other organisms that live in the gastrointestinal tract of the mosquito can also have a major impact on their ability to transmit, and we can also evaluate that population now with new methods in sequencing. So vector competence is a method that has evolved and continues to evolve as we learn more about these complex interactions.