Anita Saraf investigates mosquito saliva to grasp how viruses like dengue and West Nile are transmitted, utilizing mass spectrometry to determine potential targets for vaccines and coverings.
You may guess it’d be powerful to extract saliva from a mosquito. And also you’d be appropriate. But, as a result of mosquitos unfold illness through saliva, it’s crucial to pattern and research.
However Anita Saraf doesn’t acquire mosquito spit herself — it will get delivered so she will be able to uncover its scientific secrets and techniques.
Saraf is the director of the Mass Spectrometry & Analytical Proteomics Laboratory on the College of Kansas, the place she makes use of shotgun proteomics to research organic samples — primary science that underpins many drug therapies.
“Gathering saliva from these mosquitoes is difficult because of the small pattern measurement, which is a typical limitation in such experiments,” she stated. “Our collaborators with the USDA Agricultural Analysis Service put the mouth of the mosquitoes right into a tube, then stimulate salivation and acquire the saliva.”
Now, with a brand new two-year grant of $250,000 per 12 months from the U.S. Division of Agriculture’s Nationwide Bio and Agro-Protection Facility, Saraf at the moment is analyzing these samples of noninfectious mosquito saliva within the battle towards “arboviruses” — the time period for viruses unfold by arthropods like mosquitoes.
These embrace West Nile, yellow fever, dengue fever, Japanese encephalitis, and different viruses that may hurt or kill individuals, animals, and crops. The USDA has an curiosity in veterinary purposes for the findings, however Saraf’s work might underpin therapies for individuals as nicely.
“The USDA desires to check how the virus impacts the immune system and adjustments on the molecular stage, significantly the proteome of mosquito saliva,” Saraf stated. “When a virus infects a mosquito, it alters the proteins and different primary elements of the mosquito, much like how malaria impacts its human host because it progresses by means of totally different phases.”
Proteomics Evaluation Strategies
Saraf’s proteomics evaluation of the saliva succeeds due to utilizing very environment friendly pattern processing strategies in tandem with shotgun evaluation. This strategy is efficient with very low volumes or quantities of pattern like, as an illustration, hard-to-obtain mosquito spit.
“At our mass spectrometry core at KU, we use shotgun proteomics, nanoscale liquid chromatography coupled with tandem mass spectrometry, or nLC-MS, which requires solely a minimal quantity of protein within the nano-gram vary, making it ideally suited for some of these organic samples,” Saraf stated. “The sensitivity of our setup is essential as a result of, with out it, we would wish a lot bigger protein portions. As soon as we obtain the saliva samples, we deal with all of the pattern processing, preparation, and digestion to make them prepared for mass-spectrometry evaluation. We’ve got the most recent tools and a particular setup, which supplies us excessive sensitivity, placing us on the forefront of this analysis.”
The info is analyzed at KU mass spectrometry core to type a sturdy understanding of how the molecular properties of mosquito saliva can change below totally different situations.
“After knowledge assortment, the evaluation requires in depth post-processing, together with interpretation and comparability, for which I’ve over 24 years of expertise,” Saraf stated. “That is how we established a collaboration with USDA.”
Based on the KU researcher, her lab’s shotgun strategy of analyzing proteomes is powerful and extra delicate than different strategies, affording way more detailed time-course research.
“We plan to check these adjustments below totally different situations, together with environmental variations within the mosquito after virus an infection,” she stated. “The main focus is on the mosquitos’ saliva, which is crucial as a result of, like with the parasite (Plasmodium) that causes malaria, arboviruses are transmitted by means of the saliva when the mosquito bites.”
Saraf and her group additionally will analyze saliva from a management group of mosquitoes not contaminated by the virus — however stored below the identical feeding situations and an infection period because the check group. The tip objective is to determine particular proteins of curiosity for future host-vector-virus interplay research, which might develop into potential targets for drug therapies.
Future Instructions and Implications
To this point, Saraf’s group has labored with uninfected mosquitoes to determine baseline knowledge. As soon as inactivation protocols are authorized and approved, the work will embrace samples from Biosafety Stage 2 (BSL-2) arbovirus-infected mosquitoes.
“We’ll determine variations and adjustments on the proteome stage by evaluating the management and contaminated samples at totally different phases,” the KU researcher stated. “The objective is to find out the protein adjustments that happen, as these can probably function candidates for vaccine growth. We’ll first want to pick candidates, which is why we’re utilizing controls below the identical situations with out an infection. We should rigorously load equal quantities of protein from each to make sure correct comparisons — primarily, we’ll be capable of examine ‘apples to apples.’”
Saraf’s work product is a listing of the protein adjustments, each qualitative and quantitative, to strengthen the proof out there to fellow researchers and drug builders.
“Final time, our collaborators at USDA discovered the listing very attention-grabbing, and it aligned with what has been reported within the literature,” she stated. “Deciding on candidates for additional research would be the objective.”
The research was funded by the U.S. Division of Agriculture.