Anita Saraf investigates mosquito saliva to know how viruses like dengue and West Nile are transmitted, utilizing mass spectrometry to establish potential targets for vaccines and coverings.
You may guess it’d be powerful to extract saliva from a mosquito. And also you’d be right. But, as a result of mosquitos unfold illness by way of saliva, it’s crucial to pattern and research.
However Anita Saraf doesn’t accumulate 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 as a result of small pattern dimension, which is a standard limitation in such experiments,” she mentioned. “Our collaborators with the USDA Agricultural Analysis Service put the mouth of the mosquitoes right into a tube, then stimulate salivation and accumulate the saliva.”
Now, with a brand new two-year grant of $250,000 per yr from the U.S. Division of Agriculture’s Nationwide Bio and Agro-Protection Facility, Saraf presently is analyzing these samples of noninfectious mosquito saliva within the struggle 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 vegetation. The USDA has an curiosity in veterinary functions for the findings, however Saraf’s work might underpin therapies for individuals as effectively.
“The USDA needs to review how the virus impacts the immune system and adjustments on the molecular degree, significantly the proteome of mosquito saliva,” Saraf mentioned. “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 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 method is efficient with very low volumes or quantities of pattern like, as an example, 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 perfect for these kinds of organic samples,” Saraf mentioned. “The sensitivity of our setup is essential as a result of, with out it, we would want 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 gear and a particular setup, which supplies us excessive sensitivity, placing us on the forefront of this analysis.”
The information is analyzed at KU mass spectrometry core to kind a sturdy understanding of how the molecular properties of mosquito saliva can change underneath totally different situations.
“After information assortment, the evaluation requires in depth post-processing, together with interpretation and comparability, for which I’ve over 24 years of expertise,” Saraf mentioned. “That is how we established a collaboration with USDA.”
In accordance with the KU researcher, her lab’s shotgun method of analyzing proteomes is strong and extra delicate than different strategies, affording rather more detailed time-course research.
“We plan to review these adjustments underneath totally different situations, together with environmental variations within the mosquito after virus an infection,” she mentioned. “The main target is on the mosquitos’ saliva, which is crucial as a result of, like with the parasite (Plasmodium) that causes malaria, arboviruses are transmitted by 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 underneath the identical feeding situations and an infection period because the check group. The tip objective is to establish particular proteins of curiosity for future host-vector-virus interplay research, which might grow to be potential targets for drug therapies.
Future Instructions and Implications
Up to now, Saraf’s group has labored with uninfected mosquitoes to ascertain baseline information. As soon as inactivation protocols are authorized and approved, the work will embrace samples from Biosafety Degree 2 (BSL-2) arbovirus-infected mosquitoes.
“We’ll establish variations and adjustments on the proteome degree by evaluating the management and contaminated samples at totally different phases,” the KU researcher mentioned. “The objective is to find out the protein adjustments that happen, as these can doubtlessly function candidates for vaccine improvement. We’ll first want to pick out candidates, which is why we’re utilizing controls underneath the identical situations with out an infection. We should fastidiously load equal quantities of protein from each to make sure correct comparisons — primarily, we’ll be capable of evaluate ‘apples to apples.’”
Saraf’s work product is a listing of the protein adjustments, each qualitative and quantitative, to strengthen the proof accessible to fellow researchers and drug builders.
“Final time, our collaborators at USDA discovered the record very attention-grabbing, and it aligned with what has been reported within the literature,” she mentioned. “Deciding on candidates for additional research would be the objective.”
The research was funded by the U.S. Division of Agriculture.