Anita Saraf investigates mosquito saliva to grasp 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 robust 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 important to pattern and examine.
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 investigate organic samples — primary science that underpins many drug therapies.
“Gathering saliva from these mosquitoes is difficult as a result of small pattern measurement, which is a standard 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 accumulate 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 present is analyzing these samples of noninfectious mosquito saliva within the struggle towards “arboviruses” — the time period for viruses unfold by arthropods like mosquitoes.
These embody West Nile, yellow fever, dengue fever, Japanese encephalitis, and different viruses that may hurt or kill folks, animals, and vegetation. The USDA has an curiosity in veterinary purposes for the findings, however Saraf’s work may underpin therapies for folks as effectively.
“The USDA needs to check how the virus impacts the immune system and modifications on the molecular stage, notably the proteome of mosquito saliva,” Saraf stated. “When a virus infects a mosquito, it alters the proteins and different primary elements of the mosquito, just like how malaria impacts its human host because it progresses via totally different levels.”
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 all 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 have now the newest tools and a particular setup, which provides us excessive sensitivity, placing us on the forefront of this analysis.”
The info is analyzed at KU mass spectrometry core to type a strong understanding of how the molecular properties of mosquito saliva can change below totally different circumstances.
“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 method of analyzing proteomes is powerful and extra delicate than different strategies, affording way more detailed time-course research.
“We plan to check these modifications below totally different circumstances, together with environmental variations within the mosquito after virus an infection,” she stated. “The main target is on the mosquitos’ saliva, which is important as a result of, like with the parasite (Plasmodium) that causes malaria, arboviruses are transmitted via the saliva when the mosquito bites.”
Saraf and her workforce additionally will analyze saliva from a management group of mosquitoes not contaminated by the virus — however stored below the identical feeding circumstances and an infection length because the take a look at group. The top objective is to establish particular proteins of curiosity for future host-vector-virus interplay research, which may develop into potential targets for drug therapies.
Future Instructions and Implications
Thus far, Saraf’s workforce has labored with uninfected mosquitoes to ascertain baseline knowledge. As soon as inactivation protocols are authorized and approved, the work will embody samples from Biosafety Degree 2 (BSL-2) arbovirus-infected mosquitoes.
“We’ll establish variations and modifications on the proteome stage by evaluating the management and contaminated samples at totally different levels,” the KU researcher stated. “The objective is to find out the protein modifications that happen, as these can probably function candidates for vaccine growth. We’ll first want to pick out candidates, which is why we’re utilizing controls below the identical circumstances with out an infection. We should fastidiously load equal quantities of protein from each to make sure correct comparisons — basically, we’ll have the ability to examine ‘apples to apples.’”
Saraf’s work product is an inventory of the protein modifications, each qualitative and quantitative, to strengthen the proof obtainable to fellow researchers and drug builders.
“Final time, our collaborators at USDA discovered the record very fascinating, and it aligned with what has been reported within the literature,” she stated. “Deciding on candidates for additional examine would be the objective.”
The examine was funded by the U.S. Division of Agriculture.