Abstract

Abstract Zika virus (ZIKV) is a re-emergent flavivirus that triggered a global health emergency from 2015 to 2017. ZIKV and other impactful flaviviruses, including dengue virus, lack the antiviral treatments and vaccines needed to diminish their threat to communities world-wide. This is in part due to gaps in our understanding of the flavivirus lifecycle; despite numerous investigations into flavivirus non-structural (NS) protein function. I hope to forge a deeper understanding of crucial NS protein functions and interactions throughout the ZIKV replication cycle. I will use alpaca-derived variable heavy-chain-only (VHH) antibody fragments, isolated from an alpaca immunized with ZIKV NS proteins, to perturb the replication cycle and therefore identify key NS protein epitopes used during replication. I individually purified NS1, NS2b, NS3, NS4b, NS5 for use in alpaca immunization, phage display panning, and enzyme-linked immunosorbent assays (ELISAs). From the alpaca peripheral blood I isolated RNA and amplified VHH genes, enriched for NS-protein binders with iterative phage display, and screened potential high binders via ELISA. I have isolated VHH sequences that interact with the ZIKV helicase, NS3, and the RNA-dependent RNA polymerase, NS5. I plan to purify each VHH and express them in ZIKA-permissive A549 cells as stable lines. Using purified VHH, I will analyze their binding ability in-vitro by ELISA and bio layer interferometry, and quantify in-vitro inhibition of NS3 protease and helicase activity for NS3 binders, and NS5 polymerase and methyltransferase activity. With the stable cells lines I will investigate each VHHs ability to impede ZIKV virus production and formation of replication complexes in infected cell lines. Support from training grant "Interactions at the Microbe-Host Interface" T32AI007472

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