Utilization of Alfa-Tag System for Studying Zika Virus Life Cycle In Infected Host CellsSolares, Juan RodrigoZika VirusAlfa-Tag SystemNanobodyMolecular cloningConfocal fluorescence microscopyViral ReplicationTransfectionPlaque AssayNS2A ProteinCapsid ProteinBiochemistry and Molecular BiologyB SOrthoflaviviruses, such as Zika virus (ZIKV) and Dengue virus (DENV), are single-stranded RNA viruses that can cause diseases like congenital Zika syndrome and dengue fever, respectively. Their viral life cycle in infected cells, especially the fate of viral mRNA and the dynamics of transcription and replication, is currently not well understood. In this study, we employed the ALFA-Tag system and fluorescence nanobodies against the ALFA-Tag to investigate the early events of ZIKV infection in human cells using confocal fluorescence microscopy. We genetically engineered recombinant ZIKV cDNA clones expressing the ALFA-Tag and created nanobody (NbALFA-RFP-His) expression constructs under the control of CMV and inducible Tet promoters, respectively. Our findings suggest that the ALFA-Tag system is a viable tool for visualizing early ZIKV infection events. However, the introduction of ALFA-Tag on ZIKV affected virus formation in host cells. We also observed leaky expression of the nanobody in the Tet-inducible system. Future studies will focus on reintroducing the ALFA-Tag on different locations of the ZIKV genome to avoid disruption of virion formation, optimizing nanobody expression control in the Tet-inducible system, and optimizing co-transfections with ZIKV ALFA-Tag clones and NbALFA-RFP-His to better understand the viral life cycle. Joyce Jose, Thesis SupervisorScott E. Lindner, Thesis Honors Advisoropen_access2025-04-22T21:48:04Zhttps://honors.libraries.psu.edu/catalog/9764jxs7049