Executive Summary : | Viruses are simple pathogens that enter host cells and utilize the host's machinery for their replication. Early-replication steps include cellular attachment, penetration of the plasma membrane, and release of the viral genome. Understanding these steps is crucial for developing effective antiviral therapy against emerging or re-emerging and highly mutating viruses. Some prevalent and highly mutating respiratory viruses, such as Influenza A viruses (IAVs) and respiratory syncytial virus (RSV), are orthomyxovirus and paramyxovirus. These viruses must go through attachment to the plasma membrane, membrane fusion, and release of viral RNA in the cytosol. The exact receptors or attachment factors involved in membrane attachment vary from virus to virus. To track the pathway of cellular attachment to membrane fusion to viral genome release of two respiratory RNA viruses, H3N2 and RSV, a fluorescence imaging platform will be established at a single-cell level. This platform will be used to develop specialized fluorescence labelling (single-color and dual-color) of the viruses, employing total internal reflection fluorescence (TIRF) microscopy and confocal microscopy techniques to track labelled virus particles on/in live and fixed cells in a time-dependent manner. Three major significances of this imaging platform are (1) spatiotemporal detection of the membrane attachment, fusion, and viral RNA release events of H3N2 and RSV; (2) the imaging platform at the single-cell and single-virus level will reveal the heterogeneity of these steps, allowing correlation between successful virus replication pathways; and (3) understanding these key points will allow comparison of early-replication pathways of IAVs and RSV, aiding in antiviral drug repurposing and designing broad-spectrum antiviral candidates. |