(622ag) Quantitative Single Particle Tracking for Characterizing Virus Entry of H3 Influenza Strains

Class I membrane-enveloped viruses, such as influenza, infect host cells through the endocytotic pathway. For this class of viruses, one essential viral coat protein, hemagglutinin (HA), is responsible for both the attachment of the virus to the host cell (receptor binding) and the fusion of the viral membrane with the host endosomal membrane. Membrane fusion is necessary for genome release into the cytosol, with the timing of release being triggered by the increasingly acidic conditions during endosome maturation. Using single particle tracking (SPT) techniques, individual membrane fusion events can be observed under specific conditions, which gives detailed information regarding HA pH sensitivity, acid stability, and the rate and extent of membrane fusion. This approach provides a comparative way to characterize and distinguish fusion properties among virus strains. We used SPT to quantify the fusion properties of three H3 influenza strains: A/Aichi/68/H3N2 (X:31), A/Udorn/72/H3N2 (Udorn) and A/Brisbane/07/H3N2 (Brisbane). Among these strains, all utilize an H3-type HA protein for entry, but the X:31 and Udorn strains have been adapted to grow well in chicken eggs, while the Brisbane strain is a clinical isolate. Chicken egg culture is the standard method for the production of large quantities of virus for research purposes and vaccine production. However, typical vaccine efficacy is only about 70%. Our aim was to determine if any changes in the fusion process exist between egg-adapted and clinical strains that may provide some rationale for the reduced efficacy. We found that the rate of fusion for the most clinically relevant strain, Brisbane, is generally insensitive to decreasing pH, while fusion of the egg-adapted strains, Udorn and X:31, are strongly dependent on pH (and faster) as pH decreases. All strains exhibit similar acid stability (the length of time they remain fusogenic in an acidic environment) at higher pH’s, but the egg-adapted strains become less acid stable at lower pH’s. Thus, it appears that the egg-adapted H3 strains tested may have evolved to compensate for the faster HA deactivation at low pH with a commensurate increase in the rate of fusion and number of proteins facilitating fusion, relative to the Brisbane strain. The implications of these changes are in the timing of the release of the genome, which impacts its trafficking to the nucleus for reproduction. Thus, there may be some connection with fusion rates and properties with overall infectivity. With this single particle fusion assay, we now have the tools available to better quantify the membrane fusion process for better understanding of the fundamental steps in viral infection, which will enable development of antiviral drugs that specifically target the fusion process as well as improve vaccine strategies.