Membrane Rigidity‐Tunable Fusogenic Nanosensor for High Throughput Detection of Fusion‐Competent Influenza A Virus

Abstract

The emergence of fatal viruses that pose continuous threats to global health has fueled the intense effort to develop direct, accurate, and high-throughput virus detection platforms. Current diagnostic methods, including qPCR and rapid antigen tests, indicate how much of the virus is present, whether small fragments or whole viruses. However, these methods do not indicate the probability of the virus to be active, capable of interacting with host cells and initiating the infection cycle. Herein, a sialic acid-presenting fusogenic liposome (sLipo-Chol) nanosensor with purposefully modulated membrane rigidity to rapidly detect the fusion-competent influenza A virus (IAV) is developed. This nanosensor possesses virus-specific features, including hemagglutinin (HA) binding and HA-mediated membrane fusion. It is explored how the fusogenic capability of sLipo-Chol with different membrane rigidities impacts their sensing performance by integrating Förster resonance energy transfer (FRET) pairs into the bilayers. The addition of an intact virus led to instant FRET signal changes, thus enabling the direct detection of diverse IAV subtypes-even in avian fecal samples-within an hour at room temperature. Therefore, the sensing approach, with an understanding of the cellular pathogenesis of influenza viruses, will aid in developing bioinspired nanomaterials for evolution into nanosystems to detect infection-competent viruses.

A Fluorogenic Pseudoinfection Assay to Probe Transfer and Distribution of Influenza Viral Contents to Target Vesicles

“…12 The pseudoinfection model recapitulates two fundamental steps of the endosomal escape process, mixing of lipid compartments and transfer of viral contents (Figure 1B). 3 While lipid mixing between virus and vesicle membranes is a rapid way to test that the viral fusion protein is functional, 13 many such events are halted at the intermediate hemifused state where the outer leaflets of the two membranes merge but not the inner leaflets, and consequently, no fusion pore is formed. 14 On the other hand, a content mixing experiment provides more information, namely, the presence of a functional fusion protein, formation of a fusion pore, and possible presence of the viral genome, though the fusion pore may not necessarily be large enough to accommodate passage of the vRNPs (viral genome).…”