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Enveloped viruses, such as HIV, Ebola and Influenza, are among the most deadly known viruses. Cellular membrane penetration of enveloped viruses is a critical step in the cascade of events that lead to entry into the host cell. Conventional ensemble fusion assays rely on collective responses to membrane fusion events, and do not allow direct and quantitative studies of the subtle and intricate fusion details. Such details are accessible via single particle investigation techniques, however. Here, we implement nano-infrared spectroscopic imaging to investigate the chemical and structural modifications that occur prior to membrane fusion in the single archetypal enveloped virus, influenza X31. We traced in real-space structural and spectroscopic alterations that occur during environmental pH variations in single virus particles. In addition, using nanospectroscopic imaging we quantified the effectiveness of an antiviral compound in stopping viral membrane disruption (a novel mechanism for inhibiting viral entry into cells) during environmental pH variations.