Substrate Adhesion Method (SAM): Ultracentrifugation-Free Electron Microscopy of Influenza A virus (H3N2) Morphology and Disinfectant-Induced Changes

Electron microscopy (EM) is crucial for resolving viral ultrastructure, but conventional ultracentrifugation used for sample concentration can induce structural artifacts, particularly in enveloped viruses like influenza A virus. To overcome this, we developed and validated the Substrate Adhesion Method (SAM). This simple technique bypasses ultracentrifugation by utilizing the natural adhesion of influenza A virus (H3N2) to cell culture substrates during infection, enabling effective multimodal EM (SEM, TEM, STEM) visualization of progeny virus particles without prior concentration. SAM-enabled SEM revealed a distinct topographical "wavy" pattern on H3N2 virus particle surfaces, which is interpreted as a preparation-induced artifact arising from critical point drying (CPD) shrinkage. Ultra-high-voltage electron tomography confirmed that spike-containing envelope structures contribute to this observed morphology. Thus, SAM-facilitated SEM offers wide-field morphological insights complementing TEM's high-resolution internal structural analysis. Virus particle adhesion to the substrate via SAM was significantly enhanced by infected cells, suggesting a key role for cell-associated factors in this immobilization. Furthermore, SAM's compatibility with negative staining allowed direct visualization of ethanol-induced H3N2 virus inactivation via distinct envelope deformations such as disruption and roughening. This feature makes SAM a valuable tool for direct morphological assessment of disinfectant efficacy, complementing infectivity assays. In conclusion, SAM provides a simple, rapid, artifact-mitigating approach for detailed morphological studies of H3N2 virus and antiviral agent evaluation, opening new perspectives in virological EM.