Comparative Analysis of Extracellular Vesicle and Virus Co-Purified Fractions Produced by Contemporary Influenza A and B Viruses in Different Human Cell Lines

Influenza virus is one of the most frequent causes of respiratory infection in humans. Recent studies suggest that extracellular vesicles (EVs)-small particles released by cells during influenza virus infection-can influence the immune response and viral pathogenesis. However, during viral replication, infected cells can also release EVs, which may include different subtypes. This study aimed to purify and characterize viral preparations and EVs using sequential ultracentrifugation methods. Influenza A/H1N1, A/H3N2, and B virus strains were produced in human Calu-3 and A549 cell lines. Viral supernatants then underwent a series of differential ultracentrifugation steps at 3000× <i>g</i>, 17,000× <i>g</i>, and 100,000× <i>g</i>. Dynamic light scattering analysis (DLS) validated size heterogeneity for all three types of EVs. Measurement of infectious virus particles for all three pellets showed virus enrichment at 17,000× <i>g</i> and 100,000× <i>g</i>. Dot blot analysis confirmed the enrichment of virus particles in these fractions and the presence of EV protein. This study demonstrates the presence of EVs in virus preparations and highlights the need for improved separation methods to characterize them better and explore their role in viral infection pathogenesis.