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The growing demand of viral products for gene therapy and as oncolytic agents underscores the importance of optimizing virus production processes in the biopharmaceutical industry. Beyond established bioprocess parameters such as infectious titer, additional analytical methods are required to gain deeper insights into these processes. Electron microscopy, a well-established technique in virology, is rarely utilized in bioprocess design. In this study, we aim to identify potential targets for bioprocess optimization by combining ultrastructural changes of infected producer cells with the replication kinetics of a therapeutic virus. As a model system, we used an oncolytic recombinant vesicular stomatitis virus (rVSV). Replication kinetics were assessed by tissue culture infectious dose (TCID50), quantitative polymerase chain reaction, and optical cytometry. Transmission electron microscopy of high-pressure frozen, freeze-substituted samples was used to analyze the ultrastructure of rVSV-infected cells at different timepoints. Using this approach, we identified three key areas for bioprocess optimization. First, a decrease of the diameter of producer cells over the course of rVSV infection offers potential for online monitoring. Second, clusters of virions attached to the cell membrane of producer cells suggest the presence of cellular restriction factors, which could be overcome by strategies enhancing virus release. Finally, the observation of inclusion bodies surrounded by endoplasmic reticulum indicates a surplus of genomic copies within the producer cells, representing a bottleneck that could be targeted to increase particle assembly after genome replication.<b>IMPORTANCE</b>New biopharmaceutical products like oncolytic viruses demand the development of a broad analytical panel. Besides establishing new assays to characterize the product, development efforts can benefit from transferring advanced analytical technologies from related fields. Here, we showcase possible tie-ins for electron microscopy, a well-established technique for visualizing virus-host-cell interactions, in the biopharmaceutical development of an oncolytic virus. We report (i) a diameter decrease in producer cells alongside a destabilization of the inner cell architecture; (ii) intracellular accumulations of virions, a possible target for further optimizing virus harvest strategies; and (iii) accumulation of surplus genetic material in structures inside the cell, that is not assembled into infectious virions. These three observations illustrate how advanced microscopy techniques can open the door for new approaches in bioprocess design.