Label-free quantification of early virus-induced cellular responses by digital holographic tomography.

Early virus-induced cellular responses are a fundamental diagnostic feature of viral infections; however, early virus-host interactions, particularly during viral entry, remain difficult to study in living cells because many commonly used markers and antibodies require fixation or can interfere with cell viability. Although virus-induced cellular responses constitute a fundamental diagnostic feature of viral infections, their detection by microscopy typically relies on later-stage effects. Microscopic observation, therefore, serves as a complementary approach to molecular techniques, but the time required for visible changes to develop limits its utility for rapid assessment. Here, we propose a label-free digital holotomography (DHT) approach for the detection of early virus-induced cellular responses during the first hours of infection, including equine arteritis virus (EAV), equine herpesvirus 1 (EHV-1), and equine rhinitis B virus (ERBV). Using this method, we show that as early as 1-2 h post-infection, holotomography is capable of capturing virus-induced alterations in cellular density based only on changes in the refractive index (RI), which reflects local variations in cellular mass density and biochemical composition. In addition to global RI changes, we focused on subcellular alterations occurring at the early stages of infection, including modifications in lipid droplet-like structures and nucleoli. Our results reveal that these changes become detectable rapidly and can be captured with high sensitivity, displaying both cell type- and virus-dependent patterns. Our findings suggest that DHT may provide a rapid, label-free, and quantitative platform for detecting virus-induced subcellular changes as early as 1-2 h post-infection. IMPORTANCE: Digital holotomography (DHT) facilitates the early, label-free detection of infection and serves as a complementary diagnostic tool, allowing the rapid initiation of treatment. This method is universal and can be broadly applied to the investigation of primary host interactions, including general cellular and biomolecular optical density changes. These applications provide a strong foundation for viral entry inhibitor testing. Moreover, this approach highlights the critical importance of studying viral infections in natural host systems rather than relying solely on well-established experimental models. In this context, we employed equine lung cells (ELCs) as a biologically relevant and appropriate model to better reflect natural host-virus interactions. These findings should be interpreted in the context of an exploratory imaging data set and will require confirmation in replicate-based and longitudinal single-cell studies.