First contact: biophysical insights into non-enveloped viruses from VLPs to packed virions

Precise biophysical characterization of non-enveloped viruses and virus-like particles (VLPs) is essential for evaluating assembly, structural integrity, and sample heterogeneity — key parameters that influence infectivity, manufacturability, and therapeutic performance. Analytical ultracentrifugation, light scattering, and mass photometry provide complementary information on particle size distributions, empty/full ratios, and assembly quality. Native and charge-detection mass spectrometry extend this, enabling accurate mass determination and genome-loading assessment. Solution-based structural techniques, such as SAXS and spectroscopy, provide dynamics. High-resolution X-ray crystallography, cryogenic electron microscopy, and tomography reveal global architectures, quaternary arrangements, and conformational switching essential for entry and maturation. Besides these methods, atomic force microscopy also allows nanoscale visualization in conjunction with mechanical profiling, linking physical stability with functional behavior. Thermal, chemical, and mechanical stress assays offer insight into unfolding pathways, pH-dependent disassembly, and genome-driven capsid pressurization, informing both viral biology and formulation science. Finally, mass spectrometry-based methods enable site-specific mapping of post-translational modifications and conformational perturbations. Together, these approaches establish a comprehensive framework for characterizing viral particles and VLPs, supporting rational engineering of stable, homogeneous, and functional assemblies for vaccines, gene delivery, and nanobiotechnology. This review summarizes current label-free methods for assessing size, mass, structural organization, and stability of intact viral particles.