Uncovering the mechanisms of virus infection and assembly is crucial for preventing the spread of viruses and treating viral disease. The technique of single-virus tracking (SVT), also known as single-virus tracing, allows one to follow individual viruses at different parts of their life cycle and thereby provides dynamic insights into fundamental processes of viruses occurring in live cells. SVT is typically based on fluorescence imaging and reveals insights into previously unreported infection mechanisms. In this review article, we provide the readers a broad overview of the SVT technique. We first summarize recent advances in SVT, from the choice of fluorescent labels and labeling strategies to imaging implementation and analytical methodologies. We then describe representative applications in detail to elucidate how SVT serves as a valuable tool in virological research. Finally, we present our perspectives regarding the future possibilities and challenges of SVT. CONTENTS a Maximum excitation wavelength. b Maximum emission wavelength. c Extinction coefficient. d Fluorescence quantum yield. e The relative brightness values were calculated from the product of the molar extinction coefficient and quantum yield, divided by the value for EGFP. f Not determined.

This work reported a mild and easily operated approach to labeling the viral envelope with streptavidin-modified QDs by intercalating biotinylated lipid directly into lipid membranes, which allows us to label enveloped viruses within 2 h with high specificity and efficiency, facilitating the widespread use of SVT for uncovering complex infection mechanisms.

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Single-virus tracking (SVT) offers the opportunity to monitor the journey of individual viruses in real time and to explore the interactions between viral and cellular structures in live cells, which can assist in characterizing the complex infection process and revealing the associated dynamic mechanisms. However, the low brightness and poor photostability of conventional fluorescent tags (e.g., organic dyes and fluorescent proteins) greatly limit the development of the SVT technique, and challenges remain in performing multicolor SVT over long periods of time. Owing to the outstanding photostability, high brightness and narrow emission with tunable color range of quantum dots (QDs), QD-based SVT (QSVT) enables us to follow the fate of individual viruses interacting with different cellular structures at the single-virus level for milliseconds to hours, providing more accurate and detailed information regarding viral infection in live cells. So far, the QSVT technique has yielded spectacular achievements in uncovering the mechanisms associated with virus entry, trafficking and egress. Here, we provide a detailed protocol for QSVT implementation using the viruses that we have previously studied systematically as an example. The specific procedures for performing QSVT experiments in live cells are described, including virus preparation, the QD labeling strategies, imaging approaches, image processing and data analysis. The protocol takes 1-2 weeks from the preparation of viruses and cellular specimens to image acquisition, and 1 d for image processing and data analysis.

Nuclear trafficking of viral genome is an essential cellular process in the life cycles of viruses. Despite substantial progress in uncovering a wide variety of complicated mechanisms of virus entry, intracellular transport of viral components, virus assembly, and egress, the temporal and spatial dynamics of viral genes trafficking within the nucleus remains poorly understood. Herein, using single-particle tracking, we explored the real-time dynamic nuclear trafficking of influenza A virus (IAV) genes packaged as the viral ribonucleoprotein complexes (vRNPs) by combining a four-plasmid DNA transfection system for the reconstruction of green fluorescent protein (GFP)-labeled vRNPs and a spinning disk super-resolution fluorescence microscope. We found that IAV infection significantly induced the formation of actin microfilaments (F-actin) in the nucleus. In combination with the fluorescent protein-tagged nuclear F-actin probe, we visualized the directed movement of GFP-labeled vRNPs foci along the nuclear F-actin with a speed of 0.18 μm/s, which is similar to the microfilaments-dependent slow directed motion of IAVs in the cytoplasm. The disruption of nuclear F-actin after treatment with microfilament inhibitors caused a considerable decrease in vRNPs motility and suppressed the nuclear export of newly produced vRNPs, indicating that the slow, directed movement plays a crucial role in facilitating the nuclear egress of vRNPs. Our findings identified a nuclear F-actin-dependent pathway for IAV vRNPs transporting from the nucleus into the cytoplasm, which may in turn uncover a novel target for antiviral treatment.