Rabies Under the Microscope: Insights into Virus Entry

Rabies is a deadly viral disease that spreads through the bite of infected animals, targeting the nervous system with near-universal fatality once symptoms appear. Despite vaccines and post-exposure treatments, understanding how the virus enters and infects cells remains crucial for developing better therapies and prevention strategies.

Examining rabies at the microscopic level provides valuable insights into its structure, behavior, and interactions within host cells. Researchers use advanced imaging techniques to track the virus in real time, shedding light on key stages of infection.

Morphological Features Under The Microscope

Rabies virus, a member of the Lyssavirus genus within the Rhabdoviridae family, has a distinctive bullet-shaped morphology. This structure, approximately 180 nm in length and 75 nm in diameter, is enveloped by a lipid bilayer derived from the host cell membrane. Embedded within this envelope are glycoprotein spikes, which play a fundamental role in host cell recognition and viral entry. These surface projections, measuring around 10 nm, are densely arranged, giving the virus its characteristic studded appearance. The internal structure consists of a tightly coiled ribonucleoprotein (RNP) complex, which houses the single-stranded, negative-sense RNA genome. This genome is encapsidated by the nucleoprotein (N), forming a helical structure that interacts with the phosphoprotein (P) and the large polymerase protein (L), both essential for viral replication and transcription.

The matrix protein (M) serves as a bridge between the envelope and the RNP complex, maintaining virion integrity while regulating viral assembly and budding. Cryo-electron tomography studies have shown that the M protein forms a lattice-like arrangement beneath the lipid envelope, reinforcing stability and facilitating release from infected cells. This structural rigidity distinguishes the rabies virus from other rhabdoviruses with more flexible shapes. The bullet-like form is not just a morphological trait but an adaptation that enhances the virus’s ability to navigate the extracellular environment and efficiently fuse with host membranes.

In infected cells, Negri bodies—cytoplasmic inclusions composed of viral RNPs and host-derived components—serve as replication sites. These structures, ranging from 2 to 10 µm in diameter, are often found in neuronal cells, particularly in the hippocampus and Purkinje cells of the cerebellum. Their formation, driven by interactions between the N and P proteins, enhances replication efficiency by concentrating viral machinery in a confined space, shielding it from host defenses.

Techniques Used For Visualization

To study rabies at a microscopic level, researchers use imaging techniques that reveal its structure, entry mechanisms, and interactions with host cells. Among the most commonly used methods are fluorescence microscopy, electron microscopy, and confocal microscopy, each offering unique advantages.

Fluorescence Microscopy

Fluorescence microscopy tracks rabies virus components in infected cells by labeling viral proteins with fluorescent dyes or genetically encoded tags. This technique uses fluorophores that emit light upon excitation, allowing visualization of viral particles in live or fixed cells. Immunofluorescence assays, for example, use antibodies conjugated to fluorophores to detect viral proteins such as the nucleoprotein (N) or glycoprotein (G), revealing their distribution within the cytoplasm.

Live-cell imaging with fluorescently tagged rabies virus has provided insights into viral entry pathways, showing that the virus exploits endocytic vesicles for internalization. Studies employing green fluorescent protein (GFP)-tagged rabies virus have demonstrated its movement along microtubules toward the perinuclear region, where replication occurs. Super-resolution fluorescence microscopy techniques, such as stimulated emission depletion (STED) microscopy, further refine visualization at nanometer-scale resolution, revealing the precise organization of viral proteins within infected cells.

Electron Microscopy

Electron microscopy (EM) offers high-resolution imaging of the rabies virus and its interactions with host cells. Transmission electron microscopy (TEM) is particularly useful for visualizing the bullet-shaped virions in fine detail, showing the arrangement of the ribonucleoprotein complex and the surrounding lipid envelope. TEM images reveal that the viral glycoproteins form a dense, uniform layer on the surface, essential for receptor binding and membrane fusion.

Scanning electron microscopy (SEM) provides three-dimensional images of viral particles on the surface of infected cells, capturing the process of viral budding. Cryo-electron microscopy (cryo-EM) has advanced rabies virus studies by preserving its native structure in a near-physiological state. Recent cryo-EM research has elucidated the organization of the matrix protein (M) beneath the viral envelope, highlighting its role in maintaining virion stability. These high-resolution imaging techniques also identify structural changes during viral entry and uncoating, deepening our understanding of the infection process.

Confocal Microscopy

Confocal microscopy provides three-dimensional imaging of rabies virus-infected cells by capturing optical sections at different depths. This technique eliminates out-of-focus light, enhancing image clarity. By using fluorescently labeled antibodies or genetically encoded markers, researchers can track viral proteins in relation to cellular structures such as the cytoskeleton and organelles.

Time-lapse confocal microscopy has been used to monitor the intracellular trafficking of rabies virus in live cells, revealing its movement along actin filaments and microtubules. Studies show that the virus utilizes the dynein motor protein to travel toward the nucleus, a process essential for efficient replication. Additionally, confocal imaging has visualized the formation of Negri bodies, the cytoplasmic inclusions that serve as replication sites. By combining confocal microscopy with fluorescence resonance energy transfer (FRET) techniques, researchers have also studied protein-protein interactions within infected cells, shedding light on the molecular mechanisms driving viral assembly and egress.

Real-Time Observations Of Virus Entry

Tracking rabies virus entry in real time has provided insights into how the pathogen navigates the cellular landscape to establish infection. Live-cell imaging techniques allow researchers to observe viral particles as they engage with host receptors, undergo endocytosis, and traffic toward replication sites. These observations reveal that rabies virus does not rely on a single entry mechanism but exploits multiple pathways depending on host cell type and environmental conditions.

Fluorescently tagged viral particles have visualized the initial binding process, where the viral glycoprotein interacts with receptors such as the nicotinic acetylcholine receptor (nAChR) on neuronal cells. This interaction facilitates attachment and triggers conformational changes that prime the virus for internalization.

Once bound to the membrane, the virus is engulfed into endocytic vesicles, a process observed through total internal reflection fluorescence (TIRF) microscopy. This technique captures events at the plasma membrane with high temporal resolution, revealing that viral internalization occurs within seconds of receptor engagement. The virus is then transported through early and late endosomes, where pH-dependent fusion events release the ribonucleoprotein complex into the cytoplasm. High-speed live imaging has demonstrated that intracellular trafficking is mediated by microtubule-associated motor proteins, with viral particles moving directionally toward the perinuclear region.

Single-particle tracking studies have mapped the movement of individual virions using quantum dots, revealing alternating phases of rapid transport and transient pausing. These pauses likely correspond to interactions with cellular checkpoints that regulate viral progression. Super-resolution microscopy has captured the moment when the viral genome is released from endosomal compartments, showing that fusion occurs preferentially in acidic vesicles near the nucleus. This localization ensures that the viral RNA is delivered in close proximity to replication machinery, optimizing infection.

Cellular Changes In Infected Samples

Rabies virus induces profound alterations in host cells, reshaping intracellular structures to create an environment favorable for viral propagation. One of the most striking changes is the formation of Negri bodies, cytoplasmic inclusions that serve as replication sites. These structures, composed of viral ribonucleoproteins and host-derived factors, emerge within neuronal cells, particularly in regions such as the hippocampus and cerebellum. Their presence disrupts normal cellular organization, sequestering essential proteins and organelles, which can interfere with neuronal function.

Beyond the formation of inclusion bodies, infected cells undergo significant cytoskeletal remodeling. The virus exploits microtubule networks to facilitate intracellular transport, leading to reorganization of actin filaments and microtubule-associated proteins. This restructuring enhances viral movement but also disrupts normal cellular trafficking, affecting processes such as synaptic vesicle transport in neurons. As the infection progresses, mitochondrial dysfunction becomes evident, with studies showing a decline in mitochondrial membrane potential and increased oxidative stress. This dysregulation contributes to energy depletion and neuronal degeneration, hallmarks of rabies pathology.