From Fear to Future: The Rabies Virus

In a hospital room in India, a boy of about nine years old lays in bed, thrashing his arms and legs in the air. His head sways from side to side as his mouth froths with saliva. The nurses call out his name from a distance, telling him to stop, but he does not respond; he continues behaving aggressively, gasping for air in between the motions of his swinging head as he struggles to breathe. The boy’s symptoms had only begun a few weeks prior after a stray dog had bitten his leg and infected him. However, at this stage of his disease, the boy’s condition was almost certain to result in death.

The child’s abnormal behavior may seem like a scene from a zombie movie, but his condition is the result of a real disease: rabies. Rabies is caused by a virus known as the Rabies lyssavirus, which attacks the nervous system to produce a range of worsening physical and psychological symptoms [1]. It is one of the deadliest viruses to exist, nearing an almost 100% fatality rate in humans [1]. Rabies is zoonotic, meaning it can spread between different animal species, and it is generally transmitted through the saliva from animal bites from mammals such as dogs, cats, foxes, bats, and raccoons [2]. More than 99% of rabies infections in humans worldwide are from dog bites, but in the United States and other Western countries, public health campaigns have mostly controlled the disease through the mass vaccination and increased surveillance for rabies among dogs and other animals, both domesticated and wild [1,2]. However, rabies remains a neglected disease in many developing countries, and it is estimated to cause 60,000 deaths per year worldwide [2].

When an animal with rabies bites a human, particles of the rabies virus first enter the bite wound from the animal’s saliva [3]. The rabies virus glycoprotein, which is a biomolecule expressed on the surface of the rabies virus, then enables its entry into muscle cells and neurons by binding to specific receptors on them [3,4]. Though the rabies virus causes disease by attacking the nervous system, the muscles serve as an important location for initial viral replication [3]. These viruses that are newly produced within the muscle tissue can then be released into the neuromuscular junction, where they can penetrate the neurons that synapse with muscle cells, called motor neurons [3]. The rabies virus preferentially uses these neuromuscular junctions to infect the nervous system, exclusively entering motor neurons to do so [3]. 

After entering the axon terminals of motor neurons, the virus travels up the neuron’s axon and into its cell body [5]. In the cell body, the rabies virus replicates its genome and transcribes it to produce the viral proteins that are needed to make additional copies of the virus [5]. The newly assembled viruses then travel from the motor neuron cell body up into its dendrites, from where they bud off the neuron and enter the synapse that is formed between motor neuron dendrites and the terminals of spinal cord neurons [3]. In this synapse, the rabies virus then uses its glycoproteins to bind to receptors on the spinal cord neurons and enter them [3, 5]. The virus continues to replicate in the central nervous system and spread throughout the spinal cord and brain, traveling from neuron to neuron in a process known as trans-synaptic spread [3].

Usually, neurons can recruit immune system mechanisms to detect and destroy viruses. However, the rabies virus has proteins that allow it to evade immune responses by, for instance, disrupting the functioning of interferons and other proteins that can detect the virus inside neurons [6]. This allows the rabies virus to seamlessly travel throughout the nervous system without causing widespread neurological inflammatory responses or neuronal death [6]. Furthermore, as the rabies virus travels solely between the body’s neurons, it enters the brain without going through the blood-brain barrier, a selective filter between the brain’s blood vessels and its neuronal environment that is one of the strongest protections our brain has against pathogens and other harmful substances [3]. As such, the rabies virus can “silently” invade the nervous system and spread throughout it without being detected by the immune system.

 As the virus disperses throughout the central nervous system, it causes a range of symptoms commonly associated with the disease. For instance, when the virus infects the spinal cord, it can impair the functions of both the motor and sensory nerves that connect to it [3]. This can result in muscle weakness and paralysis as well as altered sensations of pain, tingling, and itchiness in different parts of the body [3]. As the virus accumulates in the central nervous system, it can also spread "centrifugally" to the peripheral nerves and the numerous organ systems they regulate, including the heart, salivary glands, and gastrointestinal tract [7]. The virus can then infect and replicate within these tissues and organs, causing them to function aberrantly [7]. The salivary glands are especially an important target of the rabies virus, which hypersalivate to facilitate the transmission of the virus since it is the main route by which it exits the body to infect others [3].

In the brain, though the rabies virus infiltrates all of its regions roughly equally, its infection of the limbic system is primarily responsible for the behavioral changes characteristic of rabies [3, 7]. The limbic system is a system of brain regions involved in the regulation of emotion, memory, and survival behavior [8]. Any damage to it can cause significant alterations in our personality and behavior, and this may explain why individuals become more aggressive, agitated, and hyperexcited after being infected with rabies [8, 9]. Infected individuals often also suffer from neurological symptoms like seizures and psychological symptoms like confusion and hallucinations [7]. They may also experience involuntary muscle spasms after sensing certain environmental stimuli, most peculiarly, water - a phenomenon known as hydrophobia [3]. Nonetheless, it is not yet fully understood how the rabies virus affects the central nervous system without causing any signs of physical damage to its neurons [3, 10]. 

As the virus spreads throughout the whole brain, it eventually affects one’s consciousness, which often fluctuates throughout the later stages of the disease [3]. Individuals ultimately fall into a coma, a state of full unconsciousness [2]. From here, their condition deteriorates and they succumb to death as a result of cardiac arrest or respiratory failure [3]. 

Despite the lethal nature of the rabies virus, bioengineered versions of it have been used as viral vectors to further study the complexities of the nervous system and treat the various diseases that affect it. Viral vectors are genetically modified forms of a virus that retain their overall structure but have their disease-causing genetic properties removed and some genetic material of interest, known as a gene-insert, added to the virus’ genome [11]. The makeup of this gene-insert depends on the target application, whether it be for research or clinical treatment [12]. The gene-inserts will be deposited into specific cells in a tissue or along a pathway depending on the type of glycoproteins a viral vector has, which can be modified [11]. As such, the use of viral vectors affords a wide range of flexibility and versatility. 

Among the numerous neurotropic viruses that target the nervous system, the rabies virus has many properties that make it unique. For instance, the rabies virus glycoprotein enables it with a high specificity for neurons and a strict spreading pattern that is solely trans-synaptic in the early stages of the infection [3]. If used as a viral vector, this property would minimize the spillover of the vector into non-neuronal tissue and prevent the gene-insert from being introduced in unintended areas or regions. Additionally, the rabies virus does not trigger cellular defenses that cause neuron inflammation and death, unlike other neurotropic viruses [6]. This enables rabies virus vectors to simply deposit the gene-insert to target neurons without causing any significant immune response that would damage them. Furthermore, since the rabies virus travels through the nervous system predominantly across synapses, rabies virus vectors could deliver genes to specific neurons without having to cross the blood-brain barrier, which is a major concern in the development of drug delivery and gene therapy mechanisms [3, 13]. These properties together make the rabies virus a promising tool as a viral vector. 

There have been many applications of non-pathogenic strains of the rabies virus as viral vectors in research, although most of it has been limited to studies with animal models such as rodents and monkeys. Mostly, rabies virus vectors have been utilized as a “tracer” in research to visually mark neural pathways that lead from nerve endings up through the peripheral nerves and into the spinal cord and brain [14]. To trace these pathways, a gene that codes for a fluorescent protein, such as Green Fluorescent Protein, is added to the vector [14]. Researchers then inject these vectors into the nerve endings in tissues and organs like muscles, enabling the neurons they infect along certain pathways to produce the corresponding fluorescent proteins, creating a “tracing” [14]. These tracings yield important information to researchers, as they can help determine the region of the brain or spinal cord that is involved in controlling the behavior of a certain tissue or receiving sensory information from it [15]. Tracers can also help in mapping the various neural circuits in the brain that contribute to certain complex behaviors and perceptual processes [16]. Additionally, tracers can identify specific neurons along a pathway that may be diseased or injured in animal models of certain illnesses, such as motor neuron diseases, thereby enhancing our understanding of various neurological conditions [16].

Different properties of the rabies virus can additionally be used to treat these neurological diseases by offering the capability for targeted gene therapies. One such instance is in the treatment of spinal muscular atrophy (SMA), a condition that impairs the ability of motor neurons to relay signals for movement to skeletal muscles as a result of having a nonfunctional or missing copy of a gene called SMN1 [17]. To treat this disease, viral vectors carrying the SMN1 gene can be injected into patients, which could travel to motor neurons and deliver a working copy of the gene to them [17]. Though adeno-associated virus vectors are currently approved to treat SMA, different properties of the rabies virus, especially its glycoprotein, are being researched to increase the effectiveness of vector-mediated gene therapies for SMA [17, 18]. Similarly, vectors based on the rabies virus may be effective in the treatment of other neurological conditions that may benefit from gene therapy, including neurodegenerative diseases like Parkinson’s and Huntington’s [19].

From a disease that brings fear, researchers have brought hope and possibility. Without rabies, it would have been nearly impossible for scientists to develop viral vectors with the same remarkable specificity, efficiency, and potential as the virus. Such scientific breakthroughs in healthcare promise a future where diseases once considered insurmountable may soon become manageable, bringing hope and healing to millions worldwide.

References

1. Bastos, V., Pacheco, V., Rodrigues, É. D. L., Moraes, C. N. S., Nóbile, A. L., Fonseca, D. L. M., Souza, K. B. S., do Vale, F. Y. N., Filgueiras, I. S., Schimke, L. F., Giil, L. M., Moll, G., Cabral-Miranda, G., Ochs, H. D., Vasconcelos, P. F. D. C., de Melo, G. D., Bourhy, H., Casseb, L. M. N., & Cabral-Marques, O. (2023). Neuroimmunology of rabies: New insights into an ancient disease. Journal of Medical Virology, 95(10), e29042. https://doi.org/10.1002/jmv.29042

2. Fooks, A. R., Cliquet, F., Finke, S., Freuling, C., Hemachudha, T., Mani, R. S., Müller, T., Nadin-Davis, S., Picard-Meyer, E., Wilde, H., & Banyard, A. C. (2017). Rabies. Nature reviews. Disease primers, 3, 17091. https://doi.org/10.1038/nrdp.2017.91

3. Davis, B. M., Rall, G. F., & Schnell, M. J. (2015). Everything You Always Wanted to Know About Rabies Virus (But Were Afraid to Ask). Annual Review of Virology, 2(1), 451–471. https://doi.org/10.1146/annurev-virology-100114-055157

4. Callaway, H. M., Zyla, D., Larrous, F., de Melo, G. D., Hastie, K. M., Avalos, R. D., Agarwal, A., Corti, D., Bourhy, H., & Saphire, E. O. (2022). Structure of the rabies virus glycoprotein trimer bound to a prefusion-specific neutralizing antibody. Science Advances, 8(24), eabp9151. https://doi.org/10.1126/sciadv.abp9151

5. Schnell, M. J., McGettigan, J. P., Wirblich, C., & Papaneri, A. (2010). The cell biology of rabies virus: using stealth to reach the brain. Nature reviews. Microbiology, 8(1), 51–61. https://doi.org/10.1038/nrmicro2260

6. Ito, N., Moseley, G. W., & Sugiyama, M. (2016). The importance of immune evasion in the pathogenesis of rabies virus. The Journal of Veterinary Medical Science, 78(7), 1089–1098. https://doi.org/10.1292/jvms.16-0092

7. Jackson A. C. (2011). Update on rabies. Research and Reports in Tropical Medicine, 2, 31–43. https://doi.org/10.2147/RRTM.S16013

8. Rajmohan, V., & Mohandas, E. (2007). The limbic system. Indian Journal of Psychiatry, 49(2), 132–139. https://doi.org/10.4103/0019-5545.33264

9. Mahadevan, A., Suja, M. S., Mani, R. S., & Shankar, S. K. (2016). Perspectives in Diagnosis and Treatment of Rabies Viral Encephalitis: Insights from Pathogenesis. Neurotherapeutics: The Journal of the American Society for Experimental NeuroTherapeutics, 13(3), 477–492. https://doi.org/10.1007/s13311-016-0452-4

10. Consales, C. A., & Bolzan, V. L. (2007). Rabies review: immunopathology, clinical aspects, and treatment. Journal of Venomous Animals and Toxins Including Tropical Diseases, 13(1), 5–38. https://doi.org/10.1590/S1678-91992007000100002

11. Bulcha, J. T., Wang, Y., Ma, H., Tai, P. W. L., & Gao, G. (2021). Viral vector platforms within the gene therapy landscape. Signal transduction and targeted therapy, 6(1), 53. https://doi.org/10.1038/s41392-021-00487-6

12. Wang, Y., Hu, Z., Ju, P., Yin, S., Wang, F., Pan, O., & Chen, J. (2018). Viral vectors as a novel tool for clinical and neuropsychiatric research applications. General psychiatry, 31(2), e000015. https://doi.org/10.1136/gpsych-2018-000015

13. Pardridge W. M. (2020). Blood-Brain Barrier and Delivery of Protein and Gene Therapeutics to Brain. Frontiers in aging neuroscience, 11, 373. https://doi.org/10.3389/fnagi.2019.00373

14. Gomme, E. A., Wanjalla, C. N., Wirblich, C., & Schnell, M. J. (2011). Rabies virus as a research tool and viral vaccine vector. Advances in virus research, 79, 139–164. https://doi.org/10.1016/B978-0-12-387040-7.00009-3

15. Ugolini G. (2011). Rabies virus as a transneuronal tracer of neuronal connections. Advances in virus research, 79, 165–202. https://doi.org/10.1016/B978-0-12-387040-7.00010-X

16. Han, Z., Luo, N., Ma, W., Liu, X., Cai, Y., Kou, J., Wang, J., Li, L., Peng, S., Xu, Z., Zhang, W., Qiu, Y., Wu, Y., Ye, C., Lin, K., & Xu, F. (2023). AAV11 enables efficient retrograde targeting of projection neurons and enhances astrocyte-directed transduction. Nature communications, 14(1), 3792. https://doi.org/10.1038/s41467-023-39554-7

17. Mendell, J. R., Al-Zaidy, S., Shell, R., Arnold, W. D., Rodino-Klapac, L. R., Prior, T. W., Lowes, L., Alfano, L., Berry, K., Church, K., Kissel, J. T., Nagendran, S., L'Italien, J., Sproule, D. M., Wells, C., Cardenas, J. A., Heitzer, M. D., Kaspar, A., Corcoran, S., Braun, L., … Kaspar, B. K. (2017). Single-Dose Gene-Replacement Therapy for Spinal Muscular Atrophy. The New England journal of medicine, 377(18), 1713–1722. https://doi.org/10.1056/NEJMoa1706198

18. Azzouz, M., Le, T., Ralph, G. S., Walmsley, L., Monani, U. R., Lee, D. C., Wilkes, F., Mitrophanous, K. A., Kingsman, S. M., Burghes, A. H., & Mazarakis, N. D. (2004). Lentivector-mediated SMN replacement in a mouse model of spinal muscular atrophy. The Journal of clinical investigation, 114(12), 1726–1731. https://doi.org/10.1172/JCI22922

19. Rittiner, J. E., Moncalvo, M., Chiba-Falek, O., & Kantor, B. (2020). Gene-Editing Technologies Paired With Viral Vectors for Translational Research Into Neurodegenerative Diseases. Frontiers in molecular neuroscience, 13, 148. https://doi.org/10.3389/fnmol.2020.00148