Rabies is an acute, often fatal viral disease that primarily affects the central nervous system. It is typically transmitted through bites from infected domestic dogs and wild carnivorous animals. All warm-blooded animals, including humans, are susceptible to rabies infection. The rabies virus (RABV), a member of the Lyssavirus genus, is commonly found in the salivary glands of rabid animals and is excreted in their saliva. When an infected animal bites, the virus enters the blood through a fresh wound. Once introduced, RABV propagates along nerve tissue to the brain, establishing itself within the central nervous system. Subsequently, it spreads via nerves to the salivary glands, often resulting in symptoms such as excessive salivation. The disease initially manifests with excitation of the central nervous system, characterized by irritability and aggression. Infected animals may be highly contagious even before symptoms manifest (1). Despite effective vaccines for pre- and post-exposure prophylaxis (PEP), rabies remains a significant public health challenge, particularly in developing countries where access to PEP is limited. Once clinical symptoms appear, rabies is almost always fatal, underscoring the urgent need for effective therapeutic interventions. Rabies is prevalent in >150 countries, mainly in Asia and Africa, where exposure without timely PEP results in a mortality rate >99%. The RABV genome encodes five essential proteins: Nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G) and polymerase (L). These proteins play critical roles in the viral life cycle, facilitating attachment to host cells, entry via endocytosis, uncoating and replication within the host cytoplasm.

Rabies poses a severe threat to human health and economic stability, particularly in developing countries. According to the World Health Organization (WHO) 2023 report, rabies causes ~59,000 human deaths annually, with 99% of cases resulting from dog bites (2). The highest mortality rates are reported in Asia and Africa, where access to PEP is often limited. India alone accounts for almost 36% of global rabies-related deaths, translating to an estimated 20,000 fatalities per year (3). By contrast, developed nations have largely controlled rabies through extensive vaccination programs and improved surveillance measures.

The economic impact of rabies is profound, resulting in over $8.6 billion in global annual losses. These losses stem primarily from the costs associated with PEP, the loss of livestock, and reduced productivity due to illness and death. The financial burden of rabies is particularly acute for low-income populations, where the average estimated cost of PEP can be prohibitive (4). This situation emphasizes the urgent need for novel therapeutic interventions, as there are currently no effective antiviral treatments available once clinical symptoms appear.

Recent epidemiological studies highlight the ongoing challenges in controlling rabies in specific regions. For instance, Malaysia has faced recurrent outbreaks of dog-mediated human rabies, with a significant association between low vaccination rates and increased incidence of cases (5). Another study conducted from 2015 to 2023 revealed that dogs accounted for 89.35% of confirmed rabies cases in animals, emphasizing the importance of vaccination campaigns (6). Therefore, rabies continues to pose a significant public health threat globally, particularly in developing regions where access to preventive measures is limited. Enhanced vaccination efforts and improved public health strategies are essential to combat this preventable disease effectively. The urgent need for therapeutic interventions for symptomatic patients further highlights the complexities involved in managing rabies as a public health concern.

Effective vaccines have been developed for PEP; timely administration can prevent rabies development following exposure. PEP includes wound cleansing, vaccine administration and equine or human rabies immune globulins (ERIG or HRIG). However, PEP is ineffective once neurological signs appear. Although there are rare reports of human rabies survivors, no established therapies exist. Advances in treatment often stem from key studies on rabies pathogenesis in animal models (7).

While vaccines have proven effective in preventing rabies transmission, there is a pressing need for therapeutic strategies to combat the disease in symptomatic patients. This need is particularly critical in resource-limited settings where vaccination coverage remains insufficient. The WHO has initiated efforts to improve access to rabies vaccines through partnerships with organizations such as Gavi, focusing on strengthening surveillance and reporting systems while encouraging multisectoral collaborations (8).

Conventional treatment methods include pharmacological therapies, surgical intervention and physical therapy. These methods have been the cornerstone of medical practice for decades, as they are often backed by extensive clinical research, providing established protocols for healthcare providers. In addition, this approach allows for direct patient assessment, enabling healthcare providers to tailor treatments based on individual patient needs (9). In a number of cases, conventional treatments can yield immediate results, such as pain relief or symptom management. On the contrary, these approaches can be resource-intensive, requiring significant time and personnel for administration and monitoring, and can sometimes be costly in resource-limited settings (10). Therefore, treatment strategies have incorporated in silico approaches, as they provide a better understanding of the treatment design, disease modelling and drug discovery, which include molecular docking, virtual screening and machine learning (ML) algorithms that analyze large datasets to predict treatment outcomes.

In silico methods can process vast amounts of data quickly, significantly reducing the time required for drug discovery and development compared to traditional laboratory methods. These approaches can easily scale to accommodate larger datasets without a corresponding increase in resource requirements. This scalability is particularly beneficial in addressing complex diseases with multifactorial causes in a cost-effective manner, while minimizing laboratory work and animal testing. However, computational predictions need to be validated through experimental studies, as there is a risk of false positives or negatives. Their effectiveness relies heavily on the availability and quality of data. They may not always accurately reflect the complexities of human biology or disease progression (11).

In recent years, computational methods have emerged as powerful tools in drug discovery and vaccine design for viral infections such as rabies. In silico approaches, such as molecular docking, homology modelling, virtual screening, molecular dynamics simulations and quantitative structure-activity relationship (QSAR) models, offer cost-effective and time-efficient means of identifying potential antiviral compounds and understanding their mechanisms of action. These methods can predict interactions between viral proteins and potential inhibitors, facilitating targeted therapy development (12). However, systematic clinical research into combination therapies faces challenges due to the sporadic occurrence of rabies cases. There is a pressing need for medical approaches that accelerate effective therapy development through veterinary care and investigational treatments of naturally infected dogs when appropriate. Understanding the pathogenesis of RABV in both humans and dogs has advanced significantly, providing critical insight into severe neurological dysfunction associated with the virus. Of note, four disease processes need to be managed: Viral propagation, neuronal degeneration, inflammation and systemic compromise (13).

The present review aimed to provide a comprehensive overview of recent advancements in in silico research focused on RABV and focusses on novel ligand identification, structural elucidation of key viral proteins, drug repurposing strategies as potential treatments for rabies, and explores the design of multi-epitope peptide vaccines. By emphasizing these developments, we aim to illustrate the potential of computational approaches in combating rabies and inspire further research in this vital area. While the biological mechanisms of rabies are well-documented, addressing its therapeutic challenges necessitates innovative approaches. Computational methods, with their cost-effectiveness and predictive capabilities, have emerged as a powerful solution in the quest for novel treatments.

Given the lack of effective treatments post-infection, structure-based drug discovery offers a promising avenue by exploiting detailed molecular insights into the key proteins of the virus. In silico methods, which encompass computer-based techniques, have become essential in drug discovery. These methods utilize computational tools and algorithms to predict, analyze and optimize interactions between ligands (potential drug molecules) and biological targets, such as proteins or nucleic acids. By simulating molecular interactions and screening extensive libraries of compounds, in silico approaches significantly expedite the identification of promising drug candidates, while reducing the costs and time associated with traditional experimental methods. Structure-based drug discovery has emerged as a promising and efficient strategy for identifying novel and potent drug candidates in a more efficient and cost-effective manner than conventional methods (14). This approach relies on the three-dimensional structures of biological protein targets to elucidate the molecular basis of diseases. To virtually identify drug candidates, several preparatory steps for proteins and compounds are essential for obtaining accurate results. Key steps include homology modelling, virtual screening, QSAR models and pharmacophore modelling, molecular docking and molecular dynamics simulations (12,15). Molecular docking predicts the preferred orientation of one molecule when bound to another to form a stable complex. By contrast, molecular dynamics simulations study the physical movements of atoms and molecules, providing a dynamic view of molecular interactions. These techniques are cost-effective and precise enough to predict mechanical characteristics, optimize structures, and simulate the natural motion of biological macromolecules (16). Structure-based virtual screening involves several computational phases, including target and database preparation, docking, post-docking analysis, and prioritization of compounds for testing. Homology modelling predicts protein structures from amino acid sequences by aligning them with known templates, constructing models and refining them through various steps (14). QSAR models establish associations between the properties of chemical substances and their biological activities to predict the activities of new chemical entities. Moreover, pharmacophore modelling identifies essential structural features necessary for a molecule to interact with a specific biological target, aiding in virtual screening and lead compound optimization (15). An overview of the in silico methods used in the drug discovery process, with their advantages and disadvantages is provided in Table I.

RABV, a member of the Rhabdoviridae family, encodes five structural proteins: Nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G) and the large RNA-dependent RNA polymerase (L or RdRp) (Fig. 1). Each protein plays a critical role in viral replication, immune evasion and pathogenesis. Understanding their structural and functional properties provides essential insight into antiviral drug targeting. The N protein encapsulates the viral RNA genome, forming the ribonucleoprotein complex crucial for viral transcription and replication, shielding the viral genome from host immune responses (17). The G protein mediates viral entry by interacting with host cell receptors, such as the nicotinic acetylcholine receptor and p75 neurotrophin receptor. It is also the primary target for neutralizing antibodies, rendering it a key focus for vaccine and antiviral drug development (18). The L protein, in association with the P protein, carries out viral genome replication and transcription, rendering it a promising target for broad-spectrum antivirals (19).

The advent of ML and AI has markedly facilitated the understanding of complex biological systems and accelerated the drug discovery process. One area where ML/AI has played a vital role is protein structure prediction, which is essential to understanding the biological mechanisms involved in the disease. DeepMind's AlphaFold is the game changer in this scenario, surpassing all other existing tools and recognized as the recipient of the 2024 Nobel Prize in Chemistry (24). Secondly, the role of AI in drug discovery has been monumental. In the field of RABV drug discovery, these technologies facilitate the analysis of genetic, epidemiological and pharmacological datasets to predict treatment efficiency, optimize antiviral drugs and identify novel therapeutic targets by significantly reducing the cost and time involved in developing new rabies treatments. AI-based computational models are speeding up the discovery of host factors and RABV proteins that can serve as therapeutic targets by analyzing viral genomes and host-pathogen interactions to detect the critical molecular pathways essential for RABV replication (25). In addition, predicting the conserved viral epitopes could enable the development of vaccines or anti-viral drugs (26). Likewise, AI also supports the identification of host proteins involved in immune evasion or viral invasion that could open new avenues in therapeutics (27).

Another area where AI is playing a transformative role is in the development of antiviral drugs for rabies. ML approaches enable rapid screening of millions of compounds to identify leads as potential inhibitors of viral replication by analyzing extensive molecular datasets and predicting drug-receptor interactions, followed by lead optimization to enhance their efficacy (28). Drug repurposing is another interesting area of research where AI can expedite the identification of FDA-approved drugs with activity against the RABV (29). AI also predicts drug-virus interactions, identifying promising antiviral candidates inhibiting replication. Additionally, the amalgamation of AI and CRISPR genome-editing technology is advancing research into RABV evolution, diagnosis, and treatment (30). Together, these efforts are paving the way for safer, more effective therapeutic options for rabies prevention and treatment.

Research into the molecular basis of rabies, which affects the central nervous system, has demonstrated that the virus targets neurons, leading to severe neurological symptoms (31). Currently, the only available treatment option is post-exposure prophylaxis, which comes with important drawbacks, including high costs and complicated administration. These limitations necessitate alternative strategies where drug repurposing could benefit rabies treatment. By re-examining existing drugs for novel therapeutic applications, the drug development process can be accelerated with reduced cost and potentially reveal new treatment pathways. Repurposed medications may provide a more rapid route to clinical application, addressing the urgent need for more accessible and effective therapies for rabies (10).

In a previous study, notable candidates, such as cidofovir, emtricitabine, famciclovir, acyclovir, and stavudine were shown to exhibit potential efficacy against the RABV glycoprotein (32). Advanced docking analyses were conducted using the CB-Dock2 program to assess these candidates. This sophisticated approach searched the database of the server for template ligands with strong topological similarity (FP2 ≥0.4) through an integrated homologous template-based blind docking mechanism (32). Following this, cavity detection was performed using an in-house technique known as FitDock to aid molecular docking with specific templates. The docking analyses identified five potential binding cavities for Emtricitabine within the target protein, with volumes ranging from 7209 ų to 1703 Å with significant binding affinities, varying from -5.0 to -6.1; lower scores suggest stronger binding affinity. The root means square deviation values were observed around 10 Å, and the hydrogen bond count fluctuated around 25, indicating stable interactions with limited conformational deviation and persistent hydrogen bonding (32).

In addition to computational methods for drug repurposing, the validation of these compounds is crucial for establishing their efficacy against rabies. Repositioning already approved medications can significantly lower drug development costs and timelines, while reducing the likelihood of unexpected side-effects. Computational repositioning allows for the rapid screening of candidates in silico, which renders it an attractive option for prioritizing potential treatments. In a previous study, the ChEMBL library was filtered from 2,354,965 compounds down to 6,554 that had passed through any phase of clinical trials to avoid cytotoxic inhibitors (33). The cryo-electron microscopy (cryo-EM) structure of the respiratory syncytial virus (RSV) strain A2 polymerase (PDB: 6UEN) was prepared for AutoDock Vina docking studies. Compounds with flexible ligand structures or poor binding affinities were eliminated, narrowing the selection to 4,919 candidates. Protein-Ligand Interaction Profiler was used for interaction analyses with micafungin, trombopag, trovafloxacin, azlocillin Na, trospium, amiodarone HCl, perflubron, mephenesin and methadone. Among these candidates, micafungin demonstrated exceptional promise with a binding affinity of -14.32 kcal/mol and an inhibition constant of 0.0317 nM, outperforming existing inhibitors, such as AZ-27 (0.47997 nM), ALS-8112 (2.56 nM) and ribavirin (6.98 nM) (33). In vitro assays further confirmed the efficacy of micafungin by reducing RSV polymerase activity to only 29.6% of the control levels; by contrast, verubecestat (97.4%), ribavirin (98.4%) and ALS-8112 (89.5%) retained markedly higher levels of activity compared with the control. These findings collectively demonstrate that computational approaches can effectively identify promising repurposed compounds for rabies treatment while validating their efficacy through rigorous screening processes (33).

Phthalazine derivatives have gained attention in recent times due to their pharmacological properties (34). Their unique structural characteristics make them promising candidates in combating viral infections. High-throughput screening methods have played a key role in discovering compounds that inhibit RABV replication, while maintaining low cytotoxicity. Comprehending the mechanisms of action of these compounds is vital for augmenting their therapeutic potential. Phthalazine and its derivatives, nitrogen-containing heterocyclic compounds, have gathered attention in medicinal chemistry due to its versatile pharmacological properties. Of note, one phthalazine derivative compound, 7671954, was found to be particularly effective against RABV infections across different Lyssavirus species with low cytotoxicity (IC50 values <30 µM) (34). Experiments established that these compounds inhibited viral replication by targeting the replication complex (34). Furthermore, these derivatives interact with host receptor, such as the p75 neurotrophin receptor and nicotinic acetylcholine receptor, both of which play critical roles in viral entrance, potentially increasing immune responses while limiting viral interactions with host cells (18).

In summary, drug repurposing provides a cost-effective and accelerated path for rabies treatment, identifying promising candidates such as emtricitabine, famciclovir, acyclovir, stavudine and cidofovir. Computational screening and high-throughput assays have enabled the rapid identification of drugs with potent antiviral potential against rabies. These approaches address the urgent need for affordable and accessible therapies beyond current post-exposure prophylaxis, which remains costly and limited in availability. The continued research and validation of these repurposed drugs could transform rabies management and save countless lives globally.

Generally, drug discovery focuses on treatment post-infection, whereas vaccines provide proactive protection. Computational methods have facilitated vaccine design through precise epitope mapping. Since viral entry is the key to infection, the RABV glycoprotein (RABV-G) is mainly targeted for vaccine development. Several immunoinformatic tools are utilized to identify the antigenic sites and further design multiepitope peptide vaccines (35).

The Immune Epitope Database (IEDB) is employed to predict epitopes based on the Bepipred Linear Epitope Prediction 2.0 method. Initial sequence analysis that includes antigenicity, allergenicity and virulence assessments is carried out using AllerTOP v2.0, DeepTMHMM, VirulentPred and VaxiJen. This is followed by BLASTP analysis to ascertain no cross reactivity with human proteins. Physico-chemical properties and structure prediction studies are performed using Protparam, PSIPRED and GalaxyRefine followed by the final prediction of immune response prediction using C-ImmSim server. Studies were carried out using this approach for RABV proteins (36,37).

In a previous study, AR16 and hPAB, two conserved RABV peptides, were combined with Gp96 adjuvant to create an oil-in-water emulsion. By day 14, the vaccine candidates produced virus-neutralizing antibodies and were safe. By day 28, they peaked at 5-6 IU/ml in mice and 7-9 IU/ml in beagles. The promise of multi-epitope peptide vaccines for rabies prevention was demonstrated by the 70-80% protective efficacy against a virulent rabies strain in mice (36). In another study, using computational immunoinformatic methods, B-cell epitopes from the RABV glycoprotein and nucleoprotein were found (37). The eukaryotic vector pcDNA3.1(-) was used to express the highly antigenic areas after they were put together into a multi-epitope construct. Oral delivery was facilitated by adopting food-grade Escherichia coli as a carrier. Experiments conducted both in vitro and in vivo demonstrated that the vaccination could produce a robust immune response in mice, markedly increasing survival following viral challenge without causing any negative side-effects. This method demonstrates a viable, scalable approach to rabies prevention, especially in populations of wild animals (37).

In recent times, natural forms of therapy have gained importance based on popular beliefs, skills, and indigenous practices owing to their no-side-effect advantage. The role of traditional medicine has taken a center stage, primarily including plants as the source of innumerable compounds with enormous therapeutic benefits (38). Although this approach is used for rabies management, there are limited data available to support the efficacy of these methods. Phytochemicals provide an alternative approach for antiviral treatment as they are known to inhibit viral replication (39).

Compounds such as coumarins, lignans, alkaloids, flavonoids and terpenoids exhibit antioxidant activity and may suppress viral genome activity. A previous study identified anti-rabies phytochemicals from Salix subserrata and onion, screened for toxicity using SwissADME and Protox-II servers. Among the candidates, (S+)-catechin and kaempferol exhibited promising binding interactions with RABV glycoprotein based on docking results. (+)-Catechin displayed an improved binding affinity, full fitness and estimated ΔG values than kaempferol with the formation of conventional hydrogen bonds with the target protein, rendering it a potential natural inhibitor against rabies; this demonstrates the role of natural product-based drugs (40).

Likewise, dodecandrin from Phytolacca dodecandra L'Hérit., a ribosomal inactivating protein, is expected to have an antiviral effect by depurinating the α-sarcin/ricin loop of the ribosomal RNA, thereby halting protein synthesis and inhibiting viral replication (41). Datura metel Linn. contains anticholinergic agents such as atropine, which can affect nervous system function by acting as competitive antagonists of muscarinic acetylcholine receptors. This blockade can regulate nervous system activity, perhaps relieving neurological signs of rabies (42). Salix subserrata and Silene macroselen were also previously investigated for the ability of their extracts to extend the survival rates of mice infected with rabies, although no specific bioactive compounds were reported (40). Although these plants have exhibited some potential in laboratory settings, they have not been proven effective for human rabies treatment, setting the stage for additional pharmacological and clinical studies (9). Additionally, salviifoside A from Alangium salviifolium, was previously demonstrated to exhibit potential in selectively inhibiting RVG interactions with promising binding scores derived from docking analyses against RVG protein structures obtained from UniProt (Accession no. A3RM22) (43). That study utilized homology modelling with SWISS-MODEL to create potential structures for docking analysis.

In summary, phytochemical-derived compounds present an encouraging and diverse pool of natural antiviral substances with possible activity against RABV targets; however, their therapeutic promise has yet to be definitively confirmed by thorough pharmacological and clinical trials to convert these initial results into efficient human therapies. An overview of the type of ligands, their targets and the mechanism of action is presented in Table II.

RNA-based therapeutics have emerged as a promising strategy for the prevention and treatment of rabies, leveraging advanced vaccine platforms such as mRNA and self-replicating RNA (srRNA) technologies.

Understanding the genetic diversity of the RABV (Table III) is crucial for designing effective vaccines. A previous study analyzing 50 dog-brain samples from suspected rabies cases in India provided insight into regional viral variations (53). PCR screening targeting the nucleoprotein (N) and glycoprotein (G) genes, followed by sequencing, revealed that six isolates from Mumbai belonged to a single Arctic lineage which traced back to an Indian lineage I from 2006. Bayesian coalescent analysis estimated the time to the most recent common ancestor (TMRCA) for these sequences to be 1993, indicating long-standing geographic clustering. These phylogenetic data underscore the importance of tracking viral evolution for epidemiological studies and vaccine development (53). Sequence analysis further revealed amino acid polymorphisms in G-protein epitopes, influencing antigenicity and pathogenicity. These findings highlight the necessity for region-specific vaccine strategies to account for genetic variations in circulating RABV strains (53).

Computational studies play a critical role in decoding the intricacies of RABV pathogenesis. Research into intrinsically disordered protein regions of the RABV proteome has yielded key insights (51). High intrinsic disorder levels in the phosphoprotein (P-protein) and nucleoprotein (N-protein) are linked to Negri body formation and host immune suppression. Proteomics analysis of highly purified RABV (RABV) has revealed 47 host proteins that become entrapped inside viral particles upon assembly. Of these, 11 are highly disordered. A comprehensive study on five of these highly disordered mouse proteins, neuromodulin, Chmp4b, DnaJB6, Vps37B and Wasl, utilized bioinformatics tools such as FuzDrop, D2P2, UniProt, RIDAO, STRING, AlphaFold and ELM to investigate their intrinsic disorder propensity (54). These disordered host proteins appear to play a major role in RABV pathogenicity, immune evasion, and possibly resistance to antiviral drugs. That study highlights the complex interplay between virus and host, in which intrinsic disorder plays a key role in viral pathogenic processes. This suggests that these intrinsically disordered proteins and their corresponding host interactions might be good candidates for therapeutic targets (54).

In addition, dysregulation in the matrix (M) protein could play a role in viral budding and transmission, affecting the capacity of the virus to spread. Phylogenetic analysis, comparing RABV isolates from different host species and geographic regions, has revealed clear patterns of clustering consistent with host-specific adaptation of the virus (55). These patterns highlight the ecological niche of Lyssavirus, which is strongly shaped by their mammalian hosts, with implications for transmission dynamics and vaccine development.

Addressing rabies at the therapeutic level remains a major scientific and public health challenge, particularly in regions with limited access to post-exposure interventions. While vaccines have substantially reduced transmission, effective treatments for symptomatic cases are still lacking. The present review highlighted how computational tools are transforming the drug discovery landscape for rabies by enabling the exploration of novel targets and mechanisms. Notably, in silico platforms have facilitated the rational design of peptide vaccines, the identification of antiviral leads, and re-evaluation of existing drug libraries with improved precision and efficiency. However, the success of these approaches centers on their integration with experimental validation pipelines. Future efforts are required to prioritize translational studies, interdisciplinary collaborations and capacity building in endemic regions to harness the full potential of bioinformatics-driven research. Ultimately, a data-driven, systems-level approach may provide the most promising route toward effective and equitable rabies control.