WHY IS RABIES TOUGH TO CURE?

WHAT IS RABIES?

Rabies is one of the oldest and most dreaded of human diseases. It continues to be a major public health problem in developing countries. An acute, fatal encephalitis, it is caused by a highly neurotropic RNA virus, taxonomically belonging to genus Lyssavirus, family Rhabdoviridae. Mammalian reservoirs for this exclusively neurotropic virus include Carnivora and Chiroptera, but transmission by rabid dogs still poses the greatest hazard worldwide. In India, dogs are the main vectors, accounting for 95 % of the reported cases. 

Despite continued attempts at medical intervention, rabies remains an infectious diseases with the highest case fatality ratio. The disease does not discriminate with regard to age, sex, geography, or occupation. According to World Health Organization (WHO) estimates, 50,000 human deaths are reported worldwide every year, with 60 % of these cases being reported from India. Human rabies continues to be endemic in India, except for the islands of Andaman, Nicobar, and Lakshadweep.

The rabies viral infection produces 2 distinct well-recognized clinical syndromes in humans: furious and paralytic rabies. The former, dominated by limbic symptoms, is the most recognized form of the disease, with prototypic symptoms of hydrophobia, aerophobia, and aggressive behavior. The paralytic form, however, presenting with ascending paralysis without “hydrophobic” symptoms, was initially recorded by Gamaleia in 1887 but was not widely recognized until several decades later. The less common bat-related rabies has clinical features distinct from those of dog-related rabies. Most of our present knowledge is derived from studies on experimental animals, mostly rodents, infected with fixed (attenuated) laboratory strains of the rabies virus that have a different biology and does not truly reflect natural infection by the street virus (virulent) strains that causes infection in humans and animal. The virus has a unique mode of entry, spread, and pathogenesis, completely different from all other viruses. It successfully evades immune system detection to reach the central nervous system (CNS) without a viremic phase, and this poses considerable limitations to both diagnosis (lack of antibodies/virus in circulating blood make it impossible to detect by serological/molecular biological tests) and treatment. 

HOW IS IT TRANSMITTED?

Rabies is an infection of domestic and wild animals that spreads to humans by 3 main modes: bites, mucous membranes exposure, and, less commonly, aerosol inhalation.

The virus is excreted in saliva and inoculation of virus-laden saliva through the skin into muscle and subcutaneous tissues of the victim following bite of a rabid animal is the most common mode of infection. Most infections (90 %) are transmitted by bites of domestic animals like cats and dogs, owing to their close association with humans. The virus cannot cross intact skin. Scratches infected with saliva are a less common source of transmission, as the risk of infection is 50 times lower. Bat rabies virus is more infectious than dog rabies virus as it replicates more rapidly in non-neuronal cells and at lower temperatures. Percutaneous infection may occur during skin exposure following a minute bite that escapes attention. The exact mode of viral entry from dermal nerves into the CNS by the bat rabies virus is unclear.


Casual contact with a person infected with symptomatic rabies (touching unbroken skin or contact with non-infectious tissues or bodily fluids) cannot transmit the rabies virus to another person. It may be possible to transmit the virus from a symptomatic rabies patient through mouth-to-mouth contact or kissing.

WHAT ABOUT ENTRY AND INCUBATION PERIOD?

The incubation period (IP) is highly variable, ranging from 6 days to as long as 6 years akin to a slow virus disease.The extreme variability in IP is attributed to host and viral factors, and hiding within safe sanctuaries in the host for prolonged incubation periods. The extreme variability in IP is attributed to host and viral factors, and hiding within safe sanctuaries in the host for prolonged incubation periods.

Receptors are important in viral entry, cell tropism, and spread. The most important receptors incriminated in rabies viral entry are the nicotinic acetylcholine receptors (nAChR) at the neuromuscular junction. In vitro studies demonstrate two more putative receptors: neural cell adhesion molecule, expressed at the neuromuscular junction, and the p75 neurotrophin receptor, with a probable role in infection and viral spread.
The localization of rabies viral antigen areas with rich cholinergic innervation, such as cerebral cortex and limbic structures, including amygdala, hippocampus, thalamus, hypothalamus, and basal ganglia, as seen in in vivo studies in experimental animals, confirms its cholinergic affinity. 

The nAChR are localized to neuromuscular junctions in the periphery, where they modulate skeletal muscle contraction. α-Bungarotoxin is a competitive antagonist of nAChRs, with high affinity and near irreversible binding. In the CNS, the major nicotinic receptor subtype that binds this toxin is the alpha7 subunit, a ligand-gated calcium channel found in anatomical regions of the brain essential for cognition, including cerebral cortex and hippocampus. The nAChR has been used therapeutically in a variety of conditions where localized inhibition of neuronal and/or muscle denervation is desirable, for example various dystonias, spasmodic torticollis, strabismus, blepharospasm, and other conditions with neuromuscular involvement, as also in the cosmetic treatment of facial wrinkles. Recombinant technology has helped develop modifications of the toxin with the same efficacy as its native form, but with varying receptor binding specificity, drug delivery routes, and long-term release preparations without side effects like paralysis, autonomic involvement, and so on. Whether it can be used in conjunction with passive immunization remains to be explored in humans. Knowledge of both the region of the virus involved in binding, and the binding domain on the receptor, may be helpful in developing new therapeutic strategies, especially for rabies virus that infects the CNS after successfully evade the host immune defenses.

However, the nAChR may not be solely responsible for viral spread in the CNS, as noncholinergic areas are also highly susceptible to rabies viral infection. Cellular sialic acid, galactose, mannose and N-acetylglucosamine, and gangliosides, but not fucose, are shown to be involved in the binding of rabies virus to the host cells. Variability in the receptors involved in viral entry could influence the IP, by altering the neuronal groups involved, and this mechanism needs to be evaluated as alternate therapeutic targets.

WHAT ABOUT THE LIFE IN CNS?

Rabies virus propagates to the CNS from its site of inoculation via axonal transport in a retrograde fashion. Travel to the CNS via the peripheral axons occurs at a fairly constant rate of 12 to 24 mm per day. Fast axonal transport is mediated by microtubules, and slow axonal transport is actin mediated. Colchicine, a microtubule-disrupting agent, effectively inhibits fast axonal transport, preventing rabies viral spread in vitro. Colchicine, which effectively functions as a “mitotic poison”, has an anti-inflammatory property that is used in humans in treatment of gout, Behçet’s disease, and relapsing polychondritis.

(Note:In experimental rats, stereotaxic inoculation of colchicine into the striatum produced inhibition of intra-axonal transport of the rabies virus, but the inhibition was reversible. Use of osmotic pumps to enhance the duration of colchicine-mediated inhibition by delivering the drug continuously in the rat brain was also attempted. Its use in human rabies has remained unexplored.)

Although all available data support centripetal spread of rabies virus by axonal transport from the site of inoculation to the CNS, the mode of virus spread within the CNS appears to be more complex. At present, 3 potential pathways are postulated:

1) virus dissemination within extracellular spaces;

 2) intraaxonal transport via fast axonal transport; and

 3) cell-to-cell transmission between contiguous cells and their processes.

Determining the exact mode of spread may help design new therapeutic targets, such as monoclonal antibodies, that inhibit viral spread within the CNS.

Wide dissemination in extracellular spaces is an efficient and rapid mechanism of virus dissemination within the CNS but is more important in rapid progression of fixed strain of the rabies virus

Transit via the axonal route is important in the progression of the disease by street virus, allowing it to effectively circumvent the host immune responses. The importance of long ascending and descending tracts as a route of virus spread within the CNS, as described in skunks, was observed in human and canine studies from our center (unpublished data). Interruption of axoplasmic flow by alkaloid drugs such as colchicine and vinblastine have succeeded in preventing the ascent from the site of inoculation to CNS. Interestingly, in vivo studies by intracortical inoculations of rabies virus into primates, demonstrated that transneuronal transfer is strictly unidirectional, proceeding in a retrograde direction.

Directional transport of various proteins into axons or dendrites is governed by molecular motors. Anterograde transport is mediated by the kinesin superfamily, whereas retrograde transport is dependent on dyneins. Retrograde dynein-mediated axonal transport of rabies virus enables its spread over long distances in the CNS. Though active rabies virus transport is widely believed to be unidirectional, visualization by live microscopy in infected dorsal root ganglia neurons revealed fast anterograde axonal transport of rabies virus via kinesin-dependent transport machineries.

Cell-to-cell transmission of virus between contiguous cells and their processes, particularly at synaptic junctions, is operative in experimental animals, and less frequent in human rabies

The virus replicates in the dorsal root ganglion and trigeminal ganglion (sensory neuron) and anterior horn cells (motor neurons) of spinal cord. Anterograde transport in dorsal root ganglia is dependent on virus glycoprotein G. Mutant rabies virus lacking glycoproteins fails to move anterogradely within the axon. The complete enveloped virus particles or cotransport of virus ribonucleoprotein and G-containing vesicles occurred from dorsal root ganglion neurons.

As the rabies virus receptors are detected only on motor endplates and motor axons, uptake and transmission to the CNS is believed to occur exclusively through motor axons. Involvement of sensory and autonomic ganglia occurs by centrifugal propagation from the CNS to the periphery. Virus is therefore detectable from the end organs only in terminal stages of the disease. Valuable insights into the viral pathogenesis of rabies come from transneuronal tracing studies in primates and rodent models prior to the development of clinical disease. These studies have shown that rabies virus propagation occurs at chemical synapses and not via gap junctions or cell-to-cell spread. Infected neurons remain viable, both morphologically and functionally as they continue to express neurotransmitters. Retrograde transmission causes all neuronal groups of the same synaptic order to be infected simultaneously, irrespective of their neurotransmitter class, strength of synaptic connections, or distance from entry.

Components of the rabies virus modulate its neuroinvasiveness and neurovirulence. The rabies virus glycoprotein has been shown to play a key role in several steps of rabies pathogenesis—viral uptake from site of inoculation, axonal transport, and trans-synaptic spread in CNS, as well as the viral distribution in the nervous system. The rabies viral glycoprotein (G) is critical for both neutralizing antibody production and the initiation of cell-mediated immune response. Differences in G protein affect G protein receptor interactions with nAChR at the periphery, P75 neurotrophin receptor, and antiglycolipid or ganglioside in the CNS. Minor variations in G protein such as substitution of arginine at position 333 affects neuroinvasiveness by use of different neuronal pathways. Interaction between the phosphoprotein of rabies virus, and LC8—the cytoplasmic dynein light chain that regulates microtubule-mediated transport—is implicated in viral transport. However, mutant viruses with deletion in phosphoprotein encompassing LC8-interacting motif retained their neuroivasiveness and virulence.  Neuroattenuation required simultaneous substitution of arginine at position 333 of viral glycoprotein with deletion of LC8, reinforcing that rabies viral glycoprotein plays a more important role than phosphoprotein.  Several viruses such as HIV, poliovirus, herpes simplex virus, African swine flu, and adenovirus use dynein for intracellular transport of the virus prior to viral replication in the perinuclear area in the microtubule organizing center, where the new virions are assembled. Small peptide inhibitors that disrupt this high-affinity binding domain between dynein and viral protein were shown to interfere with African swine fever viral replication, suggesting a possible novel therapeutic approach for rabies infection also.

Interaction of rabies virus phosphoprotein with microtubules is identified as a unique mode of viral antagonism of interferon (IFN) responses disabling the IFN-signaling pathway that is vital to the innate antiviral host immune response. This unique mechanism of rabies viral subversion of IFN signaling is critical to its pathogenicity, and also suggests novel targets for the development of antiviral drugs or attenuated viruses for vaccine applications.

The rabies viruses have evolved mechanisms to hijack their host cell’s transportation system for movement within cell cytoplasm to reach paranuclear zones for viral replication and subsequent movement towards the plasma membrane for egress from the cell surface. Microtubule-destabilizing agents such as nocodazole, vinblastine, and taxol have shown antiviral effects in in vitro studies, but are highly toxic.

WHY IS RABIES DIFFICULT TO TREAT?

The fundamental barrier to treating infection with RABV lies in its tropism for cells of the nervous system. Indeed, for decades it has been proposed that the replication of RABV within the nervous system enables the virus to evade the myriad of host responses triggered during non-neuronal infection.

Viral infections can usually be treated using anti-viral drugs, which inhibit virus development. Rabies virus uses a myriad of strategies to avoid the immune system and hide from antiviral drugs, even using the blood brain barrier to protect itself once it has entered the brain. The blood brain barrier is a membrane that prevents cells and large molecules from entering the brain. During infection of the brain, the permeability of the barrier can increase, allowing immune cells and antibodies through to help clear the infection. However, during infection with rabies virus, the blood brain barrier locks down, meaning nothing can get through, even antiviral drugs.

The virus goes even further to continue infection and manipulates the immune system to destroy itself instead of targeting infected nerve cells. This manipulation of host responses has made finding strategies to treat rabies following infection difficult for researchers, with many potential antivirals showing promising results in in vitro, laboratory tests being unsuccessful in more complex, in vivo systems.  

Lyssaviruses have a single envelope glycoprotein that mediates attachment and entry, with infection occurring via G protein-mediated binding to host cell receptors. For RABV, three primary receptors have been defined for cellular entry, including nicotinic acetylcholine receptors (nAchR), neuronal cell adhesion molecules (NCAM) and p75 neurotrophin receptor (p75NTR). However, the mechanisms of neuronal entry of RABV at peripheral

inoculation sites remain undefined and utilization of other host receptors is likely. Following receptor binding, the virus likely enters most efficiently through clathrin-mediated endocytosis on motor neuron end-plates. Then, once into the peripheral nervous system (PNS), virions spread transynaptically in a retrograde fashion towards the central nervous system (CNS).

No clinical outcomes are considered to be pathognomonic for RABV infection, with only laboratory diagnosis, either ante- or post-mortem, able to determine lyssavirus infection. This inability to conclusively

diagnose rabies infection without laboratory confirmation can be, alongside cost and availability of PEP, a major factor in cases of delayed treatment.

One key feature demonstrated to influence the outcome of rabies in experimental models is the permeability of the blood–brain barrier (BBB) and PIT. The BBB is a feature of the neurovasculature that acts to separate the brain from macromolecules in the blood. The BBB also plays major roles in infection, forming a physical barrier to protect the brain.

While the permanent opening of the BBB is associated with many pathologies, a transient increase in permeability has been shown to be a major factor in CNS clearance of RABV, as this facilitates the infiltration of both antibodies and immune cells. However, wild-type street strains of RABV have been shown to employ various

mechanisms to counteract the opening of the BBB, and the subsequent infiltration of immune cells. For example, during experimental infection with street rabies strains the BBB becomes refractory to permeabilizing signals and CNS inflammation does not occur.

Indeed, in vivo models with attenuated rabies strains, in contrast to wild-type strains, have demonstrated significantly enhanced BBB permeability due to the extensive inflammation and immune activation caused by attenuated RABV infection. This comes as a result of a reduction in the immune evasion mechanisms employed

by attenuated strains in comparison with wild-type RABV strains that are associated with limited G protein expression, reduced replication rates, modulation of theBBB integrity and a reduction in inflammatory chemokine/cytokine stimulation.

When immune cells do infiltrate past the BBB, rabies can actively promote apoptosis through both caspase-dependent and independent mechanisms ,or downregulate antigen presentation by inducing the production of calcitonin and vasoactive intestinal peptide (VIP) from infected cells. Furthermore, the impermeability of the BBB during clinical rabies infection is also proposed to be a  major hurdle in PIT using antibod ybased PEP. The BBB has also proved to be a major obstacle to direct-acting PIT for rabies.