Rabies – epidemiology, pathogenesis, public health concerns and advances in diagnosis and control: a comprehensive review

Figures & data

Table 1. Highlights of this rabies virus review.

Title and subtitles	Salient points
Introduction	• Rabies has created a havoc globally especially in the developing countries of Asia and Africa despite of worldwide extensive efforts and enforcement of successful control and strategic public health campaigns.
History	• French scientist Louis Pasteur demonstrated the neurotropism of rabies virus in 1881. • In 1885, the first rabies vaccine given to a French boy Joseph Meister, who was bitten severely by a rabid dog.
Epidemiology	• Though rabies is prevalent throughout the globe, many countries and islands have got rabies-free status due to strict quarantine or by virtue of their water locked geographical location.
The virus and genome organization	• Rabies virus is a bullet shaped enveloped virion (180 nm x 75 nm in size) belongs to Lyssavirus genus and Rhabdoviridae family. • Seven distinct genotypes of rabies virus are known to occur. • The classical rabies virus (RV-genotype 1) and its field strains are known worldwide and causes rabies in humans and animals. • Rabies virus genome contains 5 highly conserved monocistronic genes related to five viral proteins glycoprotein (G), nucleoprotein (N), matrix protein (M), non-structural protein (NS) and RNA-dependent RNA polymerase protein (L). • The group specific N protein exhibits precipitating, complement fixing, immunoflourescent and ELISA activity and thus plays a major role in diagnosis and virus identification. • M protein is responsible for the assembly and budding of virus by regulating the virus transcription and replication. • G protein (serotype specific) acts as protective and neutralising antigen. It is responsible for binding with the target cell receptors. • NS and L proteins are vital for the survival and replication of rabies virus.
Mode of transmission	• Most common way of entry of rabies virus into the body is either through saliva or infected neural tissue via bite wounds or open cuts in the skin or mucous membrane and not through the intact skin. • Dog slaughtering and the process of handling, catching, loading, transportation, holding and keeping in the cages increases the risk of transmission to the butchers. • Non-bite exposure methods are inhalation of aerosolized rabies virus, cornea/organ transplants and contamination of abrasions, open wounds, mucous membranes with rabies virus laden saliva or with infectious material such as brain tissue from a rabid animal.
Species affected and reservoirs	• All mammals including wild animals are susceptible and can transmit the rabies virus, but the primary reservoir is carnivorous mammals. • The virus circulates in two interrelated epidemiological cycles i.e. urban cycle and sylvatic cycle. • The urban cycle is mostly maintained by stray and community dogs. • Sylvatic cycle involves mainly pet dogs, cats and wild mammals as reservoirs.
Pathogenesis of rabies	• Incubation period or eclipse phase is highly variable from 2 weeks to 6 years (average: 2 to 3 months) according to the concentration of the virus, inoculation site and density of innervation. • Bites on the head, face, neck and hands with bleeding offer the greatest risk and are generally associated with shorter incubation period due to decreased length and greater number of neurons. • Virus gets attached through G-protein coupled receptors to the target cells (myocytes, local sensory and motor neurons) and amplifies in the muscle cells and macrophages and then through nerve spindles of sensory nerves or neuromuscular junction of motor nerves the virus ascends centripetally along the nerves to reach the central nervous system. • Virus travels by rapid retrograde fast axonal transport at a rate of 12- 100 mm per day towards the CNS. • Receptors are nicotinic acetylcholine receptor, neural cell adhesion molecule (CD56) and low-affinity nerve growth factor receptor (NTR75). • Spread of rabies virus away from the CNS (centrifugal spread) along neuronal pathways, particularly via the parasympathetic nervous system, which is responsible for infection of the salivary glands and skin. • No immune response is detectable in most cases of the human rabies at 7–10 days after the onset of clinical signs and immunosuppression has no effect on the outcome of rabies. • Inhibition of apoptosis could be a strategy of RABV to favour its progression through the nervous system.
Immunopathology	• In rabies, almost complete lack of an inflammatory response within the CNS. • IFN-γ secreted by infiltrating activated T cells could initially induce astrocytes and microglia to express class I and class II major histocompatibility complex (MHC) antigens and also to prime these class I and class II MHC antigens for subsequent cytokine production. • Other mediators in the CNS such as prostaglandin, cytokines like IFN-α, IFN-β and IFN-γ, endogenous neuropeptides like norepinephrine and vasointestinal peptides can suppress immune responses by inhibiting both class I and class II MHC expression and cytokine production by glial cells. • Defective neurotransmission involving neurotransmitters like acetylcholine, serotonin and gamma-aminobutyric acid (GABA) could be important in the pathogenesis of rabies. • Nitric oxide induced neurotoxicity can also mediate neuronal dysfunction.
Evasion of rabies virus from host immune responses	• T cells are inefficient for restriction of rabies virus infection in CNS due to their inactivation by the virus. • T cells are protective during an abortive strain of rabies infection but not during encephalitic strain due to the level of T-cell activation in the periphery. • Rabies virus utilizes various intrinsic factors secreted by the non-infected neurons like calcitonin-gene-related peptide, vasointestinal peptide, norepinephrine etc. to dampen the function of migratory T cells. • RABV infected neurons up regulates immune subversive molecules like B7-H1, FasL and HLA-G, which triggers death signalling in activated T cells resulted in killing of migratory T cells. • Dampening the IFN response also favours rabies infection. • Limited neuroinflammation during rabies resulted in impermeability of T cells via blood brain barrier into the CNS. • Restricted entry or specific destruction of migratory B cells could also contribute to immunoevasive strategy. • During rabies virus replication in the nervous system, the neuronal cell bodies are not damaged by premature apoptosis and neural integrity is preserved. • Sequestration of TLR3 into Negri bodies with an attempt of the virus to protect the infected neuron against apoptosis.
Pathology of rabies	• Brain, spinal cord and peripheral nerves show ganglion cell degeneration, perineural as well as perivascular infiltration of mononuclear cells and neuronophagia. • Vascular lesions like thrombosis and haemorrhages in the brain stem, hypothalamus and limbic system may be noticed. • Degeneration of the salivary and lacrymal glands, pancreas and adrenal medullae are observed focally outside the nervous system.
Diagnosis of rabies	• Based on clinical signs: Prodromal symptoms of rabies in humans include itching, pain or parasthesia at the site of bite wound and gastrointestinal upset. • Furious rabies is characterized by irritability, agitation, hyperaesthesia, autonomic disturbances with pathognomonic symptom of hydrophobia, aerophobia and generalised flaccid paralysis. • Magnetic resonance imaging (MRI), diffusion-weighted imaging, diffusion tensor imaging and biomarkers are recently in use. • Traditional rapid Seller's staining and histopathological (HP) methods are still in use; however, fail to detect positive rabies cases in the absence of Negri bodies. • Isolation of rabies virus in mice (3-4 weeks old or 2 day-old new-born mice) or in cell lines like mouse neurobrastoma cell line (Neuro2a/CCL 131), baby hamster kidney (BHK) 21/C13, Vero cells and McCoy cells are the most reliable methods. • Direct fluorescence antibody test (dFAT) is the most widely recommended test by OIE and WHO for diagnosis of rabies. It is the gold standard test for diagnosis of antigen in fresh brain tissues of dogs. • Nucleic acid based tests and DNA microarray are also in use.
Therapeutic approaches	• Human and equine rabies immunoglobulins are currently available for passive immunization. • Small interfering RNAs (siRNAs) targeting rabies virus nucleoprotein (N) and polymerase (L) genes as antiviral agents have also been developed.
Prevention and control	• It requires enhanced surveillance, accurate and timely diagnosis with proper reporting. • Pre-exposure vaccination of dogs and cats, elimination of stray animals and public health education, etc. are the components of animal rabies control. • Preventive immunization is recommended by the World Health Organization (WHO) for all the staffs involved in handling of materials that are infected or suspected. • Three injections are included in the immunization protocol at day 0, 7 and 28. Booster vaccination is recommended for every year. • In Europe, rabies virus strains like SAG 2, SAD Bern and SAD B19 were used for oral immunization in wildlife for rabies control programmes. • Post-exposure management includes strict quarantine of animals for 6 months which are exposed to a confirmed or suspected rabid animal. • It is suggested to euthanize animals which are developing clinical signs of rabies and head must be shipped for testing. • Post-exposure prophylaxis (PEP) is accompanied by anti-rabies immunoglobulin injection of either human or equine origin. • Enhancement of laboratory-based surveillance in wild and domestic animals are necessary. • Awareness campaigns through mass media outlets and workshops. • Collaboration between the medical and veterinary sectors is necessary for controlling the disease effectively.
Conclusion and future perspectives	• There is a need to enhance the surveillance for rabies virus variants currently circulating in animals particularly in wildlife. • The continuous monitoring of less common non-reservoir species is also important for detecting newly introduced rabies virus variants. • Reporting to the physicians by all in-contact individuals and giving importance to post-exposure prophylaxis is essential. • Suitable vaccination along with monitoring the antibody titres in every 2 years is necessary for the control of rabies in endemic areas.

Table 2. Journey of rabies virus.

Year	Scientist	Discovery
Twenty-third century BC	–	The first references to animal rabies in the laws of Eshunna in Mesopotamina
Before 2300 BC	Aristotle	Rabies was first recognized in Egypt and Greece by Aristotle
Sixth century BC	–	Canine rabies was described in the Avesta (Persia)
Fourth century BC	–	Canine rabies was described in Greece
First century BC	–	Canine rabies was described in the Susrutasamhita (India)
1804	Zinke	Infectious nature of saliva from infected dogs was recognised
1881	Louis Pasteur	Demonstrated the neurotropism of rabies virus
1885	Louis Pasteur	Developed the first RABV vaccine from the spinal cord of rabbits infected with rabies that was air-dried for inactivation.
1885	Louis Pasteur	First rabies vaccine administered to a boy, Joseph Meister, bitten severely by a rabid dog
1903	Remlinger and Riffat-Bay	Identified the rabies virus structure
1920	–	United Kingdom was officially announced free from rabies
1940s	–	RABV emerged in red foxes (Vulpes vulpes) in the Kaliningrad area and then spread to Central and Western Europe
1978	–	The first rabies oral vaccination campaign for wildlife was conducted in Switzerland, and then subsequently to other European countries.
1988	–	A field trial of oral vaccination campaigns and compulsory vaccination to dogs in the outbreak area was initiated using SAD B19 bait in Finland
1991	–	Finland declared free from rabies
2002	–	One death occurred in an unvaccinated bat conservationist in Dundee who contracted an RABV (European bat lyssavirus type 2) and did not receive post-exposure prophylaxis. This is first report after UK announced free from rabies in 1920

Figure 1. Gross and histopathological lesions in rabies. (A) Marked congestion of blood vessels in sulci of swollen cerebral hemispheres. (B) Massive perivascular infiltration with mononuclear cells (lymphocytes and macrophages) around dilated blood vessels in white matter of camel brain. H&E x400. (C) Dense eosinophilic and sharply outlined Negri bodies (arrow) of various sizes in the cytoplasm of intact neuron in camel brainstem section. H&E x400. (D) Brain section showing degenerated neurons, severe cuffing with mononuclear cells and edema. A Negri body is also visible in the degenerated neuron (arrow) having mild diffuse gliosis. H&E x200.

Figure 2. Diagnostic techniques for rabies. (A) Brain impression smear showing oval or round magenta coloured Negri bodies (arrow) in the cytoplasm of neuron. Seller's stain x1000. (B) Intense brown colour signals of rabies virus antigen in the cytoplasm of pyramidal neurons in experimentally infected mice brain with challenge virus standard (CVS) strain of rabies virus. IHC-DAB-MH x200. (C) Brain impression smear showing bright apple green dusty fluorescent signals of rabies virus antigen in the cytoplasm of neurons in a spotted deer. Inset showing plenty of specific signals in the cytoplasmic processes and stroma of neuron. dFAT x200. (D). Bright apple green fluorescent signals of rabies virus antigen in the smear of saliva in a dog. dFAT x200.

Table 3. Type of contact, exposure and recommended post-exposure prophylaxis.

Category	Type of contact	Type of exposure	Recommended post-exposure prophylaxis
I	✓ Touching or feeding of animals ✓ Licks on intact skin	None	None, if reliable case history is available
II	✓ Nibbling of uncovered skin ✓ Minor scratches or abrasions without bleeding	Minor	• Administer vaccine immediately • Stop treatment if the animal remains healthy throughout an observation period of 10 days or is proved to be negative for rabies by a reliable laboratory using appropriate diagnostic techniques. • Wound management
III	✓ Single or multiple transdermal bites or scratches, licks on broken skin ✓ Contamination of mucous membrane with saliva (i.e. licks) ✓ Suspect of contacts with bats	Severe	• Wound management • Rabies immunoglobulin • Anti-rabies vaccine