▶The History of Virus Discovery
Rabies disease has been well-known since antiquity. It is a zoonotic acute infection of central nervous system that is inevitably fatal.
The first breaking success in the fight against rabies was achieved in 1884 by Louis Pasteur and his outstanding colleagues Emile Roux, Charles Chamberland, and Louis Thuillier, who created the efficient antirabies vaccine. Since that time this mortal disease has begun to retreat.
V. Babes in 1887 and A. Negri in 1903 described specific inclusion bodies in neurons of animals, dead from rabies. These inclusions were referred to as Babes-Negri bodies.
The viral etiology of rabies was proved in 1903 by P. Remlinger, E. Riffat-Bay, and A. di Vestea, who isolated rabies virus.
Although the number of human cases is small, rabies is a major public health problem because it is broadly spread among animal reservoirs.
▶Classification of Rabies Virus
This virus pertains to the order Mononegavirales, family Rhabdoviridae, genus Lyssavirus, and species rabies virus.
To date 7 genotypes and 5 serotypes of rabies virus are established. Overall, lyssaviruses demonstrate the lowest genetic variability in comparison with other rhabdoviruses.
▶Structure of Virus
Rabies viruses are rod- or bullet-shaped particles of about 75×180 nm. Type of symmetry of virions is helical.
Viruses are surrounded by lipid envelope with protruding spikes. Ribonucleocapsid is confined inside the envelope. Viral genome includes single-stranded, non-segmented negative-sense RNA.
Genomic RNA encodes 5 structural proteins.
Nucleocapsid proteins N (nucleoprotein), P (phosphoprotein), and L (RNA polymerase) account for replication of viral genome and viral mRNA transcription.
Matrix M protein is located under lipid envelope; it takes active part in viral budding.
Supercapsid glycoprotein G makes spikes in lipid envelope. It plays an essential role in rabies pathogenesis.
First, it specifically binds to nicotinic acetylcholine receptors (nAChR) on membranes of neurons and muscle cells promoting viral attachment and membrane fusion. Also protein G stimulates apoptosis of infected cells.
Furthermore, superficially located protein G activates host immune response in the course of infection. It elicits the synthesis of virus-neutralizing antibodies.
According to structural variations of G proteins, rabies virus is divided into 5 serotypes. However, all viral serotypes are enough similar and induce the formation of cross-reactive neutralizing antibodies. Thus, it became possible to use only 1 serotype of vaccine virus for rabies vaccination.
When freshly isolated in the laboratory from external source, the viral strain is designated as rabies street virus. These viruses show long and variable incubation periods (usually 21-60 days in dogs) and regularly produce cytoplasmic inclusion bodies. Sequential brain-to-brain passages in rabbits primarily made by L. Pasteur yielded a “fixed” virus. This pathogen lost the ability to multiply in extraneural tissues. Fixed mutant virus propagates rapidly, and its incubation period has been shortened to 4-6 days. Inclusion bodies are found rarely in this infection.
Rhabdoviruses demonstrate generally low resistance. Rabies virus is inactivated rapidly by exposure to ultraviolet radiation or sunlight, and by heating (1 hour at 50°C or 1 minute at 100оС).,
Nevertheless, it remains long-time infectious at low temperatures, e.g. it stays viable at 4°C for weeks.
Rabies virus is sensitive to the commonly used biocides (e.g., sodium hypochlorite and other halides, detergents, aldehydes, ethanol, ether, and others). The virus loses viability at рН<3 or рН>10.
▶Viral Replication Cycle
Rabies virus attaches to cells via its glycoprotein spikes. Spike G protein binds to nicotinic acetylcholine receptor (nAChR) facilitaing viral entry by endocytosis. Molecules of nAChR are expressed on the membranes of neurons and muscle cells, so these cells are the primary targets for virus.
Acidification of endosome content activates G proteins. It results in envelope-membrane fusion and viral penetration into cytoplasm followed by uncoating.
Rabies virus replication occurs in cytoplasm of infected cells.
Single-stranded RNA genome is transcribed by virion-associated RNA polymerase L to mRNA. Messenger RNAs code for five structural virion proteins: nucleoprotein (N), polymerase (L), phosphoprotein (P), matrix (M), and receptor glycoprotein (G).
Negative-sense genomic RNA is transcribed via complementary positive-sense RNA intermediate. Newly synthesized genomic RNAs associate with N, L, and P proteins with formation of ribonucleocapsids. They further interact with matrix M proteins in cytoplasm.
The nascent virions acquire an envelope and external spikes, when released by budding through the cell plasma membrane, where G proteins were primarily embedded.
Rabies virus is readily cultured in brain tissues of laboratory mice, syrian hamsters, or rabbits; the infected animals display encephalitis with paralysis.
Also the virus is adapted to various cell lines (Vero cells, BHK cultures and others). Acidophylic inclusions (Babes-Negri bodies) are detected in cytoplasm of infected cells.
Cytopathic effect of rabies virus is not observed.
▶Pathogenesis, Clinical Findings and Immunity in Rabies
Rabies is an acute zoonotic neuroinfection developing after the bite of a rabid animal and followed by progressive CNS damage with lethal encephalitis.
Without urgent vaccination rabies disease demonstrates 100% fatality.
Rabies virus has a broad host range. All warm-blooded animals, including mammals, can be infected.
Susceptibility of many mammalian species is very high (e.g. foxes, wolves, racoons and raccoon dogs, cats, rats and many others.)
Recovery from infection in animals is extremely rare except certain bats species. For instance, vampire bats may transmit the virus for months without any signs of disease.
Thus, the source of infection is a rabid animal.
The virus is usually transmitted to humans via the rabid animal bite or by contact with infected saliva through the skin or mucosal lesions.
Incubation period varies strongly depending on host’s immune status, the amount of inoculum, and the distance the virus should move from the point of inoculation to the central nervous system. The shortest incubation period (about 7-10 days) is observed in patients bitten on the face, head or neck; the longest occurs in cases of bites on feet (1-3 months). Moreover, some documented cases of rabies had the incubation period of more than 10 years. Thus, rabies is regarded as slow viral infection.
When entered into the tissues, rabies virus binds to nicotinic acetylcholine receptor (nAChR) on the membranes of neurons and neuromuscular junctions.
Next pathogenesis of infection follows two basic ways.
If the virus locally multiplies in muscle tissue at the site of inoculation, it may stay long and propagate in primary location up to 2 months.
By contrast, if the virus binds to neuronal transport proteins dynein or neurotropin in axoplasm of peripheral nerves, it undergoes fast retrograde axonal transport that delivers virus directly to bodies of neurons in spinal cord and brain. The velocity of viral movement is equal to 50-100 mm/daily that substantially shortens the incubation period.
Next the virus multiplies in the nuclei of CNS in spinal cord, brainstem, hippocampus, thalamus, cerebellum and other CNS parts.
The progeny viruses spread further through peripheral nerves to the salivary glands and other tissues. The highest concentration of rabies virus is observed in submaxillary salivary gland. Viruses are also found in skeletal muscles, retina and cornea, heart, kidneys and other inner organs. However, rabies virus is not isolated from patient’s blood.
The virus produces characteristic eosinophilic cytoplasmic inclusions, Babes-Negri bodies, within infected neurons. This finding is pathognomonic on rabies.
The disease manifests as acute fulminant fatal encephalitis.
During the acute neurologic phase the patients demonstrate deep neurologic disorders. Patients exhibit hydrophobia (fear of water), photophobia (fear of light), aerophobia, noise phobia. They feel a profound thirst; their swallowing is impaired. Severe sympathetic hyperactivity results in hypersalivation, increased perspiration and lacrimation. The patients often show aggressiveness.
In case of paralytic forms of infection initial local paralysis progresses into generalized disease.
The late phase of rabies is followed by coma and death, usually 2-7 days after the disease onset. The major cause of death is respiratory paralysis.
▶Laboratory Diagnosis of Rabies
All the animals indicated as “rabid or suspected rabid” should be sacrificed immediately for laboratory examination of nervous tissue. Other animals should be observed for 10 days. If they demonstrate any signs of encephalitis, or unusual behavior, they should be killed and the neural tissues examined.
Post-mortem diagnosis of rabies is highly specific.
The specimens of brain tissues taken from died animals or patients are used for laboratory diagnosis. The virus is most rapidly identified by immunofluorescence or immunoperoxidase staining using antirabies antibodies Cytological examination of slides reveals Babes-Negri bodies in the neurons of brain or spinal cord of affected person.
Reverse transcription-PCR can be used to detect rabies virus genome in brain tissue.
For viral isolation the tissue samples are inoculated intracerebrally into suckling mice. Infection in mice results in encephalitis and next death. Animal CNS tissues are examined for viral antigen or Babes-Negri bodies.
Similarly the virus can be inoculated into laboratory cell lines with further identification by immunofluorescence, cytology, ELISA or PCR.
Antemortem (intravital) diagnosis of rabies is applied in cases of atypical infections in humans and, more often, for rapid diagnosis of animal infection needs to urgent vaccination of affected individuals.
Tissue biopsies of bite sites and back of neck skin, buccal and corneal epithelium, cerebrospinal fluid or saliva are examined by immuno-fluorescence or PCR.
Detection of serum specific antibodies by ELISA might be helpful in monitoring of humoral response in vaccinated persons.
▶Principles of Rabies Prophylaxis and Treatment
There is still no treatment for clinical rabies.
Thus, post-exposure rabies prophylaxis is lifesaving intervention and should be initiated without any delay. The medications include rabies vaccination, administration of rabies immune globulin if required, and perfect surgical management of bite wounds.
If the vaccine or specific antibodies are timely administered, virus propagation arrests, and the virus can’t invade the central nervous system. Passively administered antibodies lower the concentration of virus, providing additional time for a vaccine to stimulate active antibody production, thereby preventing viral entry to CNS.
All the vaccines for humans contain inactivated rabies virus. The most commonly administered is human diploid cell vaccine.
Vaccine is injected 5-6 times into deltoid muscle. It confers long-term immunity, postvaccinal complications are rare.
Live attenuated vaccine can be used for animal vaccination.
For passive post-exposure prophylaxis specific immunoglobulins are administered.
Equine rabies immunoglobulin is obtained from horses hyperimmunized with rabies vaccine virus.
Human anti-rabies immunoglobulin is a globulin fraction prepared from the plasma of vaccinated humans. It renders fewer side effects in comparison with equine antibodies.
Passive prophylaxis is administered prior to vaccination in case of multiple bites on upper limbs, neck, or head, where the incubation period is short.