Rabies is an infectious viral disease caused by the Rabies virus. It affects humans as well as domestic and wild animals.
Rabies virus is an enveloped, single-stranded RNA virus with a bullet-shaped or rod-shaped morphology. It is a member of the family Rhabdoviridae and the genus Lyssavirus.
Structure of Rabies virus
Shape: Rabies virus is rod or bullet-shaped (75 x 180 nm); one end conical and the other planar (concave).
Envelope: Present. Virus envelope contains glycosylated G-protein spikes embedded in a lipid membrane derived from the host cell. This envelope doesn’t cover the planar end.
Matrix (M) protein: The membrane or matrix (M) protein layer lies beneath the envelope. Virus nucleoprotein (N) which tightly binds the viral RNA to form the nucleocapsid core lies inside to matrix protein.
Viral genome: The core of the virion consists of helical RNP (group-specific antigen). The genome is unsegmented, linear, ss (-) sense RNA ( 12kb, MW 4.6 X 106) and the virion contains an RNA-dependent RNA polymerase.
- survives at 4°C for weeks and at -70°C for years.
- Sensitive to ethanol, iodine- preparations, 40– NH4+ compounds, soap, detergents, and lipid solvents (ether, chloroform, acetone).
- Inactivated by phenol, formalin, ß- propiolactone( BPL), UV, or sunlight; thermal inactivation at 50°C in 1 hr, and at 60°C in 5 minutes.
- Inactivated by CO2, so, for storage in dry ice, it should be sealed in glass vials.
- Attachment: Rabies virus attaches to host cells via its glycoprotein spikes; nicotinic acetylcholine receptor may serve as a cellular receptor (in neurons).
- Entry: Entry is by endocytosis.
- Replication and Assembly:
- Viral RNA polymerase transcribes the viral genome into 5 mRNA species; the monocistronic mRNA species code for 5 virion proteins: nucleocapsid (N), polymerase proteins (L, P), matrix (M), and glycoprotein (G).
- Genome RNP is a template for complementary (+) sense RNA, which is responsible for a generation of (-) sense progeny RNA. The same viral protein serves at polymerase for viral RNA replication as well as transcription.
- newly replicated genomic RNA associates with viral transcriptase and nucleoprotein to form RNP cores in the cytoplasm; M- protein is important in packaging the RNA.
- Release: The newly formed virus particles acquire an envelope by budding through the plasma membrane.
Animal Susceptibility and Growth of Virus
Rabies virus has a wide host range; all warm-blooded animals including humans can be infected. Susceptibility varies;
- high (fox, wolves, bats, cats)
- moderate (dogs, sheep)
- low ( opossums).
The virus is widely distributed in the nervous system, saliva, urine, lymph, milk, and blood of infected animals.
Street Virus: When freshly isolated in the lab, strains are referred to as “street virus”; these cause fatal encephalitis in lab animals following inoculation by any route, after a long and variable incubation period ( 1-12 weeks) and regularly produce intracytoplasmic inclusion bodies (Negri bodies).
Fixed Virus: Serial brain-to-brain passage in rabbits of the street virus yields a fixed virus that no longer multiplies in extraneural tissues; after intracerebral inoculation, it produces fatal encephalitis after a short and fixed incubation period of 6-7 days; Negri bodies usually not demonstrable.
There is a single serotype of the rabies virus; however, there are strain differences among viruses isolated from different species.
- substitution at amino acid position 333 of gp results in loss of virulence, where Arg has replaced Gln or Ile in the more pathogenic variant.
- purified spikes containing viral gp elicit neutralizing( protective) antibodies; thus providing a safe and effective subunit vaccine.
- antiserum prepared against purified nucleocapsid Ag used in diagnostic IFT, the antibody is not protective and is group-specific.
- virus possesses haemagglutinating activity (property of gp spike), optimally seen with goose erythrocytes at 0-4°C and PH 6.2.
- GP spikes are inactivated by heat ( 56°C for 30-60 min), ether, trypsin, deoxycholate, or tween 80, but not by BPL.
Rabies is primarily a disease of lower animals; it spreads to humans by bites of rabid animals or by contact with saliva. Human to human transmission is very rare, only documented cases involve transmission by corneal transplants. The virus does not penetrate intact skin, and if deposited, it is inactivated due to time and drying. Human cases due to non-bite exposures to rabies are very rare. Uncommon routes for transmission are:
- Inhalation while in a bat-infested cave
- Aerosols released during centrifugation of infected materials in the lab
- Ingestion of flesh of rabid animals (high dose would be necessary)
Consuming unpasteurized milk from a rabid animal may cause rabies (theoretically possible), but no documented cases so far.
Rabies virus multiplies in muscle or connective tissues at the site of inoculation for 48 to72 hrs → enters peripheral nerves at neuromuscular junction → spreads up the nerves to CNS → multiplies in the CNS and progressive encephalitis develops → and spreads centrifugally along the peripheral nerve trunk to various body parts including the salivary glands where it multiplies and is shed in saliva; organ with the highest titer of a virus is a submaxillary gland. Other organs where the virus has been found include the pancreas, kidney, heart, retina, and cornea.
Rabies virus has not been isolated from blood.
Susceptibility to infection and incubation period depends on
- hosts age, genetic background, and immune status;
- viral strain involved,
- amount of inoculum,
- the severity of laceration, and
- the distance between the point of entry and the central nervous system.
The virus produces a specific eosinophilic cytoplasmic inclusion, the Negri body in infected nerve cells, which are round or oval, purplish-pink structure and varies in size from 3-27 µm; the Negri bodies are filled with viral nucleocapsids. The presence of such inclusions is pathognomonic of rabies, but it is not observed in at least 20% of cases; therefore the absence of Negri bodies does not rule out rabies as a diagnosis.
The incubation period of rabies viral disease is typically 1-2 months; maybe as short as 1 week or as long as years. The clinical spectrum can be divided into 3 phases;
prodrome lasting 2-10 days may show malaise, anorexia, headache, photophobia, nausea and vomiting, sore throat, and fever; there is a profound sense of apprehension, anxiety, agitation, irritability, insomnia, or depression.
Acute neurologic (encephalitic) phase
- During the acute neurologic phase (2-7 days), there are signs of nervous system dysfunction which begins with hyperactivity → bouts of bizarre behavior, agitation, or seizures.
- The pathognomic feature is difficulty in drinking, together with intense thirst; attempts to drink may bring painful spasms of pharynx/ larynx producing choking and gagging, patients develop a dread of even the sight or sound of water (hydrophobia).
- Generalized convulsions follow death occurs within 1-6 days due to respiratory arrest during the convulsion
Rabies may present as:
- furious rabies: a predominantly encephalitic disease with neurologic dysfunction.
- dumb rabies: paralytic illness; with symmetrical ascending paralysis followed by coma and death, occurs in about 20% of patients.
The pathogenicity of a strain is related to its capacity to induce cell fusion in neuroblastoma cells. Observable damage to nerve cells in the brain appears minimal; non-specific changes include parenchymal microglial response and perivascular cuffing, with lymphocyte and plasma cell infiltration in the grey matter of the brain stem and spinal cord.
Laboratory Diagnosis of Rabies Virus Infection
Laboratory diagnosis of human or animal rabies has to be based on the following findings:
- Demonstration of virus antigens (Ag) or nucleic acid from the brain, spinal cord, salivary glands, saliva, cornea, or skin by means of immunofluorescence or polymerase chain reaction (PCR).
- Postmortem demonstration of Negri bodies in brain tissue.
- Isolation of virus from brain tissue and/or saliva.
- Antibodies (Ab) cannot usually be demonstrated before the manifestation of disease, and in many cases, serological tests are also negative throughout the clinical course.
Rabies antigens or nucleic acid
Tissues infected with rabies virus are currently identified most rapidly and accurately by means of immunofluorescence or immunoperoxidase staining using anti-rabies monoclonal antibodies. A biopsy specimen is usually taken from the skin of the neck at the hairline. Impression preparations of a brain or cornea tissue may be used.
A definitive pathologic diagnosis of rabies can be based on the findings of Negri bodies in the brain or the spinal cord. They are sharply demarcated, more or less spherical, and 2-10 µm in diameter, and they have a distinctive internal structure with basophilic granules in an eosinophilic matrix. Negri bodies contain rabies virus antigens and can be demonstrated by immunofluorescence. Both Negri bodies and rabies antigens can usually be found in animals or humans infected with rabies, but they are rarely found in bats.
Reverse transcription- polymerase chain reaction (RT-PCR) testing can be used to amplify parts of a rabies virus genome from fixed or unfixed brain tissue. Although unusual as a diagnostic test, sequencing of amplified products allows identification of the infecting virus strain.
Available tissue sample is inoculated intracerebrally into suckling mice. Infection in mice results in encephalitis and death. The central nervous system of the inoculated animal is examined for Negri bodies and rabies antigen. In specialized laboratories, hamster and mouse cell lines can be inoculated for rapid (2 to 4 days) growth of rabies virus; This is much faster than virus isolation in mice. An isolated virus is identified by fluorescent antibody tests with specific antiserum. Virus isolation takes too long to be useful in making a decision about whether to give a vaccine.
Serum antibodies to rabies can be detected by immunofluorescence or neutralisation( Nt) tests. Such antibodies develop slowly in infected persons or animals during the progression of the disease but promptly after vaccination with cell-derived vaccines. Antibodies in cerebrospinal fluid are produced in rabies-infected individuals but not in response to vaccination.
All animals considered rabid or suspected rabid should be sacrificed immediately for laboratory examination of neural tissues. Other animals should be held for observation for 10 days. If they show any signs of encephalitis, rabies, or unusual behavior, they should be killed and the tissues examined in the laboratory. If they appear normal after 10 days, decisions must be made on an individual basis in consultation with public health officials.