Rabies Virus: Structure, Pathogenesis, and Clinical Findings

–         enveloped, ss RNA viruses with bullet- shaped or rod- shaped morphology
–         2 genera: lyssa virus → rabies virus and vesiculovirus → vesicular stomatitis virus

Structure of Rabies Virus

– rod or bullet- shaped (75 x 180 nm) ; one end conical, other planar ( concave).

– Membranous envelope with protruding spikes or peplomers (10 nm) which are composed of trimers of viral gp (G) spikes don’t cover the planar end.
– Beneath the envelope is the membrane or matrix (M) protein layer
– membrane may project outwards from planar end of some virion forming a bleb.
– The core of the virion consists of helical RNP ( group specific antigen).
– Genome is unsegmented, linear, ss(-)sense RNA ( 12kb, MW 4.6 X 106).
– virion contain an RNA dependent RNA polymerase.

– survives at 40C for weeks and at -700C 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 500C in 1 hr, and at 600C in 5 minute.
– inactivated by CO2, so, for storage in dry ice, it should be sealed in glass vials.
– virus attaches to cells via its gp spikes; nicotinic acetylcoholine receptor may serve as cellular receptor ( in neurons).
– Entry is by endocytosis.
– genome transcribed by viral RNA plymerast to 5 mRNA species; the monocistronic mRNA species code for 5 virion proteins; nucleocapsid (N); polymerase proteins (L,P); matrix (M), and gp (G).
– Genome RNP is a template for complementary(+) sense RNA, which is responsible for generation of (-) sense progeny RNA
– same viral protein serves at polymerase for viral RNA replication as well as transcription.
– newly replicated genomic RNA associates with viral transcriptase and nucleo- protein to form RNP cores in the cytoplasm; M- protein important in packaging the RNA and protein N and linking it to the envelope.
– The particles acquire an envelope by budding through the plasma membrane.
– rabies virus has a wide host range; all warm- blooded animals including human can be infected.
– Susceptibility varies; high (fox, wolves, bats, cats), to moderate ( dogs, sheep) to low ( opossums).
– Virus is widely distributed in the nervous system, saliva, urine, lymph, milk and blood of infected animals.
– when freshly isolated in 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).
– 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 produce fatal encephalitis after a short and fixed incubation period of 6.7 days; negri bodies usually not demonstrable.
– there is a single serotype of rabies virus; however there are strain differences among viruses isolated from different species.
– substitution at AA 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 elict neutralizing( protective) antibody; thus provide a safe and effective subunit vaccine.
– antiserum prepared against purified nucleocapsid Ag used in diagnostic IFT, the antibody are not protective and are group specific.
– virus passesses haemagglutinating activity ( property of gp spike), optimally seen with goose erythrocytes at 0-40C and PH 6.2.
– gp spikes are inactivated by heat ( 560C for 30-60 min), ether, trypsin, deoxycholate or Tween 80, but not by BPL.
–         virus multiplies in muscle or connective tissues at the site of inoculation of r 48- 72 hrs → enters peripheral nerves at neuromuscular junction → spreads up the nerves to CNS → multiplies in the CNS and progressive encephalitis develops → 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 highest titer of virus is submaxillary gland.
–         Other organ where virus has been found include pancreas, kidney, heart, retina, and cornea; virus has not been isolated from blood.
–         Susceptibility to infection and incubation period depend on hosts age, genetic background and immune status; viral strain involved, amount of inoculum, severity of laceration, and the distance between point of entry and CNS.
–         Virus produces a specific eosinophilic cytoplasmic inclusion, the Negri body in infected nerve cells, which are round or oval, purplish pink structure and vary in size from 3-27 µm; the Negri bodies are filled with viral nucleocapsids.
–         Presence of such inclusions is pathognomonic of rabies, but it is not observed in at least 20% pf cases; therefore absence of Negribodies doesnot rule out rabies as a diagnosis.
–         Disease is acute, fulminant, fatal encephalitis, primarily a disease of lower animals; spread to humans by bites of rabid animals or by contact with saliva.
–         Incubation period is typically 1-2 month; may be as short as 1 week or as long as yrs
–         Clinical spectrum can be divided into 3 phases
1. short prodrome. 2. acute neurologic( encephalitic) phase 3. coma / death
• prodrome lasting 2-10 days may show malaise, anorexia, headache, photophobia, nausea and vomiting, sore throat, fever; there is profound sense of apprehension, anxiety, agitation, irritability, insomnia or depression.
During the acute neurologic phase( 2-7 days), there are sign of nervous system dysfunction which begins with hyperactivity → bouts of bizarre behaviour, agitation or seizures.
• pathognomic feature is difficulty in drinking, together with intense thirst; attempts to drinking 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 with in 1-6 days due to respiratory arrest during convulsion.
–         Rabies may present as:
– furious rabies→ predominantly encephalitic disease with neurologic dysfunction.
– dumb rabies → paralytic illness; with symmetrical ascending paralysis followed by coma and death, occurs in about 20% patients.
–         Pathogenicity of a strain related to its capacity to induce cell fusion in neuroblastoma cells.
–         Observable damage to nerve cells in brain appears minimal; non- specific changes include parenchymal microglial response and perivascular cuffing, with lymphocyte and plasma cell infiltration in grey matter of brain stem and spinal cord.
Lab diagnosis
– now that survival from established rabies has been demonstrated in rare instances, it is necessary to be able to make lab distinct between rabies and other form of encephalitis.
1)      Detection of viral antigen and nucleic acid
•tissues infected identified rapidly by IFT or immunoperoxidase staining using anti- rabies monoclonal antibody.
•specimens: salivary, corneal or conjunctival smears or skin biopsy from nape of the neck ( ante mortem).
or impression smears of cut surface of salivary gland, hippocampus, brain stem or cerebellum ( post mortem).
– RT- PCR to amplify parts of rabies virus genome; useful in molecular epidemiology to trace the source of virus in cases with out a history of exposure.
2)      Viral isolation
available tissue inoculated intracerebrally into suckling mice this results in encephalitis and death; CNS of inoculated animal examined for Negri bodies and rabies antigen ; cytopathic effect is minimal.
isolated virus identified by fluorescent Ab- tests with specific antiserum
3)      Serology
– serum antibody detected by IFT or Nt tests; such antibody develop slowly (7-10 days) since the onset of illness).
– Antibody in CSF present in rabies – infected individuals, but not in response to vaccination.
– a latex agglutination test on saliva from rabid dogs gives 99% specificity and 95% sensitivity when compared with IFT on brain smears.
– All animals considered rabid or suspected rabid should be sacrificed immediately for lab examination of neural tissues; other animal should be held for observation for 10 days.
– The brain of infected animal may be removed carefully and 2 portions
•one in 50% glycerol saline ( for biological test) and
• other in jenkers fixative ( for microscopy).
– Examination of salivary glands by IFT for demonstration of viral antigen.
– impression smears of brain stainded by sellers technique ( basic fuchsin and methylene blue in methanol) to find Negri bodies.
§            99% infection who develop symptoms end fatality.
§         It is essential that individuals at high risk receive protective immunization → introduction of cell culture vaccines which are free from serious complication has made pre –exposure immunization in humans safe and feasible.
§         Postexposure prophylaxis consists of local treatment, administration of rabies immunoglobulin and a vaccination regimen.
• local treatment : prompt cauterization of wound destroys the virus
– wound should be thoroughly cleaned with soap solution / detergent and running water for 5 minute, followed by application of 40-70% alcohol or tincture of iodine or 40– NH4 compound ( cetavlon).
– scrubbing should be avoided and suturing delayed if possible.
•passive treatment with antirabies immunoglobulin (Ig) preferably human, 20 IU/ Kg body weight, given half in an around the wound and half in the gluteal muscle.
active immunization: with inactivated whole virus vaccine ( cell – culture grown), containing atleast 2.5 IU/ dose, given into deltoid muscle.
§         Immunogenic vaccine or specific antibody administered promptly to depress viral multiplication and to prevent invading of virus to CNS; action of passively administered antibody is to neutralize some of inoculated virus and lower the concentration of virus in the body, providing additional time for a vaccine to stimulate active antibody production.
all vaccines for human use contain only inactivated rabies virus.
o        Neural vaccines( suspension of nervous tissues of animals eg sheep, goat or mouse, infected with the fixed rabies virus)
– associated with serious risk of neurological complications ( postvaccination encephalitisP due to the presence of encephalitogenic factor ( a basic protein associated with myelin)
– low potency per dose; poor immunogen.
– complete treatment involves upto 23 painful injections.
A.    Semple vaccine → 5% suspension of sheep brain infected with fixed virus inactivated with phenol at 370C, leaving no residual live virus.
B.      Beta- propiolactone ( BPL) vaccine→ BPL used as inactivating agent.
C.    Infant brain vaccine→ prepared in brains of suckling mice; inactivated by UV, BPL, or phenol; the encephlitogenic factor scanty or absent.
o       Non neural vaccines:
 1.egg vaccine: duck embryo vaccine → discontinued due to poor immunogenicty
live attenuated vaccine → prepared in chick embryos ( eg flury strain)
– used for animals but not for humans
– low egg passage of 40-50 (LEP) for dogs.
– High egg passage of 180 (HEP) for cattles and cats.
2. Tissue culture vaccines
a. Human diploid cell vaccine (HDCV): Virus adapted to growth in WT- 38 human normal fibroblast cell line.
– virus preparation concentrated by ultrafiltration and inactivated with BPL
– highly antigenic and free from serious side effects; high cost.
b. rabies vaccines, adsorbed ( RNA): Made on diploid cell line derived from fetal rhesus monkey lung cells; inactivated with EPL and concentration by adsorption to aluminum phosphate.
c. purified chick embryo cell vaccine ( PCEC) prepared from fixed rabies virus strain flury LEP grown in chicken fibroblasts; inactivated with BPL and further purified by zonal centrifugation.
–         other cell culture vaccines include 10- cell culture vaccines grown on hamster kidney and dog kidney cells, and continuous cell culture vaccines grown on vero cell line.
3. Sub unit vaccine:
Recombinant viral vaccine consisting of vaccinia virus carrying the rabies surface gp gene has successfully immunized animals following oral administration.
Types of rabies antibody
Rabies immunoglobulin Human ( HIRG)
– prepared by cold ethanol fractionation from plasma of hyperimmunized humans
–         fewer adverse reaction than to equine IG.
 Antirabies serum, equine
– concentrated serum from horses hyper immunized with rabies virus.
  •  indicated for persons at high risk ( researchers, lab workers, veterinarians )
  • To attain an antibody level presumed to be protective by means of vaccine administration prior to any exposure.
  • Two injections of 1 ml vaccine given into deltoid muscle 4 weeks apart; re- inforcing dose given at 12 months.
    decision to administer rabies antibody, rabies vaccine or both depend on
1)       Nature of biting animal
2)      Availability of biting animal for lab examination.
3)      Existence of rabies in the area
4)      Manner of attack
5)      Severity of bite
6)      Advice from local public health officials.
–         5 IM 1- ml doses (2.5 IU/ dose) on days 0,3,7, 14 and 30 and optionally 90
–         Give protection for atleast 5 years.
–         Passive immunization: should be given before or simultaneously with the first injection of the vaccine, but not after it.
–         Vaccine failure not uncommon with neural vaccines, while they are extremely rare when immediate local treatment has been followed by rabies IG and a full course of a cell culture vaccine.
–         Vaccine for animals: conc. Cell culture vaccines containing inactivated virus given as single IM injection at 12 weeks of age and repeated at 1-3 yr. intervals.
–         10th most common cause of death in humans due to infections.
–         About 10 million persons given post exposure prophylaxis annually.
–         All person bitten by bats and wild animals must receive post exposure prophylaxis
–         Virus does not penetrate intact skin, and if deposited it is inactivated due to time and drying.
–         Uncommon routes for transmission
    → Inhalation while in bat- infested cave
→ aerosols released during centrifugation of infected materials in lab.
→ Ingestion of flesh of rabid animals ( high dose would be necessary)
– Human to human transmission is very rare.
– Only documented cases involve transmission by corneal transplants.
– no successful treatment for clinical rabies; symptomatic treatment may prolong life, but outcome is almost always fatal.
– preexposure vaccination is desirable for all persons who are not at high risk of contact with rabid animals, but this doesnot eliminate the need for prompt post exposure prophylaxis if an exposure to rabies occur.
–         In countries where dog rabies exists, stray animals should be destroyed and vaccination of pet dogs and cats should be mandatory.

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