History of Rabies

Rabies is one of the oldest recognized infectious diseases

Rabies also historically known as hydrophobia (fear of water)

Louis Pasteur and Emile Roux, pioneers of rabies vaccine
Dried strips of spinal cords removed from rabid animals
Pasteurs first vaccine testing on dogs
1885 first human subject injected with Pasteurs vaccine

Pasteur Institute founded to treat rabies victims

Rabies comes from rabhas which means to do violence



Italian scholar Girolamo Fracastoro first described rabies disease in
1546 (350 years before Pasteur and Roux developed rabies vaccine)




The patient can neither stand nor lie down, like a mad man he
flings himself hither and thither, tears his flesh with his hands, and
feels intolerable thirst. This is the most distressing symptom, for he
so shrinks from water and all liquids that he would rather die than
drink or be brought near to water. It is then they bite other
personns, foam at the mouth, their eyes look twisted, and finally
they are exhausted and painfully breathe their last.
Rabies
Canine or street (wild-type) rabies has afflicted mankind for
centuries

Mad dog symbolized mans fear of rabies for centuries.
Figure 13.1: An 1826 cartoon depicting
citizens trying to protect themselves from
a rabid dog.
National Library of Medicine
Virus Classification
Order: Mononegavirales


Family: Rhabdoviridae


Genus: Lyssavirus



Rabies Virus Structure
Adapted from the CDC
Unique bullet-shaped appearance

75 nm diameter by 180 nm in length

Surface of the particle is covered with glycoprotein (G) spikes (trimers)

Matrix (M) protein inside of particle links nucleocapsid with envelope

Nucleocapsid = viral RNA bound to the N protein (nucleocapsid also termed ribonucleoprotein)

Viral RNA dependent RNA polymerase (RdRp) proteins L and P bound to nucleocapsid
1. Attachment glycoprotein G binds to nicotinic acetylcholine
receptors (can also use alternate receptors)

2. Entry/Uncoatinglow pH dependent fusion with endosomal
membranes
- Acidic interior of the endosome allows the viral
nucleocapsid (N + RNA) to escape into the cytoplasm
- M protein dissociates from the nucleocapsid


The Life Cycle of Rabies Virus
5. Genome replication
- Full-length + ssRNA copies (antigenomes) made from strands
genomes
- Anti-genomes (+) RNA serve as templates for progeny
(-ssRNA) genomes which will be packaged into the viral particle
3. Transcription of Viral Genes
- L (RNA dependent RNA polymerase) begins to transcribe the
viral genome

4. Viral Protein Synthesis
- viral mRNAs translated by host cell ribosomes
6. Virus Assembly/Exit
- As soon as -ssRNAs, N, M, P, L proteins have accumulated within
infected cells, assembly occurs
- The mature infectious particle buds through host cell plasma
membrane
Rabies Pathogenesis
Most common route of entrybreak in skin

Virus replicates in the muscle cells surrounding the wound

Virus attaches to nicotinic acid receptors of the peripheral nerve
cells of the neuromuscular junction

Virus journeys within axons of nerves at a rate of 0.5 to 15 inches per
day

It may take weeks before the virus reaches the spinal cord motor
neurons of the CNS

This is why post-exposure vaccination is possible

Antibodies prevent the spread of the virus to the CNS, stopping the
disease

Sequential Steps of
Rabies Pathogenesis
Figure 13-11a
Adapted from A. C. Jackson and W. H. Wunner, eds. Rabies.
Academic Press, 2002
Clinical Signs and Symptoms of Rabies
2 forms of human rabies
Furious (encephalitic)
Paralytic (or dumb)

With either form, incubation period
varies
4 days to 6 years (rare)
Average 18-21 days post-exposure

Death occurs on average 18 days
after onset of symptoms
Figure 13.8: Two dogs afflicted with dumb rabies.
Courtesy of CDC
Symptoms During Prodromal Period
Headache

Malaise

Fever

Anorexia

Nausea

Vomiting
Secondary Symptoms of Furious Rabies
Hydrophobia
Difficulty swallowing
Agitation
Anxiety
Hallucinations
Hypersalivation
Bizarre behavior
Biting
Jerky and violent contractions of the diaphragm
Rabies patient experiencing hydrophobia
Secondary Symptoms of Paralytic Rabies
Lack of hydrophobia
Lack of hyperactivity
Lack of seizures
Weakness and ascending paralysis

These symptoms also apply to animals
Rabies Diagnosis in Humans
Laboratory tests are rarely done in developing countries

Several routine antemortem tests available in the U.S.
Virus isolation

RT-PCR to detect viral RNA
Saliva
Tears
CSF

Serum
Antibodies to rabies (appear in 2nd week of illness)

Skin punch biopsies at the nape of the neck
Rabies antigen in cutaneous nerves at the base of hair follicles
Human Postmortem direct fluorescent antibody (dFA) tests
Performed on brain of victim

Fixed brain tissue sections stained with
anti-N antibodies that are linked to a
fluorescent marker

Any rabies present in the section there
will be a fluorescent signal

Gold standard for rabies diagnosis


Rabies Infection of the Brain
Electron Micrograph of Infected Neuron
Figure 13.9
Micrograph from F.A. Murphy, School of Veterinary Medicine, University of California, Davis
Classical pathological lesions called Negri Bodies
Results of an Autopsy
Little evidence of brain
damage even though
fluorescent antibody labeling
and electron microscopic
analysis of brain tissues show
all the neurons are infected

Fixed tissues stained with
histological dyes may show
the appearance of Negri
bodies in the cytoplasm of
brain neurons
Figure 13.12: Photomicrograph of hematoxylin- and
eosin-stained brain tissue from a rabies encephalitis
patient.
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Human Rabies
Without post-exposure prophylaxis, rabies almost
always fatal after secondary symptoms develop (virus
in brain)

8 survivors (after CNS involvement) recorded in the
scientific literature

Half of the survivors had severe, permanent
neurological disorders

Rabies in Animals
Disease of mammals

In the U.S. rabies is
primarily an infectious
disease of wildlife

Worldwide: dogs are a
major reservoir


Distribution of Terrestrial Wildlife Reservoirs of Rabies, U.S.
Figure 13.3: Distribution of major terrestrial reservoirs of rabies in the United States in 2005.
Adapted from the CDC, Animals and Rabies, Rabies: Just for Kids, http://www.cdc.gov/ncidod/dvrd/kidsrabies/Animals/animals.htm.
Rabies in Bats

Figure 13.4: Distribution of bat rabies in the United States in 2001.
Since 1990, over 90% of human cases in the U.S.
were associated with bat bites
Important Animal Rabies Reservoirs Worldwide
Asia, Africa, South America, Mexico
Dogs

Europe, Canada, Alaska, former Soviet Union
Foxes

Siberia, Japan, northern India, Europe
raccoon dogs

Thailand
dogs (95% of cases)

Caribbean Islands
Mongooses

Mexico and South America
vampire bats

U.S. Rabies Testing in Animals
Gold standard is direct fluorescent antibody test (dFA)
Post-mortem test
Done on animals behaving abnormally or show consistent
rabies signs if human or other animal exposure have
occurred

Rabies is present in nervous tissue
Brain is the ideal test tissue2 or 3 samples tested
Brain stem (medulla)
Cerebellum
Hippocampus

dFA test is rapid (30 minutes to 4 hours)


U.S. Rabies Management Programs
Vaccination programs to control
rabies in animals began in the
U.S. during the 1940s and 1950s

Wildlife Services Program, part
of the USDA distributes vaccines

Over 10 million oral rabies
vaccine baits distributed in the
U.S. and Canada in 2003
Figure 13.5: Biologists collect blood and other biological samples from trapped
animals to evaluate the effectiveness of the oral rabies vaccine.
Figure 13.6: Merial Raboral V-RG rabies vaccine bait.
Photos courtesy of APHIS/USDA
Management of Human Rabies
All high-risk individuals should be vaccinated against rabies
virus:

Veterinarians
Animal handlers (e.g. zookeepers)
Dog catchers
Mail carriers
Speleologists
Trappers and hunters in rabies endemic areas
Laboratory workers who work with rabies virus
Individuals who are involved in the production of rabies vaccines
Peace Corps workers
International travelers who go to places that have endemic dog
rabies
Rabies Vaccine Shot Schedule
Dose3 X 1 ml shots injected intramuscularly
into the forearm or shoulder on:
Days 0, 7, 21 or 28
Usually takes 7 -14 days to induce immunity
Immunity lasts approximately 2 years

Individuals who are at high-risk (e.g. vets) must
be tested every six months for rabies
antibodies

A booster will be administered if necessary

Wound Care
Middle of the 20th century
cauterize wounds caused by
rabid animals

Today
wounds washed thoroughly
with 20% soap, 70% ethanol, or
2% benzalkonium chloride

Also irrigated with a virucidal
agent such as povidone-iodine.
What Should You Do if You Are Bitten by an Animal?
Provide the following information to a physician
Animal involved
Whether the bite was provoked or unprovoked
Vaccination status of the animal (if known)
Whether the animal can be safely captured for rabies
testing

If the animal shows signs of rabies and can be
captured, it will be euthanized and the head
shipped to a qualified laboratory for testing

Postexposure Prophylaxis
Human Rabies Immune Globulin (HRIG, Hyperab)

Provides passive immunity until active antibodies are
induced by the vaccine

Unvaccinated individuals who experience a bite from a rabid
animal will receive immune globulin and a shot of vaccine on
day 0

HRIG is expensive and not available in all countries

Many countries use ERIG (of equine or horse origin)
It is considerably cheaper.
Rabies Virus Attaches to the Nicotinic Acetylcholine
Receptors and Journeys Within Axons of Nerves
Figure 13-11b
Immunity
No antibody responses detected during the
incubation period of rabies virus infection.

Antibodies appear in serum and later in CSF
after symptoms appear

Some patients (who experience encephalitic
furious rabies) mount a cell-mediated
response
Course of Rabies Infection
While in the neurons, the virus is hidden from the
bodys immune surveillance system

Rabies viruses spread to many tissues via the
peripheral nervous system

Virus particles predominately bud out of the cells
of the salivary glands

Viruses exit from the body via saliva
13.6 Genetic Variation
Fixed strains
Laboratory strains used in research
Street strains
Strains isolated from patients or rabid animals
The rabies L protein does not possess RNA
repair/proofreading ability.
Mutation rate is 10
-4
to 10
-5
per nucleotide per
cycle of replication
Virus File 13-1 Why Did Jeanna Geise
Survive?
15-year-old Jeanna Giese was
bitten by a rabid bat in Fond du
lac, WI
Bite was washed with hydrogen
peroxide but no medical
treatment was sought
Rabies symptoms began 1 month
later
Neither rabies vaccine nor rabies
immune globulin were
administered because Giese had
already developed an antibody
response toward the virus.
Figure VF 13-1a
Morry Gash/AP Photos
The Milwaukee Protocol
Combined strategy
Antiexcitatory drugs (induce coma, brain protectant)
Geise was intubated and put into a drug-induced coma with
aketamine and midazolam for 7 days
Antivirals (ribavirin and amantidine)
Aggressive supportive care

Antiexcitatory drugs were tapered after 7 days
Antiviral drugs were discontinued
Timeline of Gieses Hospital Stay
Figure VF 13-1b
Adapted from R. E. Willoughby, Jr., et al., N. Engl. J. Med. 352 (2005): 2508-2514.
Geise discharged from the hospital on the 76th day
after rehabilitation.
Geise is now in college.
To date, not 100% recoverywider gait, numbness of
bitten finger, alteration of tone in her left arm
Her recovery drew global interest.
http://content.nejm.org/cgi/content/full/357/9/94
5

Flash video of Geise 18 months after exposure to
rabies
http://content.nejm.org/cgi/content/full/357/9/945/DC1

Why Did Geise Survive
To date, the Milwaukee Protocol is not reproducible
Patients in the U.S., Thailand, Germany and India all
died after administration of this protocol.
Did she get exposed to an attenuated bat strain of
rabies?
No virus was recovered from Geises bodily fluids to
test this hypothesis.
Potential animal research
Apply Milwaukee protocol to rabid animals and
systematically test what parts of the protocol are critical.
Vet schools have not permitted these studies.
Concerns over rabid animals in their ICUs.
Postexposure Prophylaxis
Vaccination
3 categories of vaccine
Nerve tissue vaccines
Avian embryo vaccines
Cell culture vaccines
Early vaccines made of nerve tissues
Produced serious side effects such as Guillian Barre-like
syndrome, paralysis, CNS disease, meningoencephalitis
Semple vaccine1 in 200 recipients experience serious
side effects, up to 14% of recipients die
Nerve tissue vaccines are still being used in Asia, South
America and Africa
Viral Replication: Genome Organization
Genome consists of a 11,932 nucleotides

ssRNA of negative polarity

5 genes and a remnant gene or pseudogene ()
Figure 13.14: Genome organization of rabies virus.
Adapted from S. J. Flint, et al. Principles of Virology: Molecular Biology, Pathogenesis, and Control of Animal Viruses, Second Edition. ASM Press, 2003.
Vaccine
s
Three
vaccines
used in the
U.S.
HDCV, RVA,
PCECV