Unit 3 - Rabies Virus

Question 1

What is the history of Rabies?

Answer 1

• One of the oldest known diseases

Earliest record of rabies from Babylon (23rd century BC)
Homer likens Hector to a “raging dog” in The Illiad (800-700 BC)

• Rabies derived from the Sanskrit word rabhas (“to do violence”) and the Latin word rabere (“to rave”)

Rabid animals often appear angry or in a rage

• Italian scholar Girolamo Fracastoro was the first to describe the true nature of rabies disease (1546)

350 years before Pasteur and Roux developed the rabies vaccine

Question 2

Describe rabies in the 19th Century

Answer 2

• Rabies was termed hydrophobia (fear of water)
• Canine or street rabies was everywhere, especially Europe.
• The fear of rabies was almost irrational, due to the significant number of vectors and lack of any efficacious treatment
• “Mad” dog symbolized the human fear of rabies for centuries
• Suspected victims often killed themselves or were killed (shot, poisoned, suffocated) or condemned to die
• France passed law in 1810 making it illegal to murder individuals suffering from rabies, hydrophobia or any disease causing fits, convulsions, or madness

Question 3

Describe the history of rabies when the vaccine was discovered.

Answer 3

• Louis Pasteur and Emile Roux, pioneers of rabies vaccine

Dried strips of spinal cords removed from rabid animals
Pasteur’s first vaccine tested on dogs (50/50 survived)
1886: first human subject injected with Pasteur’s vaccine; 9-yr old boy

• Pasteur’s success made him a hero

Pasteur Institute founded in 1887 to treat rabies victims
Institute widely respected for its research in microbial diseases

Question 4

What is rabies in animals?

Answer 4

Disease in mammals:

• primarily wildlife (wild dogs, raccoons, skunks, bats, etc.)
• Less frequent in domesticated animals (cats, dogs, livestock)

Question 5

What are the worldwide important Animal Rabies Reservoirs?

Answer 5

• Asia, Africa, South America, Mexico: wild dogs (threatening over 3 billion people)
• Europe, Canada, Alaska, former Soviet Union: foxes
• Siberia, Japan, Northern India, Europe: raccoon dogs
• Thailand: wild dogs (95% of cases)
• Caribbean Islands: mongooses
• Mexico and South America: vampire bats

Question 6

What are the U.S. Rabies Management Programs?

Answer 6

• Vaccination programs to control rabies in animals bean in the U.S. during the 1940s and 1950s.
• Wildlife Services Program, part of the USDA distributes vaccines via hand distribution and plane distribution
• Over 10 million oral rabies vaccine baits distributed in the U.S. and Canada in 2003

Question 7

What are the Oral Rabies Vaccine for wildlife?

Answer 7

• Raboral V-RG vaccine by Merial, Inc.
• In use in the US since 1990
• Recombinant vaccine

Attenuated vaccinia virus

Gene encoding rabies glycoprotein G

Question 8

What is Human Rabies?

Answer 8

• 15 million post-exposure vaccinations

most common in children aged under 15

90% of human cases in the US were associated with bat bites.

• 55,000 rabies - related human deaths yearly:

dogs source of 99% human rabies death

Question 9

What is involved between rabies and organ transplantation?

Answer 9

• Infections by corneal transplant have been reported in Thailand, India, Iran, the US, and France.
• June 2004: three organ recipients in US died from rabies transmitted in the transplanted kidneys and liver
• Donor died of brain hemorrhage/unidentified brain disorder
• February 2012: Maryland man died of rabies; received a kidney from an infected donor in 2011

Question 10

How are U.S. soldiers affected by rabies?

Answer 10

• In August 2011 a U.S. soldier stationed at a military base in New York became ill with symptoms compatible with rabies.
• three months following active deployment in Afghanistan
• variant associated with Afghani dogs
• Soldiers returning from active duty abroad may have unreported animal exposures

Question 11

What is World Rabies Day?

Answer 11

• Occurs each year on September 28, the anniversary of the death of Louis Pasteur
• Aims to raise awareness about the impact of rabies on humans and animals

promoting government involvement in rabies prevention and control programs
increasing the vaccination coverage of pets and community dogs
improving the educational awareness

Question 12

What are the clinical signs and symptoms of rabies?

Answer 12

Two forms of Human Rabies

1. Furious (encephalitic)
2. Paralytic (or dumb)

With either form, incubation period varies

• 4 days to 6 years (rare)
• Average 18-21 days post-exposure

With either form, course of diesase 2-14 days before coma supervenes

• Death occurs on average of 18 days after onset of symptoms

Question 13

What are the symptoms of rabies during the prodromal period?

(the time of onset specific symptoms)

Answer 13

Heachache

Malaise

Fever

Anorexia

Nausea

Vomiting

Question 14

What are teh secondary symptoms of Furious Rabies?

Answer 14

• Hydrophobia (fear of water)
• Difficulty swallowing
• Agitation
• Anxiety
• Hallucinations
• Hypersalvation
• Bizarre behavior
• Biting
• Jerky and violent contractions of the diaphragm

Question 15

What are the Secondary symptoms of Paralytic rabies?

Answer 15

• Lack of hydrophobia
• Lack of hyperactivity
• Lack of seizures
• weakness and ascending paralysis
• These symptoms also apply to animals:
• Animals that are predators show signs of furiuos rabies.
• Nonpredators display paralytic or dumb rabies symptoms

Question 16

What is U.S. rabies testing in animals?

Answer 16

• 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 occured

• Rabies is present in nervous tissue

Brain is the ideal test tissue: 2 or 3 samples tested

• Brain stem (medulla)
• Cerebellum
• Hippocampus

Question 17

What is Direct Fluorescent Antibody Test (dFA)?

Answer 17

• Ideal tissue to test for rabies antigen is brain.
• Fluorescently labeled antibodies directed against the viral nucleoprotein.
• dFA test is rapid (30 minutes to 4 hours)

Question 18

How is rabies diagnosed in humans?

Answer 18

• 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 second week of illness)

• Skin punch biopsies at the nape of the neck

Rabies antigen in cutaneous nerves at the base of hair follicles

Question 19

What is Human Postmortem dFA tests?

Answer 19

• Performed on brain of victim
• Long-needle biopsies (collect 2 or more samples)

Brain Stem

Cerebellum

• Followed by RT-PCR, virus isolation in cluture, or suckling mous inoculation

Histologic, immunnocytochemistry

Electron mircroscopy

Question 20

Who are included in Human Rabies Surviviors?

Answer 20

• Seven survivors recorded in the scientific literature before use of the Milwaukee Protocol
• All 7 received pre- or post-exposure rabies passive antibodies, immunoglobulin, or vaccine
• Half of the survivors had severe, permanent neurological disorders.
• 2004: Jeanna Geise, Wisconsin, Milwaukee Protocol Survivor
• Bat bite
• No post-exposure vaccine
• Milwaukee protocol treatment (see Virus File 13-1)

Question 21

What is the Milwaukee protocol?

Answer 21

• Experimental course of treatment of an acute infection of rabies in a human being
• Patient into a chemically induced coma (ketamine, midazolam, & phenobarbital)

Most rabies deaths are caused by temporary brain dysfunction with little to no damage occurring to the brain

• Antiviral drugs ribavirin and amantidine
• Aggressive supportive care
• 15 yr-old Jeanna Giese (2004; Virus File 13-1)

Contracted rabies from bat bite; hospitalized 1 month after bite
Brought out of coma once signs of immune response were detected
Declared virus free after 31 days
Discharged from hospital 76th day after rehabilitation
Remarkable recovery; graduated college in 2011

Question 22

Why did the Girl Deise Survive?

Answer 22

• To date, the success of the initial Milwaukee Protocol has been difficult to replicate.
• 4 survivors out of 35 patients
• Patients in the United States, 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 Geise’s 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.

Question 23

Are there animal surivivors?

Answer 23

A few descriptions of “spontaneous recovery” in the literature

• Pasteur laboratory animals
• 1980: Fekadu and Baer reported two beagles recovering spontaneously after experimental infection

Question 24

What is the pathogenesis of Rabies?

Answer 24

• Most common route of entry is a break in the 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.

Question 25

What are the sequential steps of rabies transmission?

Answer 25

Question 26

Rabies Virus attaches to the nicotinic acetylcholine receptors and journeys within axons of nerves.

Answer 26

Virus hidden from immune surveillance while in neurons

Question 27

What happens during the course of rabies infection?

Answer 27

* Rabies viruses spread to many tissues via the peripheral nervous system. Cardiac muscle, kidney, pancreas, cornea, salvary glands * Virus particles predominately bud out of the cells of the salivary glands. Viruses exit from the body via saliva. Intermittent shedding

Question 28

What are the results of an autopsy?

Answer 28

* _Little_ evidence of brain damage even though fluorescent antibody labeling and electron microscopic analysis of brain tissues show all the neurons are infected. Neural dysfunction through inhibition of neurotransmitters? * Fixed tissues stained with histological dyes may show the appearance of **_Negri_** bodies in the cytoplasm of brain neurons.

Question 29

What are Negri Bodies?

Answer 29

* Inclusion bodies found in cytoplasm of nerve cells. * Contain ribonuclear proteins produced by virus.

Question 30

What is the immunity?

Answer 30

* _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. * Virus can _inhibit_ type I and II **interferon** responses

Question 31

What is the management of Human Rabies?

Answer 31

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

Question 32

What is the Rabies Vaccine shot schedule?

Answer 32

* Dose: 3× 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.

Question 33

What is involved in the wound care?

Answer 33

* 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

Question 34

What should you do if you are bitten by an animal?

Answer 34

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 * Geographic location of the incident 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.

Question 35

What is Postexposure Prophylaxis?

Answer 35

**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. * Highly purified and safe (serum sickness rare)

Question 36

What else is involved in Postexposure Prophylaxis?

Answer 36

* Vaccination * Three categories of vaccine 1. Nerve tissue vaccines 2. Avian embryo vaccines 3. Cell culture vaccines * Early vaccines made of nerve tissues Produced serious side effects such as Guillian Barré-like syndrome, paralysis, CNS disease, meningoencephalitis Semple vaccine—1 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

Question 37

What are the vaccines?

Answer 37

Three Vaccines used in the U.S. 1. HDCV 2. RVA 3. PCECV

Question 38

What is the Rabies Life Cycle? (structure of the Rabies Virus Particle)

Answer 38

* Unique bullet-shaped appearance * 75 nm diameter by 180 nm in length * Negative strand RNA virus * Enveloped; surface covered with glycoprotein spikes * Matrix (M) protein inside of particle * Ribonucleoprotein core (RNP) * Genomic RNA tightly encased by N, P, and L proteins

Question 39

What is the viral replciation of the genome organization?

Answer 39

Genome consists of a 11,932 nucleotides Negative strand ssRNA Five genes

Question 40

What is the Virus Life Cycle?

Answer 40

* Rabies surface glycoprotein shares sequence homology to snake neurotoxins * **Attachment**—binds to nicotinic acetylcholine receptors Co-receptors? * **Entry**—low pH dependent fusion with endosomal membranes * Clathrin-coated pits * Acidic interior of the endosome allows the viral nucleocapsid to escape into the cytoplasm * **M protein** dissociates from the RNP during uncoating

Question 41

What is involved in the transcription of Viral Genes?

Answer 41

Negative sense ssRNA * Pre-packaged **L** and **P** proteins responsible for transcription * L **(RNA dependent RNA polymerase)** begins to transcribe the viral genome. * A separate +ssRNA transcript is generated for each viral gene. * Each viral transcript is capped and polyadenylated by the viral L protein.

Question 42

What is Viral Genome Replciation?

Answer 42

* After N is synthesized, it binds to leader region of viral RNA genomes. * Full-length +ssRNA copies of the viral genome are made from N-encapsidated viral RNAs. Termed anti-genome RNA Anti-genome RNA serves as a template for progeny (–ssRNA) genomes which will be packaged into the viral particle * Virus exit As soon as –ssRNAs, N, M, P, and L proteins have accumulated within infected cells, assembly occurs. The mature infectious particle buds through host cell plasma membrane.

Question 43

What are the genetic variations involved in rabies viruses?

Answer 43

* **Fixed** strains Laboratory strains used in research * **Street** (wild) strains Strains isolated from patients or rabid animals * The rabies L protein does not possess RNA repair/proofreading ability. **High frequency of mutations** Mutation rate is 10–4 to 10–5 per nucleotide per cycle of replication