RABIES

Tropical Infectious Disease Division Department of Internal Medicine Faculty of Medicine Brawijaya University / Dr. Saiful Anwar General Hoapital Malang

Rabies The Disease

Rabies has been around for centuries; described as early as 2300 B.C. Transmission is direct, primarily via inoculation by bite, with infectious virus present in saliva. The reservoir for rabies is the animal pool that circulates rabies virus (diverse species of mammals each with a specific strain). Rabies is >99% fatal once symptoms occur

Rabies is caused by a virus

Family Rhabdoviridae bullet shaped Genus Lyssavirus

Rabies Lagos bat strain Mokola Duvenhage EBL-1 EBL-2 ABLV

Picture from Centers for Disease Control and Prevention www.cdc.gov/ncidod/dvrd/rabies

Rabies Virus
Bullet Shaped Morphology Helical RNP Core RNA Structure And Organization
Envelope M protein G protein

Five proteins
Ribonucleoprotein (RNP) Core: Nucleocapsid protein (N) Nucleocapsid phosphoprotein (NS or P) RNA polymerase (L) Matrix protein (M) Glycoprotein (G)
Cross Sectional

RNP core

G protein Envelope (membrane bilayer) RNP

RNA
M protein

Illustrations from Centers for Disease Control and Prevention www.cdc.gov/ncidod/dvrd/rabies

How is rabies contracted

Rabies is most commonly spread by bite contact between the rabid animal and the recipient animal or human

In rabies infection, the virus present in the CNS and other organs
Average incubation period (the time between an initial exposure to the virus and the development of symptoms of disease) is 4 weeks

2 main ecologic cycles: Bat all over U.S. except Hawaii Terrestrial (ground animals) raccoon, skunk, fox, coyote

Map from Centers for Disease Control and Prevention www.cdc.gov/ncidod/dvrd/rabies

Rabies Infection
Virus-laden saliva or other infectious material from the rabid animal must be introduced through a break in skin (bite) or onto mucous membranes Virus binds to a nerve cell & migrates to spinal cord to brain (centripetal spread), then viral replication occurs & produces encephalitis

Rabies attacks the Central Nervous System

Watch as the rabies virus from an exposure on the leg spreads up the spinal cord to the brain and throughout the rest of the body. Rabies virus entering the body.

Transmission/Pathogenesis
Viral particles travel out from brain (centrifugal spread) via nerve cells to salivary glands, where further replication occurs & secretion in saliva, rendering the person or animal to be infectious At the time it gets to the salivary glands, this is the end stage of the disease, and death usually occurs shortly thereafter within several days Incubation period: Usually 4 weeks; can range from 10 days to a year or more (??)

Infection Cycle
1. Attachment to host cell - Main site of attachment is at the neuromuscular junction site of nerve cells - Main receptor is the nicotine acetylcholine receptor - These receptors have a high affinity for the viral G-protein Lewis 2000

Nerve terminal

www.bris.ac.uk/depts/physiology

2. Penetration via endocytosis

-the viral membrane attaches to the host cellular membrane and enters into acidic endosomes by endoycytosis

3. Uncoating of the viral envelope

-uncoating separates the nucleic acid from the envelope and nucleocapsid -this step is necessary before transcription and replication can take place -takes place in the cytoplasm
Gaudin 2000

4) Transcription
-each gene is transcribed, separately, into its complementary mRNA

5) Translation
-the five viral proteins are synthesized

6. Replication
-the RNA polymerase binds to mRNA and begins synthesis of the complementary positive strand -the newly synthesized N-protein binds to the termination synthesis of each mRNA so that a complete strand can be synthesized -the full-length positive RNA strand then serves as a template for the synthesis of the negative viral genome

7. Assembly
-the N,L, and P-protein form the nucleocapsid around the RNA strand and attaches to the cell membrane

6. Budding
-Virus buds from the cell membrane, taking some of the glycoprotein from the host to form the envelope
Wagner 1996

Diagnostic Techniques
1. Histological examination for Negri bodies
-negri bodies are cytoplasmic masses of viral nucleocapsids found in the brain tissue -problem is that negri bodies are only present in 50-80% of rabies cases

2. Direct flourescent antibody test

-uses tissue from suspected host and labeled antibodies, specific to the viral antigen -if the rabies antigen is present, the antibodies will attach; monitored by flourescent microscope -the technique poses the risk of infection to technician, but is a quick diagnosis
Jogai 2002

Negri body

http://www.med.sc.edu:85/virol/negri-bris.jpg

3. Immunohistochemical technique
-confers the presence of viral antigens in organs outside the NS (GI-tract, heart, etc.) -biopsies are stained with immunoperoxidase, to expose antigens, and treated w/ labeled rabiesspecific antibodies to detect antigens -benefits
reduces risk to technician because tissue sample are embedded in formalin-fixed paraffin can examine the spread of the virus in organs outside of the nervous system

Jogai 2000

4. Mouse inoculation test

-brain material from the patient in question is inoculated into mice to see if it leads to fatality -this procedure takes at least a week

5. RT-PCR
-Reverse Transcriptase-Polymerase Chain Reaction -can make a DNA copy of the viral genome and use PCR, with a primer specific to the rabies genome, to determine its presence

Meslin 1996

Symptoms

Headache, fever, sore throat Nervousness, confusion Pain or tingling at the site of the bite Hallucinations Seeing things that are not really there Hydrophobia Fear of water" due to spasms in the throat Paralysis Unable to move parts of the body Coma and death

Rabies Human Deaths

Annual human deaths worldwide are approximately 55,000; every 15 minutes a patient dies of rabies. 40-70% rabies victioms are children under 15 years of age Modern cell culture vaccines and animal control measures in developed countries have reduced the incidence of rabies deaths. In the United States, there has been a mean of 3 deaths per year since 1990.

Prevention steps after an animal bite:

Wash the wound well with soap and water Have the animal tested for rabies See a Doctor, even if the bite is very small. Contact your local health department and animal control officer.

Should Anti-Rabies Prophylaxis be Administered?

CONSIDERATIONS:
High or lower risk animal? Was there an exposure?

Likelihood & timing for animal capture for confinement or testing?

High Risk Animals

Raccoon Skunk Groundhog Fox Bat free-roaming cats

Intermediate Risk Animals

Dogs Cats vaccinated or nonroaming Livestock horses, cattle, pigs Other non-rodent wild animal species i.e, opossum, bear, deer, coyote, etc

Low Risk Animal

Squirrels, chipmunks Rats Mice, voles Indoor small caged pet rodents Logomorphs

WHO Definition of Exposure

Category Type of contact I II Touching or feeding of animals; Licks on intact skin; Nibbling of uncovered skin Minor scratches or abrasions without bleeding Type of Recommended treatment exposure None Minor None if reliable history is taken Administer vaccine immediately; Stop treatment if animal remains healthy for of 10 days or if animal is proven to be negative for rabies by a reliable laboratory using appropriate diagnostic techniques Administer RIG and vaccine immediately. Stop if animal remains healthy for 10 days or if animal is negative for rabies

III

Single or multiple transdermal bites or scratches, licks on broken skin; Contamination of mucous membrane with saliva (i.e. licks); Exposures to bats

Severe

Was There An Exposure?

A bite (penetration of the skin by teeth) from a known or suspect rabid animal Scratches, abrasions, open wounds (bleeding within 24 hrs), or mucous membranes (eyes) contaminated with saliva or other potentially infectious material from a known or suspect rabid animal Other contact - such as petting an animal or contact with urine, feces or skunk spray - does NOT constitute an exposure

Can The Biting Animal Be Confined & Observed?

Healthy dogs, cats, ferrets and livestock may be confined and observed for 10 14 days

Raccoons, skunks, fox, groundhogs and other wildlife may excrete rabies virus while asymptomatic for extended periods and cannot be safely confined & observed. Testing of the animal - or prophylaxis of bite victim - is always recommended

10 Day Confinement & Observation Period In domestic animals the virus usually appears in the saliva at the onset of clinical signs so if animal healthy, probably not rabid Rarely, the virus can appear in the saliva 1 to 3 days prior to onset of illness, so thus an observation period created Clinical course usually less than 7 days animal dead before end of 10 days

Rabies Vaccination Status Of Animal

Lower risk if animal has been regularly vaccinated But NO vaccine is 100% effective Put as much weight on animal behavior & health status

Animal Behavior/Health Risk Factors To Consider

Was the bite or exposure provoked? Did the animal escape in a normal manner? Rabies is characterized by abnormal behavior with neurologic impairment. There is often a period of aggression that progresses to paralysis, although aggression may not always occur

Rabies Virus Survival

If saliva or other material potentially containing the rabies virus is dry to the touch, the virus can be considered noninfectious. Stability of the virus in the environment Strong soaps, detergents, acids and alkalis all inactivate the virus Heat inactivates the virus Radiation destroys the virus Lipid solvents inactivate the virus

Pre- and Post-exposure Prophylaxis

WHO Recommended Post-exposure prophylaxis

1. Immediate flushing and washing of the wound with soap and water, or other detergent
If soap or detergent are not available, flush extensively with water

2. Passive immunization: Administration of Rabies immune globulin for Category III contacts/exposures 3. Active immunization: Administration of tissue culture vaccine according to one of WHO regimens

Rabies Postexposure (PEP)

Two biologics are administered:

Human Rabies Immunoglobulin (HRIG) confers immediate protection with antibodies vs rabies Rabies Vaccine - patient develops antibodies over a 2 to 4 week period

WHO Recommended PEP Schedule Essen intramuscular Regimen

Standard intramuscular regimen. One dose into deltoid on each of days:

5 vials 5 visits
day 0 3 7 14 28

Rabies immunoglobulin

Pre-exposure Prevention
1. Avoid contact with wild animals 2. Do not handle dead animals 3. People that work with wild or domestic animals should be vaccinated 4. Vaccination of domestic and reservoir wild animals

Preexposure Vaccination
Recommended for veterinarians, veterinary technicians, animal control officers, animal shelter workers, rabies lab personnel and person working with wildlife. Provides protection from unapparent exposures and when treatment is delayed Also recommended for persons spending 1 month or more in countries with endemic dog rabies and in which PEP would likely be significantly delayed to geographic distances/ lack of medical infrastructure

Pre-exposure Vaccination Protocol

Three doses of vaccine administered on days 0, 7 and 21 or 28 Dosage: 1.0 ml administered IM in the upper deltoid Test serum every 2 years to determine if an adequate antibody level persists. If absent, administer booster

WHO Recommended Pre-exposure Pre-exposure

3-dose series intramuscular or intradermal regimen

day

21 or 28

day 0

Exposure: No Rabies immunoglobulin needed

What is an Effective vaccine?

One that increases the number of antibodies specific to the viral antigen
Antigens are expressed on the cell surface and bind with antibody The antibody acts by neutralizing the antigen, decreasing its pathogenic ability

Acts as an inhibitor for adsorption and/or replication of the virus Is high in G- or N-viral proteins

http://www.niaid.nih.gov/publications/autoimmune/work.html

Main Types of Vaccines

1. Brain-tissue vaccine
-synthesized from infected brain tissue originally by Pasteur -use both adult and suckling mammal brain tissue (SMB) -causes neural complications
associated with immune response to neural antigens and foreign proteins contained in the vaccine these complications are lower in SMB vaccine relatively cost-effective 90% of human vaccination still uses this

Koprowski 1996

2. Cell-culture vaccine
-prepared from supernatant of virus-infected cells -two main types
a. chicken-embryo
major neurological complications due to embryo antigens not generally used in U.S. for this reason

b. human-diploid cell vaccine

virion preparations grown in human diploid cells requires fewer doses and causes fewer complications used as standard for preparation of other vaccines very costly

Meslin 1996

3. Genetically engineered vaccines

-the goal is to conserve the antigenic structure of the Gprotein, while reducing its pathogenic ability -the recombinant G-protein is modified in such a way that:
it is almost identically similar to challenge virus it decreases viral uptake, and/or prevents budding, and/or stimulates high expression of the G-protein

-induces high levels of neutralizing antibodies, allowing protection against several rabies strands -safe, potent, cost-effective -but through recombinant processes in body, wild-type virus could be regenerated

Morimoto 2001

Post-exposure Prophylaxis
1. Wash bite wound thoroughly with soap and water 2. Isolate the animal if possible 3. Seek post-exposure treatment
-same vaccines above are also used in post-exposure treatment to stimulate the development of antibodies -can also use lectins or neurotoxins that are specific to the nAchR to inhibit viral infection
these will successfully compete with the receptor, decreasing viral uptake

Marchetti 1995 and Voyles 1993

Monitoring Post-exposure Vaccines

Why?
Efficacy of the vaccine varies with individual The generation of high amounts of rabies antibodies in a short time is imperative for survival

Methods to monitor vaccine efficacy

1. Mouse Neutralization test (MNT)
Virus/serum mix at several dilutions are inoculated in mice and the mortality/survival rate is measured Time-consuming, expensive, and need constant supply of rabies conjugate

2. Rapid Flourescent Focus Inhibition Test

-mix dilute serum with constant dose of CVS -stain with flourescent antibody to detect presence of nonneutralized virus -time-consuming, expensive, and need constant supply of rabies conjugate

3. ELISA
-dilutions of sera are added to wells coated with G-or N-protein
-detection of rabies antibodies specific to viral protein by monitoring absorbance -expensive and equipment may not be readily available

4. Latex agglutination test

-new technique that is less expensive, less time-consuming, and less laborous -serum of treated patient is tested on beads that have been coated with purified glycoprotein of the rabies virus -amount of agglutination is a direct measure of the efficiency of the vaccine

Thank You!!!!!!