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Rabies

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									Suzanne M. Thomas
    Dawn Bauer
  Vivian Moughni
          Presentation Outline
1.  Some facts about rabies
2.  History of the rabies virus
3.  Transmission
4.  Symptoms
5.  Prevalence
6.  Morphology and genome
7.  Important viral proteins
8.  Replication cycle
9.  Diagnostic Techniques
10. Prevention, including pre- and post-exposure
    treatments
11. Conclusion
    Some Facts about Rabies
• Belongs to the family Rhabdoviridae
• Genus Lyssavirus (Greek root “lyssa”)
• Infects warm-blooded mammals, including
  humans
• Causes encephalitis of the central nervous
  system
• Results in death of the host
      History of Rabies Virus
• Man described the disease in 2300 B.C. in
  the Eshnunna Code of Mesopotomia.
• The origin “rabhas”, meaning “to do
  violence” comes from ancient Indian
  Sanskrit dating 3000 B.C.
• Many cultures have incorporated myths
  and legends that can be connected to the
  rabies virus.
          History of the Rabies Virus
                        •Outbreak of rabies in Europe
                         in 20th century spawned the
                         Vampire myth- linked to bats.
                        •Rabies epidemic in 13th
                         century France may have
                         spurred Werewolf myth as
                         well- linked to wolves.
                        •Rabies is commonly spread
                         by bats and wolves and other
                         wildlife like foxes and coyotes.
www.Davidsposters.com
    History of the Rabies Virus
• Since Roman times, man established the
  link between the infectivity of a rabid dogs
  saliva and the spread of the disease.
• Because there is no cure for rabies, those
  that had been bitten by a rabid dog would
  commonly commit suicide to avoid the
  painful death that would inevitably follow.
   History of the Rabies Virus
• Louis Pasteur was the first person to
  diagnose that rabies targeted the CNS.
• Also determined that nervous tissue of an
  infected human or animal also contained
  the virus.
• In 1890 created the rabies vaccine and
  saved 9 year old Joseph Meister after he
  had been bit by a rabid dog.
      Transmission of Rabies
• Is zoonotic, meaning is transmitted from
  animal-animal or animal-human.
• Rabies is most commonly contracted from
  the bite of an animal that is infected with
  the virus.
• The virus is found in the saliva of the
  infected animal.
      Transmission of Rabies
• Can also enter via any mucous
  membrane, such as the eye, nose or
  mouth.
• Handling and skinning of infected animal
  carcasses.
• Few cases of human to human
  transmission.
• Can be transmitted via aerosol, but that’s
  also uncommon.
                 Transmission of Rabies
  • Inside the U.S., important reservoirs for the
    transmission of the disease are wild animals
    such as raccoons, skunks, foxes, bats, and
    coyotes.
  • The time it takes for rabies to affect the animal
    averages 1-3 months. Sometimes may only be
    a few days, and rarely longer than a year after
    exposure.




www.cdc.gov/rabies.html
     Transmission of Rabies
• The rabies infection and the symptoms
  that accompany it is classified by five
  stages:
  1. Incubation (1-3 months)
  2. Prodromal, where first symptoms occur
  3. Acute neurological phase
  4. Coma
  5. Death or recovery
      Transmission of Rabies
1. Incubation
   -The animal or human is bitten by rabid animal.
   -Rabies enters the body via the saliva of the infected
   animal.
   -The virus spreads through the nerves via retrograde
   axoplasmic flow. The virus makes it’s way to the dorsal
   root ganglion where it replicates.
   - No symptoms are experienced at this time, and the
   disease can’t be transmitted to another animal yet.
          Transmission of Rabies
2.       Prodromal Stage
     -Virus replicates in the dorsal root ganglion of the spinal
     cord where the blood-nerve barrier is thin.
     -This is where the immune system detects the virus and
     starts creating antibodies.
     -First symptoms are felt: fever, muscle ache, agitation,
     anxiety, nausea, vomiting, ache in the bitten extremity.
     -From this point the virus replicates and travels up to the
     brain, and the immune system can’t keep up with fighting
     the virus.
           Transmission of Rabies
3.       Acute Neurological Stage
     - There is rapid proliferation of the virus in the brainstem,
     thalamus, basal ganglia, and spinal cord
     - Brain begins to swell, encephalitis
     - Fluctuation between consciousness and confusion
     - Aggressive behavior, violent episodes
     - Convulsions and seizures
     - Hallucinations
     - Hydrophobia (foaming at the mouth) induced by
     pharyngeal spasms (the most common image of rabies)
          Transmission of Rabies
3.       Acute Neurological Stage, continued
- Aerophobia
- Sensitivity to lights, sounds and smells
- Respiratory spasms.


4.       Coma
-    Spasms decrease because paralysis occurs
-    Heart arrhythmias and irregular heartbeat persist
-    Cerebral encephalitis causes major organs to collapse
-    Fixed pupils that are unresponsive to light
      Transmission of Rabies
5. Death or Recovery
   - Death results from encephalitis of the brain.
   - Heart failure along with major organ failure are the
   main causes of death.
   - Only four known cases of human recovery, and they
   were permanently brain damaged.
       Prevalence of Rabies
• In the U.S., dogs were the main reservoir
  previous to the 1950’s., where now
  raccoons are the main reservoir.
• Vaccines in domestic house pets
  drastically reduced the number of human
  rabies cases.
• Now less than 10% of reported rabies
  cases come from domestic animals.
                     Prevalence of Rabies
   • In the U.S. in 1955, raccoons accounted
     for 1% of rabies cases, where in 2001
     raccoons accounted for 40% of over 7,000
     reported cases.




www.cdc.gov/rabies
Data Summaries by the United States Public Health
Service Regarding Prevalence of Rabies in the U.S.


 5,540 Mammals tested
 In 1955- reported by the
 US Public Health Service




   3,900 Animals                   6,947 Animals tested
   Tested in 1960                  In 1994




  Cockrum, 1997
       Prevalence of Rabies
• In this century, the number of human
  deaths in the United States attributed to
  rabies has declined from 100 or more
  each year to an average of 1 or 2 each
  year after the 1960’s. Most recent rabies
  cases are linked to exposure to rabid
  wildlife.
• Animal control and vaccination programs
  begun in the 1940's have practically
  eliminated domestic dogs as reservoirs of
  rabies in the United States.
       Prevalence of Rabies
• Although human deaths in the U.S. are
  very low, the World Health Organization
  reports that 35,000 to 50,000 people die
  every year from rabies in developing
  countries, mainly India and Asia.
• Currently, dogs are the number one
  reservoir of rabies in the developing world.
• China is suffering from more rabies deaths
  than SAR’s (Chinese Ministry of Health).
Morphology,

   Replication and

      Diagnostic

          Techniques
       Morphology of rabies virus
•Negative single-stranded RNA genome
•Rod shaped with a flexible nucleocapsid tapering
at one end
  –Nucleocapsid contains important viral proteins



                                        180nm\75nm




               http://www.ncbi.nlm.nih.gov
• A membranous envelope surrounds the
  nucleocapsid
  – Envelope contains both viral and glycoproteins
  – Derived from the lipids of the host cell membrane
  – Viral proteins of envelope form spikes that aid in viral
    attachment




      http://www.microbe.org1microbes/virus_or_bacterium.asp
       Important viral proteins
• N-protein
  – makes up most of the nucleocapsid
  – catalyzes replication process


• G-protein
  – glycoprotein, come together to form the spikes of the
    viral envelope
  – mediates fusion
  – its presence triggers immune response of host
  – determines pathogenic ability of the virus
    • L-protein
          – Small part of nucleocapsid
          – Codes for RNA polymerase
          – Functions in polymerizing activity


    • P-protein
          – Also makes up a small portion of the nucleocapsid
          – Contributes to the ability of the L-protein to both bind
            and move down template strand during the
            attachment of nucleotides



Voyles 1993
Voyles 1993
                         Infection Cycle
1. Attachment to host cell
      -      Main site of attachment is
             at the neuromuscular
                                                                                Nerve terminal
             junction site of nerve cells
      -      Main receptor is the
             nicotine acetylcholine
             receptor
      -      These receptors have a
             high affinity for the viral G-
             protein
                                              www.bris.ac.uk/depts/physiology




Lewis 2000
  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
            rabies-specific 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
Pre- and Post-exposure
      Prophylaxis
     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
  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 G-
              protein, 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 non-
       neutralized 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
             Current Research
•    Though the rabies virus is an unyielding,
     generally fatal disease, it can be used for the
     good
    1. The virus is being used to map specific neuronal
       pathways in the body
    2. Through the molecular characterization of the rabies
       virus, the transmission from reservoir species to
       non-reservoir species can be monitored
             Future Research
•   For any of you future microbiology
    researchers, here are some aspects of
    rabies that need to be further addressed
    1. Need clarification of the mechanism of
       transport and how rabies actually targets
       cells after uptake and replication
    2. The evolutionary force resulting in the
       development of two or more dominant
       strains in a particular region and why with
       time one may become obsolete
Thank You!!!!!!

    Suzanne
     Dawn
     Vivian

								
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