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Thomas Dawn Bauer Vivian MoughniPresentation Outline: Presentation Outline Some facts about rabies History of the rabies virus Transmission Symptoms Prevalence Morphology and genome Important viral proteins Replication cycle Diagnostic Techniques Prevention, including pre- and post-exposure treatments Conclusion Some Facts about Rabies: 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: 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: 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.comHistory of the Rabies Virus: 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: 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: 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: 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: 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.htmlTransmission of Rabies: 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: 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: 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: 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: 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: Transmission of Rabies 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: 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: 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/rabiesSlide19: 5,540 Mammals tested In 1955- reported by the US Public Health Service 3,900 Animals Tested in 1960 6,947 Animals tested In 1994 Data Summaries by the United States Public Health Service Regarding Prevalence of Rabies in the U.S. Cockrum, 1997Prevalence of Rabies: 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: 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).Slide22: Morphology, Replication and Diagnostic TechniquesSlide23: 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.govSlide24: 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.aspImportant viral proteins: 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 Slide26: 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 1993Slide27: Voyles 1993Infection Cycle: Infection Cycle 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 Nerve terminal www.bris.ac.uk/depts/physiology Lewis 2000Slide29: 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 2000Slide30: Transcription -each gene is transcribed, separately, into its complementary mRNA Translation -the five viral proteins are synthesizedSlide31: 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 Assembly -the N,L, and P-protein form the nucleocapsid around the RNA strand and attaches to the cell membrane Budding -Virus buds from the cell membrane, taking some of the glycoprotein from the host to form the envelope Wagner 1996Diagnostic Techniques: Diagnostic Techniques 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 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 2002Slide33: Negri body http://www.med.sc.edu:85/virol/negri-bris.jpgSlide34: 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 2000Slide35: 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 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 1996Slide36: Pre- and Post-exposure ProphylaxisPre-exposure Prevention: Pre-exposure Prevention Avoid contact with wild animals Do not handle dead animals People that work with wild or domestic animals should be vaccinated Vaccination of domestic and reservoir wild animalsWhat is an Effective vaccine?: 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 proteinsSlide39: http://www.niaid.nih.gov/publications/autoimmune/work.htmlMain Types of Vaccines: Main Types of Vaccines 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 1996Slide41: 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 1996Slide42: 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 2001Post-exposure Prophylaxis: Post-exposure Prophylaxis Wash bite wound thoroughly with soap and water Isolate the animal if possible 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 1993Monitoring Post-exposure Vaccines: 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 Slide45: 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 availableSlide46: 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 vaccineCurrent Research: Current Research Though the rabies virus is an unyielding, generally fatal disease, it can be used for the good The virus is being used to map specific neuronal pathways in the body Through the molecular characterization of the rabies virus, the transmission from reservoir species to non-reservoir species can be monitoredFuture Research: Future Research For any of you future microbiology researchers, here are some aspects of rabies that need to be further addressed Need clarification of the mechanism of transport and how rabies actually targets cells after uptake and replication The evolutionary force resulting in the development of two or more dominant strains in a particular region and why with time one may become obsoleteThank You!!!!!!: Thank You!!!!!! 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