Rhabdoviruses.docx

Rhabdoviruses Learning Objectives: After reading and studying this chapter, you should be able to: ∙∙ Describe morphology of rabies virus ∙∙ Describe the following: Street virus vs. fixed virus; pathogenesis and clinical features of rabies; Negri bodies Learning Objectives ∙∙ Discuss laboratory di...

Rhabdoviruses Learning Objectives: After reading and studying this chapter, you should be able to: ∙∙ Describe morphology of rabies virus ∙∙ Describe the following: Street virus vs. fixed virus; pathogenesis and clinical features of rabies; Negri bodies Learning Objectives ∙∙ Discuss laboratory diagnosis of rabies ∙∙ Discuss prophylaxis against rabies ∙∙ Describe neural and non-neural vaccines against rabies. Introduction Bullet shaped, enveloped viruses with single stranded RNA genome are classified as rhabdoviruses (from rhabdos, meaning rod) in the family Rhabdoviridae. Classification Rhabdoviruses infecting mammals belong to two genera in Rhabdoviridae family A. Lyssavirus It contains rabies virus and related viruses (Lagos bat virus, Mokola, Duvenhage). B. Genus vesiculovirus: It contains vesicular stomatitis virus (VSV) and related viruses like Chandipur (arbelius). Rabies virus Morphology 1. Virion: The rabies virion consists of a helical nucleocapsid contained in a bullet-shaped lipoprotein envelope 180 × 75 nm, with one end rounded or conical and the other plane or concave. 2. Proteins: Protruding from the lipid envelope are approximately 200 glycoprotein (G) spikes of the virus, hemagglutinin activity. Spikes do not cover the planar end of the virion. 3. Membrane or matrix (M) protein: Beneath the envelope is the membrane or matrix (M) protein and is the major structural protein of the virus which may be invaginated at the planar end. 4. Genome: The core of the virion consists of Helically arranged ribonucleoprotein. The genome is single-stranded RNA, linear, nonsegmented, negative-sense. RNA-dependent RNA polymerase enzyme which is essential for the initiation of replication of the virus, is enclosed within the virion in association with the ribonucleoprotein core. Resistance Rabies virus is sensitive to ethanol, iodine preparations, quaternary ammonium compounds, soap, detergents and lipid solvents such as ether, chloroform and acetone. It is inactivated by phenol, formalin, beta propiolactone, ultraviolet irradiation, sunlight and heat at 50°C for 1 hour or 60°C for 5 minutes. Rabies virus survives storage at 4°C for weeks and at –70°C for years or by lyophilization It is inactivated by CO2 so on dry ice it must be stored in glass-sealed vials. Antigenic Properties There is a single serotype of rabies virus. A. Glycoprotein G: The surface spikes are composed of glycoprotein G, which is important in pathogenesis, virulence and immunity. Purified spikes containing the viral glycoprotein elicit neutralizing antibody in animals. The purified glycoprotein may therefore provide a safe and effective subunit vaccine. Hemagglutinating activity: Rabies virus possesses hemagglutinating activity, optimally seen with goose erythrocytes at 0–4 °C and pH 6.2. Hemagglutination is a property of the glycoprotein spikes. The hemagglutinin antigen is species specific and distinct from the antigens on rabies related viruses. B. Nucleocapsid protein: Complement fixing antibodies are induced by the nucleocapsid protein and are not protective. This antigen is group specific and cross-reactions are seen with some rabies related viruses. Antiserum prepared against the purified nucleocapsid is used in diagnostic immunofluorescence for rabies. C. Other antigens: Other antigens identified include two membrane proteins, glycolipid and RNA dependent RNA polymerase. Host Range and Growth Characteristics A. Animals Rabies virus has a wide host range. All warm blooded animals, including humans, can be infected. All mammals are susceptible to rabies infection, though differences in susceptibility exist between species. Susceptibility varies among mammalian species, ranging from very high (foxes, coyotes, wolves) to low (opossums); those with intermediate susceptibility include skunks, raccoons, and bats. Humans and dogs occupy an intermediate position. Pups are more susceptible than adult dogs. Experimental infection can be produced in any laboratory animal but mice are the animals of choice. They can be infected by any route. After intracerebral inoculation, they develop encephalitis and die within 5–30 days. Street Virus The rabies virus isolated from natural human or animal infection is termed the street virus. Following inoculation by any route, it can cause fatal encephalitis in laboratory animals after a long and variable incubation period of about 1–12 weeks (usually 21–60 days in dogs). Intracytoplasmic inclusion bodies (Negri bodies) can be demonstrated in the brain of animals dying of street virus infection. Fixed Virus After several serial intracerebral passages in rabbits, the virus undergoes certain changes and becomes what is called the fixed virus that no longer multiplies in extraneural tissues.The fixed (or murant) virus is more neurotropic, though it is much less infective by other routes. After intracerebral inoculation, it produces fatal encephalitis after a short and fixed incubation period of 6–7 days. Negri bodies are usually not demonstrable in the brain of animals dying of fixed virus infection. The fixed virus is used for vaccine production. B. Chick Embryos The rabies virus can be grown in chick embryos. The usual mode of inoculation is into the yolk sac. Serial propagation in chick embryos has led to the development of attenuated vaccine strains like Flury and Kelev. C. Tissue Culture The rabies virus can grow in chick embryo fibroblast, porcine or hamster kidney cells human diploid cell and vero cell cultures. Pathogenesis Rabies infection usually results from the bite of rabid dogs or other animals. The virus can also be transmitted following nonbite exposures through the inhalation of aerosolized virus (as may be found in bat caves), in transplanted infected tissue (e.g. cornea), and by inoculation through intact mucosal membranes. The virus present in the saliva of the animal is deposited in the wound. The virus appears to multiply in the muscles, connective tissue or nerves at the site of deposition. The virus remains at the site for days to months before progressing to the central nervous system (CNS). Rabies virus travels by retrograde axoplasmic transport to the dorsal root ganglia and to the spinal cord. Once the virus gains access to the spinal cord, the brain becomes rapidly infected. The virus then disseminates from the CNS via afferent neurons to highly innervated sites, such as the skin of the head and neck, salivary glands, retina, cornea, nasal mucosa, adrenal medulla, renal parenchyma, and pancreatic acinar cells. The presence of the virus in the saliva and the irritability and aggression brought on by the encephalitis ensure the transmission and survival of the virus in nature. The virus ultimately reaches virtually every tissue in the body, though the centrifugal dissemination may be interrupted at any stage by death. The virus is almost invariably present in the cornea and the facial skin of patients because of their proximity to the brain. The virus may also be shed in milk and urine. With rare exception (three known cases), rabies is fatal once clinical disease is apparent. Pathogenesis of rabies virus infection Clinical Features A. Humans Rabies is primarily a disease of lower animals and is spread to humans by bites of rabid animals or by contact with saliva from rabid animals. The infection has also been acquired from aerosols in bats’ caves. The incubation period in humans is typically 1–2 months but may be as short as 1 week or as long as many years (up to 19 years). It is usually shorter in children than in adults. Phases of Clinical Spectrum The clinical spectrum can be divided into three phases: a. Prodromal phase: The prodrome, lasting 2–10 days, may show any of the nonspecific symptoms. b. Acute neurologic phase: During the acute neurologic phase, which lasts 2–7 days, patients show signs of nervous system dysfunction such as nervousness, apprehension, hallucinations, and bizarre behavior. Hydrophobia: The pathognomonic feature is difficulty in drinking, together with intense thirst. Patients may be able to swallow dry solids but not liquids. Attempts to drink bring on such painful spasms of the pharynx and larynx producing choking or gagging that patients develop a dread of even the sight or sound of water (hydrophobia). Generalized convulsions follow. c. Coma: Patients who survive the stage of acute neurological involvement lapse into coma. Death usually occurs within 1–6 days due to respiratory arrest during convulsions. B. Rabies in Dogs Clinical Picture Rabies in dogs may manifest itself in two forms— Furious rabies and Dumb rabies. a. Furious rabies: This is the typical “mad dog syndrome”, characterized by (i) A change in behavior; (ii) Running amuck; (iii) Change invoice; (iv) Excessive salivation and foaming at the angle of the mouth; (v) Paralytic stage— Paralysis, convulsions and death follow. b. Dumb rabies: In this type, the excitative or irritative stage is lacking. The dumb form is as infectious as the furious type. Laboratory Diagnosis 1. Diagnosis of Human Rabies Tests are performed on samples of saliva, serum, spinal fluid, and skin biopsies of hair follicles at the nape of the neck. A. Rabies Antigens by Immunofluorescence The method most commonly used for diagnosis is the demonstration of rabies virus antigens by immunofluorescence. The specimens tested are corneal smears and skin biopsy (from face or neck) or saliva antemortem, and brain postmorterm. Direct immunofluorescence is done using antirabies serum tagged with fluorescein isothiocyanate. The use of monoclonal antibody instead of crude antiserum makes the test more specific. B. Virus Isolation a. Mouse Inoculation Samples of brain tissue, saliva, CSF, or urine may be injected intracerebrally into newborn mice for isolation of the virus. Infection in mice results in encephalitis and death. The inoculated mice are examined for signs of illness and their brains are examined at death or at 28 days postinoculation for Negri bodies, or by immunofluorescence rabies antigen. b. Isolation in Cell Culture A more rapid and sensitive method is isolation of the virus in tissue culture cell lines (WI38, BHK21,CER). Virus isolations is identified by immunofluorescence. A positive IF test can be obtained as early as 2–4 days after inoculation. The identity of the isolate can be established by the neutralization test with specific antirabies antibody. C. Serology Rabies antibodies can be detected in the serum and CSF of the patient by ELISA. High titer antibodies are present in the CSF in rabies but not after immunization. D. Detection of Nucleic Acid Reverse transcription-polymerase chain reaction (RT-PCR) testing can be used to amplify parts of a rabies virus genome from fixed or unfixed brain tissue for detection of rabies virus RNA. This technique can confirm dFA results and can detect rabies virus in saliva and skin biopsy samples. 2. Animal Rabies The head of the animal is cut off and sent to the nearest testing laboratory, duly packed in ice in an air-tight container. Alternatively, the brain may be removed with antiseptic precautions and sent in 50% glycerol-saline for examination and the other in Zenker’s fixative, sent for biological test and microscopy, respectively. The portion of brain sent should include the hippocampus and cerebellum as Negri bodies are most abundant there. The following tests are done in the laboratory: 1. Demonstration of rabies virus antigen by immunofluorescence This is a highly reliable and the best single test currently available for the rapid diagnosis of rabies viral antigen in infected specimens. This test can establish a highly specific diagnosis within a few hours. Examination of salivary glands by immunofluorescence is useful. 2. Demonstration of inclusion bodies (Negri bodies) A definitive pathologic diagnosis of rabies can be based on the finding of Negri bodies in the brain or the spinal cord. Negri bodies, named after the Italian physician who first discovered them. This is still the method most commonly used as facilities for immunofluorescence and biological tests are not available in many laboratories. Impression smears of the brain are stained by Seller’s technique (basic fuchsin and methylene blue in methanol), which has the advantage that fixation and staining are done simultaneously. Negri bodies are seen as intracytoplasmic, round or oval, purplish pink structures with characteristic basophilic inner granules. Negri bodies vary in size from 3–27 μm. Other types of inclusion bodies may sometimes be seen in the brain in diseases such as canine distemper but the presence of inner structures in the Negri bodies makes differentiation easy. Failure to find Negri bodies does not exclude the diagnosis of rabies. The microscopic examination for Negri bodies identifies 75–90% of cases of rabies in dogs. Failure to find Negri bodies does not exclude the diagnosis of rabies. Negri bodies contain rabies virus antigens and can be demonstrated by immunofluorescence. Both Negri bodies and rabies antigen can usually be found in animals or humans infected with rabies, but they are rarely found in bats. If impression smears are negative, the tissue should be sectioned and stained by Giemsa or Mann’s method. 3. Isolation of the rabies virus (biological test) This is done as described above, for human rabies diagnosis. 4. Corneal test Rabies virus antigen can be detected in live animals in corneal impressions or in frozen sections of skin biopsies by the flueroscent antibody test. Prophylaxis This may be considered under: A. Postexposure Prophylaxis B. Preexposure Prophylaxis A. Post-exposure Prophylaxis This consists of: a. Local treatment , b. Antirabic vaccines, c. Hyperimmune serum. a. Local treatment of wound: i. Cleansing: Immediate flushing and washing the wound(s), scratches and the adjoining areas with plenty of soap and water, preferably under a running tap, for at least 5 minutes as soap inactivates virus by destroying its envelope. ii. Chemical treatment: After cleansing wound should be inactivated by irrigation with virucidal agents - either alcohol (400–700 ml/liter), tincture or 0.01% aqueous solution of iodine or povidone iodine. iii. Suturing: Bite wounds should not be immediately sutured. iv. Antirabies Serum: The local application of antirabies serum or its infiltration around the wound has been shown to be highly effective in preventing rabies. v. Antibiotics and antitetanus measure: When indicated should follow the local treatment recommended above. b. Antirabic vaccines: Antirabic vaccines fall into two main categories: neural and non- neural The former are associated with serious risk of neurological complications and have been replaced by the latter. Rabies vaccines I. Neural vaccines 1. Pasteur vaccine 2. Fermi vaccine 3. Semple vaccine 4. Beta-propiolactone (BPL) vaccine 5. Suckling mouse brain vaccine II. N on-neural vaccines A. Duck egg vaccine B. Cell culture vaccines a. First-generation cell culture vaccine – Human diploid cell vaccine (HDCV) b. Second-generation cell culture vaccines – Purified chick embryo cell vaccine (PCEC) – Purified Vero cell rabies vaccine (PVRV) III. Third-generation rabies vaccine Poxvirus-rabies glycoprotein recombinant vaccine (undergoing clinical trials in humans) Indications for Antirabies Treatment Antirabies treatment should be started immediately: i. If the animal shows signs of rabies or dies within 10 days of observation. ii. If the biting animal cannot be traced or identified. iii. Unprovoked bites. iv. Laboratory tests (e.g. fluorescent rabies antibody test or test for Negri bodies) of the brain of the biting animal are positive for rabies. v. All bites by wild animals. Vaccination Schedules Neural Vaccines The dosage of the vaccine depends on the degree of risk to which the patient has been exposed. Accordingly, patients are classified as follows. Classification of Exposures One of the factors determining the dose of antirabies vaccine is the degree of risk of rabies to which the person is exposed. Accordingly, patients are classified as follows. Class I (Slight risk) a. Licks on healthy unbroken skin. b. Licks on intact mucous membrane or conjunctiva. c. Bites or scratches which have raised the epidermis but have not drawn blood, on all parts of the body except the head, face, neck or fingers. d. Consumption of unboiled milk or handling raw flesh of rabid animals. Class II (Moderate risk) a. Licks on definitely remembered fresh cuts or abrasions on the fingers. b. Scratches with oozing of blood. c. All bites except those on head, neck, face, palms and fingers. d. Minor wounds less than 5 in number. Class III (Severe risk) a. Licks on definitely remembered fresh cuts or abrasions on head, face or neck. b. All bites or scratches on the head, face or neck. c. All bites or scratches on the fingers which are lacerated, more than half centimeter long or have penetrated the true skin. d. All bites penetrating the true skin and drawing blood, when there are five teeth marks or more. e. All bites on any part of the body causing extensive laceration. f. Bites from wild animals—All jackal and wolf bites. g. Any Class II patient who has not received treatment within 14 days of exposure. Dosage Schedules A full schedule consists of 7–10 daily inoculations followed by 1–2 boosters. Site for Vaccination The ideal site for vaccination is the anterior abdominal wall, for this area offers enough space to accommodate the large quantity of vaccine to be injected. The injections are given deep subcutaneously. Adverse Reactions 1. General: Headache, insomnia, giddiness, palpitation, diarrhea. 2. Local: Itching irritation, pain, tenderness, redness and swelling at the site of injection. 3. Allergic: Urticaria, syncope, angioneurotic edema, anaphylactic reaction. 4. Neuroparalysis: Post-vaccinial paralysis due to sensitization. i. Preexposure Prophylaxis This is indicated for persons at high risk of contact with rabies virus such as laboratory workers handling rabies virus or with rabid animals (veterinarians). Preexposure prophylaxis requires three doses of the vaccine injected on day 0, 7, 21 or 0, 28 and 56. A booster dose is recommended after one year and then one every five years. ii. Postexposure Prophylaxis Postexposure prophylaxis requires five or six doses, on days 0, 3, 7, 14, 30 and optionally 90. The vaccine is to be given intramuscular (1M) or subcutaneous (SC) in the deltoid region, or in children on the anterolateral aspect of the thigh. Gluteal injections are to be avoided as they are found to be less immunogenic. This course is expected to give protection for at least five years, during which period any further exposure may need only one or two booster doses (on days 0, 3) depending on the degree of risk. After five years, it is advisable to give a full five injection course if exposed to infection. C. Passive Immunization Passive immunization is an important adjunct to vaccination and should be invariably employed whenever the exposure is considered of high risk. Two preparations of anti rabies serum are available for passive immunization: i. Horse Antirabies Serum: It should be given on day 0 in a single dose of 40 IU/kg of body weight subject to a maximum of 3000 Units. ii. Human Rabies Immune GlobulinHuman rabies immune globulin (HRlG) has now replaced equine antirabies serum in many countries. The dose recommended is a single administration of 20 IU per kg of body weight. Vaccine for Animals Concentrated cell culture vaccines containing inactivated virus are now available, which give good protection after a single IM injection. Injections are given at 12 weeks of age and repeated at 1–3 year intervals. B. Preexposure Prophylaxis This is indicated for persons at high risk of contact with rabies virus or with rabid animals. It is recommended that antibody titers of vaccinated individuals be monitored periodically and that boosters be given when required. Epidemiology Rabies is the classic zoonotic infection, spread from animals to humans. Rabies virus is present in terrestrial animals in all parts of the world except Australasia and Antartica, and some islands like Britain. All warm blooded animals including man are susceptible to rabies. Rabies in man is a dead-end infection, and has no survival value for the virus. Major source of virus is saliva in bite of rabid animal. Direct person-to-person transmission of rabies has not been recorded. An unusual mode of transmission of rabies has occurred in some recipients of corneal grafts. Minor source is aerosols in bat caves containing rabid bats. Epidemiological forms of rabies: Rabies exists in two epidemiological forms: a. Urban rabies: b. Wild-life or sylvatic rabies: In certain Latin American countries and parts of U.S.A. the vampire bat is an important host and vector of rabies. Reservoir of rabies—Reservoir of rabies are wild animals. . Control Human rabies can be checked by control of rabies in domestic animals, by registration, licensing and vaccination of pets and destruction of stray animals.