Rhabdoviruses

The family Rhabdoviridae comprises more than 200 viruses which infect mammals, reptiles, birds, fishes, insects and plants. It has two genera-Vesiculovirus, which causes vesicular stomatitis in horses, cattle and pigs,rarely infects humans & Lyssavirus, which comprises rabies virus. 

Rabies Virus

Morphology- bullet shaped,180 X 75 nm, with one end rounded or conical and the other planar or concave. The lipoprotein envelope carries knob-like spikes (peplomers) composed of glycoprotein G. Spikes do not cover the planar end, it can be released from the envelope by treatment with lipid solvents or detergents. Beneath the envelope is the membrane protein, M protein layer which may be invaginated at the planar end. The membrane may project outwards from the planar end of some virions forming a bleb. The core consists of helically arranged nucleoprotein. The genome is unsegmented, linear, 11-12 kb, (- sense) RNA. In the nucleocapsid,  RNA dependent RNA polymerase enzyme and some structural proteins are present.

Resistance- the virus is sensitive to ethanol, iodine preparations, quaternary ammonium compunds, soap, detergents, lipid solvents such as ether, chloroform and acetone. It is inactivated by phenol, formalin, BPL, UV. Thermal inactivation in 1 hour at 50⁰C, dies at room temperature but survive for weeks if in 50% glycerol. It can be preserved at –70⁰C or by lyophilization. For storage in dry ice, the virus has to be sealed in vials as it is inactivated on exposure to CO₂.

Antigenic Properties- the surface spikes are composed of glycoprotein G, which is important in pathogenesis. It mediates the binding of virus to acetylcholine receptors in neural tissues, induce hemagglutination inhibiting (HI) and neutralizing antibodies and stimulates cytotoxic T cell immunity. It is a serotype specific antigen. The purified glycoprotein may therefore provide a safe and effective subunit vaccine.

Rabies virus possesses hemagglutinating activity, provided by the G spikes, but can be inactrivated by heat, ether, trypsin. HI antibodies develop following infection or immunization and parallel neutralizing antibodies. Thus, HI tests provide a useful method of assaying immunity to rabies, but the low sensitivity of the test and the presence of inhibitors (nonspecific) limit its value. These inhibiors can be destroyed by treatment with acetone.

The nucleocapsid protein induces complement fixing antibodies. Antiserum prepared against the nucleocapsid antigen is used in diagnostic immunofluroscence tests. Other antigens are two membrane proteins, glycolipid and RNA dependent RNA polymerase.

Host range, Growth characteristics- (i) Animals : All mammals are susceptible to rabies infection. Cattle, cats, foxes are highly susceptible. Humans and dogs occupy an intermediate position. Pups are more susceptible than adult dogs. Experimentally infection can be done on mice, which develop encephalitis and die with in 5-30 days. The  rabies virus isolated from natural human  or animal infection is called street virus. Following inoculation by any route, it cause fatal encephalitis in laboratory animals. Negri bodies (intracytoplasmic inclusion bodies) demonstrated in the brain of dying animals. After serial intracerebral passages in rabbits, the virus undergoes certain changes and becomes fixed virus, which is more neurotropic, and less infective by other routes. The incubation period is only 6-7 days and fatal encephalitis occurs, Negri bodies is not demonstrable. It produces paralytic rather than furious symptoms. The fixed virus is used for vaccine production. (ii) Chick embryos : The usual mode of inoculation is into the yolk sac. Attenuated vaccine strains developed from the serial propagation in chick embryos. Strains adapted to duck eggs are used in the preparation of inactivated vaccines. (iii) Tissue culture : Grown in primary and continuous cell cultures such as baby hamster kidney,human diploid lung fibroblasts, chick embryo fibroblasts,Vero monkey kidney cells, but cytopathic effects and yield is low.

Pathogenicity – Rabies has been recognised from very ancient times as a disease transmitted to humans and animals by the bite of ‘mad dogs’. The virus present in the saliva of the infected animal is deposited in the wound, when a rabid dog bites. Rarely infection can also occur by licks, aerosols, transplantation of cornea or other virus infected tissues.

            After entering the body, the rabies virus replicates in injured tissue for 1-4 days, after it reaches a sufficient concentration it infects the peripheral nerves in the muscle or skin. Once within the nervefibres, it is out of reach of any circulating antibody. It progresses rapidly up the spinal cord to the brain by the flow of axoplasm through axons at a speed of 3mm per hour. In the CNS it multiplies and produces encephalitis. The incubation period in humans range from 13 days to 2 years, usually 20- 60 days. The virus then spreads outwards to variuos parts of the body through the nerves, including the salivary glands where they multiply and shed in the saliva. The length of time required for symptoms to appear is proportional to the distance between the wound and the brain and is affected by the accessibility of nerve fibres. Thus, a bite on the face which is well supplied with nerves and close to the brain, produces symptoms much more quickly than does a bite on the leg.

            The course of the disease can be classified into 4 stages- Prodrome, Acute Encephalitic Phase, Coma, Death. The onset is marked by prodromal symptoms such as fever, headache, nausea, fatigue, anorexia and partial paralysis near the bite site. These symptoms persist for 2- 10 days and then worsen, when the acute encephalitic phase ensues by hyperactivity which is intermittent, bizzare behaviour, agitation etc. Such hyperactivity may be spontaneous or by external stimuli. Hydrophobia occurs as throat muscles undergo painful spasms or contractions during swallowing. Aerophobia occurs because the skin is hypersensitive to any sensations. Confusion, hallucinations also occur. Within 10-14 days of the onset of symptoms the patient typically goes into a coma and dies. Death is due to respiratory arrest during convulsions.

            In dogs, the incubation period is usually 3-6 weeks, but may range from 10 days to a year. The initial signs are an alert, troubled air and a change in position with restlessness, snapping at imaginary objects, licking or gnawing at the site of bite. After 2-3 days of this prodromal stage, the disease develops into either the furious or dumb type of rabies. In furious rabies the dogs run and bite without provocation. The lower jaw droops and saliva drools from the mouth. Paralysis, convulsions and death follow. In dumb rabies or the paralytic form in which the animal lies huddled, unable to food. The dog may not bite but attempts to feed it are dangerous. Rabid dog usually dies in 3-5 days.

            Rabies virus ascends to the brain along the nerves and does not come in contact with immune system of the body. It is only after the virus spreads to different parts of the body that antibodies are produced. But by this time it is too late, irreversible damage of the neurons has already been caused and patient dies of respiratory paralysis. However, antibody is protective when present before exposure and prompt vaccination after exposure induces resistance which is associated with antibody production.

Laboratory Diagnosis- the rabies virus antigens can be demonstrated by Immunofluorescent Antibody Test(IFAT). Specimens tested are corneal smears, skin biopsy, saliva, brain. Direct immunofluorescence is done using antirabies serum tagged with fluorescein isothiocyanate. Diagnosis may be made by postmortem, by demonstrating Negri bodies in the brain. Detection of rabies virus RNA by reverse transcription, PCR. Isolation can be done in primary cell cultures.

In animals the whole carcass or the severed head send to the laboratory. The brain removed carefully and two portions- one in 50% glycerol saline and the other in Zinker’s fixative. The viral antigens, inclusion bodies are demonstrated.

Prophylaxis – Specific prophylaxis is ideally given before exposure to infection. In animals this is imperative, but in humans, only employed in persons at high risk such as veterinarians, dog handlers, because neural vaccines carry some risk of  serious complications. Cell culture vaccines are safe. Antirabic treatment consists of local treatment, antirabic vaccines, hyperimmune serum.

1. Local treatment- the bite of an animal (rabid or not) is treated by first thoroughly cleaning it with soap and flushing it with large amounts of water. Hyperimmune rabies serum is introduced into and around the wound to neutralize viruses before they reach the nervous system. Interferons can also be applied on the wound. Antitetenus measures and antibiotics to prevent sepsis may be used as necessary.

2. Antirabic vaccines – Two categories :  Neural & Non-neural

Neural vaccines are associated with serious risk of neurological complications. These are suspensions of nervous tissues of animals infected with fixed rabies virus,Examples are,

• Semple vaccine- developed by Semple, it is a 5% suspension of sheep brain infected with fixed virus and inactivated with phenol at 37⁰C.
• Beta PropioLactone vaccine – Modification of Semple vaccine, BPL is used as an inactivating agent. More antigenic, so that smaller doses are adequate.
• Infant Brain vaccines – The encephalitogenic factor in brain tissue is a basic protein associated with myelin. It is scanty or absent in the non-myelinated  neural tissue of newborn animals, so developed using infant mouse, rabbit.

Neural vaccines are poor immunogens as they contain nucleocapsid antigen, with small quantities of G which is the sole protective antigen. They may contain infectious agents which are not inactivated during vaccine  preparation and storage, but they are cheap, abandoned in developed countries.

            Neural vaccines dosage depends on the degree of risk to which the patient has been exposed. Accordingly patients are classified into 3 :

• Class I – Risk is estimated to be slight. Licks or direct contact of saliva on cuts or abrasions except head, face, neck, fingers, but on mucus membrane or conjunctiva, bites or scratches which have raised  the epidermis, but not drawn blood except head, face, neck, fingers and consumption of unboiled milk or handling raw flesh of rabid animals. Dosage – 2ml X 7 days(semple vaccine), 2ml X 7 days (BPL vaccine).
• Class II – Moderate risk. Licks on cuts or abrasions on the fingers, all bites scratches on the fingers not more than a ½ cm long and not penetrated the true skin, bites or scratches on all parts except head, face, neck, fingers which have drawn blood. Dosage – 5 ml X 14 days(semple vaccine), 3ml X 10 days (BPL vaccine).
• Class III – High risk. Licks on fresh cuts or abrasions on head, face, neck, all bites or scratches on head, face, neck, bites and scratches more than ½ cm long or penetrated the true skin and drawing blood with 5 teeth marks or more, all jackal and wolf bites and also Class II patients whom has not received treatment within 14 days of exposure. Dosage – 10 ml X 14 days(semple vaccine), 5 ml X 10 days(BPL vaccine)

The immunity following neural vaccination is for 6 months, and any exposure later should receive fresh treatment. The vaccine is administered subcutaneously on the anterior abdominal wall. Antirabic vaccine may cause minor local reactions to serious neuroparalytic complications. Severe exertion and the use of alcohol during vaccination increases the risk of neurological reactions.

Non- neural vaccines are less complicated.

• Egg Vaccines : Duck egg vaccine prepared from a fixed virus adapted for growth in duck eggs and inactivated with BPL, but poor immunogenicity. Live attenuated chick embryo vaccines prepared by 40- 50 passages, ie., Low Egg Passage (LEP) vaccine for dogs and by 180 passages, ie., High Egg Passage(HEP) for cattles and cats, not used now.
• Tissue Culture Vaccines : Human Diploid Cell (HDC) vaccine is prepared by purifying and the concentrated preparation of fixed rabies virus grown on human diploid cells and inactivated with BPL. It is highly antigenic, but expensive. Primary cell culture vaccines, continuous cell culture vaccines are available.
• Subunit Vaccine : The glycoprotein subunit on the virus surface, the protective antigen, cloned and recombinant vaccines produced.

Schedules -  Vaccine administered when a person has bitten, scratched or licked by a rabid animal. Animal should be observed for 10 days because virus may be present in the saliva 3-4 days before the onset of symptoms and animal usually dies within 5-6 days. If animal is healthy after this period, no risk of rabies.

3. Hyperimmune serum – Manufactured by hyperimmunisation of horses. Passive immunisation is an important adjunct to vaccination and should be employed when the exposure is considered of high risk. Passive immunisation should be given before or simultaneously with the first injection of the vaccine, not after it. Persons receiving serum and vaccine, a booster dose of cell culture vaccine on day 90 given.

Epidemiology- Two epidemiological types,(1) Urban- transmitted by domestic animals & (2) Sylvatic- involving wild animals. Carnivorous animals may acquire the infection by eating carcasses containing the virus. Another natural cycle of rabies concerns bats, transmitted by vampire bats to cattles. Rabies also occur in insectivorous and frugivorous bats. Humans may be infected by aerosols if they enter caves where infected bats colonize. Human rabies can be checked by control of rabies in domestic animals, by registration, licensing and vaccination of pets, killing of stray animals.