family rhabdoviridae

FAMILY RHABDOVIRIDAE

A Presentation By

Isaac U.M.,Dept. of Microbiology & Parasitology,

Faculty of Medicine, International Medical & Technological University,Dar-Es-Salaam, Tanzania.

Introduction

• The members of the family Rhabdoviridae (from the Greek word rhabdos, meaning "rod") include pathogens for a variety of mammals, fish, birds, and plants.

• The family contains Vesiculovirus (vesicular stomatitis viruses [VSVs]); Lyssavirus (rabies and rabieslike viruses), an unnamed genus constituting the plant rhabdovirus group; and other ungrouped rhabdoviruses of mammals, birds, fish, and arthropods.

• Rabies virus is the most significant pathogen of the rhabdoviruses.• Until Louis Pasteur developed the killed-rabies vaccine, a bite from a

"mad" dog always led to the characteristic symptoms of hydrophobia and certain death.

Unique Features of Rhabdoviruses

• Bullet-shaped, enveloped, negative, single-stranded RNA viruses that encode five proteins.

• Prototype for replication of negative-stranded enveloped viruses. • Replication in the cytoplasm.

Physiology & Structure

• Rhabdoviruses are simple viruses encoding only five proteins and appearing as bullet-shaped, enveloped virions with a diameter of 50 to 95 nm and length of 130 to 380 nm.

• Spikes composed of a trimer of the glycoprotein (G) cover the surface of the virus.

• The viral attachment protein, G protein, generates neutralizing antibodies.

• The G protein of the vesicular stomatitis virus is a simple glycoprotein with N-linked glycan.

• This G protein has been used as the prototype for studying eukaryotic glycoprotein processing.

• Within the envelope the helical nucleocapsid is coiled symmetrically into a cylindrical structure, giving it the appearance of striations.

• The nucleocapsid is composed of one molecule of single-stranded, negative-sense RNA (ribonucleic acid) of approximately 12,000 bases and the nucleoprotein (N), large (L) and nonstructural (NS) proteins.

• The matrix (M) protein lies between the envelope and the nucleocapsid.

• The N protein is the major structural protein of the virus.

Physiology & Structure

• It protects the RNA from ribonuclease digestion and maintains the RNA in a configuration acceptable for transcription.

• The L and NS proteins constitute the RNA-dependent RNA polymerase.

Figure 61-1 Rhabdoviridae seen by electron microscopy: rabies virus (left) and vesicular stomatitis virus (right). (From Fields BN: Virology, New York, 1985, Raven.)

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Replication

• The replicative cycle of VSV is the prototype for the rhabdoviruses and other negative-strand RNA viruses.

• The viral G protein attaches to the host cell and is internalized by endocytosis.

• The viral envelope then fuses with the membrane of the endosome on acidification of the vesicle.

• This uncoating releases the nucleocapsid to be released into the cytoplasm, where replication takes place.

• The RNA-dependent RNA polymerase associated with the nucleocapsid transcribes the viral genomic RNA, producing five individual messenger RNAs (mRNAs).

• These mRNAs are then translated into the five viral proteins. • The viral genomic RNA is also transcribed into a full-length positive-

sense RNA template that is used to generate new genomes.• The G protein is synthesized by membrane-bound ribosomes,

processed by the Golgi apparatus, and delivered to the cell surface in membrane vesicles.

• The M protein associates with the G protein-modified membranes.

Replication

• Assembly of the virion occurs in two phases: (1) assembly of the nucleocapsid in the cytoplasm and (2) envelopment and release at the cell plasma membrane. • The genome associates with the N protein and then with the

polymerase proteins L and NS to form the nucleocapsid. • Association of the nucleocapsid with the M protein at the plasma

membrane induces coiling into its condensed form. • The virus then buds through the plasma membrane and is released

when the entire nucleocapsid is enveloped. • Cell death and lysis occur after infection with most rhabdoviruses, with

the important exception of rabies virus, which produces little discernible cell damage.

Pathogenesis & Immunity

• Rabies infection usually results from the bite of a rabid animal. • Rabies infection of the animal causes secretion of the virus in the

animal's saliva and promotes aggressive behavior ("mad" dog), which in turn promotes transmission of the virus.

• The virus can also be transmitted 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.

• Virus may directly infect nerve endings by binding to nicotinic acetylcholine or ganglioside receptors of neurons or muscle at the site of inoculation.

• The virus remains at the site for days to months (Figure 61-2) 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 affected areas are the hippocampus, brain stem, ganglionic cells of the pontine nuclei, and Purkinje cells of the cerebellum.

Figure 61-2 Pathogenesis of rabies virus infection. Numbered steps describe the sequence of events. (Redrawn from Belshe RB, editor: Textbook of human virology, ed 2, St Louis, 1991, Mosby.)


© 2005 Elsevier


• 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.

• After the virus invades the brain and spinal cord, an encephalitis develops, and neurons degenerate.

• Despite the extensive CNS involvement and impairment of CNS function, little histopathologic change can be observed in the affected tissue other than the presence of Negri bodies.

• With rare exception (three known cases), rabies is fatal once clinical disease is apparent.

• The length of the incubation period is determined by (1) the concentration of the virus in the inoculum, (2) the proximity of the wound to the brain, (3) the severity of the wound, (4) the host's age, and (5) the host's immune status. • In contrast to other viral encephalitis syndromes, rabies rarely causes

inflammatory lesions.


• Neutralizing antibodies are not apparent until after the clinical disease is well established.

• Little antigen is released, and the infection probably remains hidden from the immune response.

• Cell-mediated immunity appears to play little or no role in protection against rabies virus infection.

• Antibody can block the spread of virus to the CNS and to the brain if administered or generated during the incubation period.

• The incubation period is usually long enough to allow generation of a therapeutic protective antibody response after active immunization with the killed rabies vaccine.

Disease Mechanisms of Rabies Virus

• Rabies is usually transmitted in saliva and is acquired from the bite of a rabid animal.

• Rabies virus is not very cytolytic and seems to remain cell associated.

• Virus replicates in the muscle at the site of the bite, with minimal or no symptoms (incubation phase).

• The length of the incubation phase is determined by the infectious dose and the proximity of the infection site to the central nervous system (CNS) and brain.

• After weeks to months, the virus infects the peripheral nerves and travels up the CNS to the brain (prodrome phase).

• Infection of the brain causes classic symptoms, coma, and death (neurologic phase).

• During the neurologic phase the virus spreads to the glands, skin, and other body parts, including the salivary glands, from where it is transmitted.

• Rabies infection does not elicit an antibody response until the late stages of the disease, when the virus has spread from the CNS to other sites.

Disease Mechanisms of Rabies Virus

• Antibody can block the progression of the virus and disease.• The long incubation period allows active immunization as a

postexposure treatment.

Epidemiology

• Rabies is the classic zoonotic infection, spread from animals to humans.

• It is endemic in a variety of animals worldwide, except in Australia.• Rabies is maintained and spread in two ways: In urban rabies, dogs

are the primary transmitter, and in sylvatic (forest) rabies, many species of wildlife can serve as the transmitter.

• In the United States, rabies is more prevalent in cats because they are not vaccinated.

• Virus-containing aerosols, bites, and scratches from infected bats also spread the disease.

• The principal reservoir for rabies in most of the world, however, is the dog.

• In Latin America and Asia, this feature is a problem because of the existence of many stray, unvaccinated dogs and the absence of rabies-control programs.

• These two factors are responsible for thousands of rabies cases in dogs each year in these countries.

• Because of the excellent vaccination program in the United States, sylvatic rabies accounts for most of the cases of animal rabies in this country.

Epidemiology

• Statistics for animal rabies are collected by the U.S. Centers for Disease Control and Prevention, which in 1999 recorded more than 8000 documented cases of rabies in raccoons, skunks, bats, and farm animals, in addition to dogs and cats.

• Badgers and foxes are also major carriers of rabies in Western Europe.

• In South America, vampire bats transmit rabies to cattle, resulting in losses of millions of dollars each year.

• The distribution of human rabies approximates the distribution of animal cases in each country.

• It is estimated that rabies accounts for between 40,000 and as high as 70,000 deaths annually worldwide, and at least 25,000 deaths in India, where the virus is transmitted by dogs in 96% of cases.

• In Latin America, cases of human rabies primarily result from contact with rabid dogs in urban areas.

• In Indonesia, an outbreak of more than 200 human cases of rabies in 1999 promoted the killing of more than 40,000 dogs on the islands.

• The incidence of human rabies in the United States is approximately one case per year, because of effective dog vaccination programs and limited human contact with skunks, raccoons, and bats.

Epidemiology

• Since 1990, human cases of rabies in the United States have been caused primarily by bat variants of the virus.

• The World Health Organization estimates that 10 million people per year receive treatment after exposure to animals suspected of being rabid.

Figure 61-3 Distribution of animal rabies in the United States, 1999. The percentages relate to the total number of cases of animal rabies. (Data from Krebs JW et al: JAVMA 217:1799-1811, 2000.)


© 2005 Elsevier

Epidemiology of Rabies Virus

• Disease/Viral Factors Virus-induced aggressive behavior in animals promotes virus

spread. Disease has long, asymptomatic incubation period.

• Transmission Zoonosis:

• Reservoir: Wild animals. • Vector: Wild animals and unvaccinated dogs and cats.

Source of virus: • Major: Saliva in bite of rabid animal. • Minor: Aerosols in bat caves containing rabid bats.

• Who Is at Risk? Veterinarians and animal handlers. Person bitten by a rabid animal. Inhabitants of countries with no pet vaccination program.

• Geography/Season Virus is found worldwide, except in some island nations. There is no seasonal incidence.

Epidemiology of Rabies Virus

• Modes of Control Vaccination program is available for pets. Vaccination is available for at-risk personnel. Vaccination programs have been implemented to control rabies in

forest mammals.

Clinical Syndromes• Rabies is virtually always fatal unless treated by vaccination. • After a long but highly variable incubation period, the prodrome phase

of rabies ensues. • The patient has symptoms such as fever, malaise, headache, pain or

paresthesia (itching) at the site of the bite, gastrointestinal symptoms, fatigue, and anorexia.

• The prodrome usually lasts 2 to 10 days, after which the neurologic symptoms specific to rabies appear.

• Hydrophobia (fear of water), the most characteristic symptom of rabies, occurs in 20% to 50% of patients.

• It is triggered by the pain associated with the patient's attempts to swallow water.

• Focal and generalized seizures, disorientation, and hallucinations are also common during the neurologic phase.

• From 15% to 60% of patients exhibit paralysis as the only manifestation of rabies.

• The paralysis may lead to respiratory failure. • The patient becomes comatose after the neurologic phase, which lasts

from 2 to 10 days. • This phase almost universally leads to death due to neurologic and

pulmonary complications.

Progression of Rabies Disease

Disease Phase Symptoms Time (days) Viral Status Immunologic Status

Incubation phase Asymptomatic 60-365 afterbite

Low titer, virusin muscle

-

Prodrome phase Fever, nausea, vomiting, loss of appetite, headache, lethargy, painat site of bite

2-10 Low titer, virusin CNS andbrain

-

Neurologic phase Hydrophobia, pharyngeal spasms,hyperactivity, anxiety, depression

2-7 High titer, virusin brain andother sites

Detectable antibody inserum and CNS

CNS symptoms: loss of coordination, paralysis, confusion,delirium

Coma Coma: cardiac arrest, hypotension, hypoventilation,secondary infections

0-14 High titer, virusin brain andother sites

-

Death - - - -

CNS, Central nervous system.

Laboratory Diagnosis

• The occurrence of neurologic symptoms in a person who has been bitten by an animal generally establishes the diagnosis of rabies.

• Unfortunately, evidence of infection, including symptoms and the detection of antibody, does not occur until it is too late for intervention.

• Laboratory tests are usually performed to confirm the diagnosis and to determine whether a suspected individual or animal is rabid (postmortem).

• The diagnosis of rabies is made through detection of viral antigen in the CNS or skin, isolation of the virus, detection of the genome, and serologic findings.

• The hallmark diagnostic finding has been the detection of intracytoplasmic inclusions consisting of aggregates of viral nucleocapsids (Negri bodies) in affected neurons.

• Although their finding is diagnostic of rabies, Negri bodies are seen in only 70% to 90% of brain tissue from infected humans.

• Antigen detection using direct immunofluorescence or genome detection using reverse transcriptase polymerase chain reaction (RT-PCR) are relatively quick and sensitive assays that are the preferred methods for diagnosing rabies.

Figure 51-3 Negri bodies caused by rabies. A, A section of brain from a patient with rabies shows Negri bodies (arrow). B, Higher magnification from another biopsy specimen. (A from Hart C, Broadhead RL: A color atlas of pediatric infectious diseases, London, 1992,

Wolfe.)

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Laboratory Diagnosis

• Samples of saliva, serum, spinal fluid, skin biopsy material from the nape of the neck, brain biopsy or autopsy material, and impression smears of corneal epithelial cells are the specimens that are examined.

• Rabies can also be grown in cell culture or in intracerebrally inoculated infant mice.

• Inoculated cell cultures or brain tissues are subsequently examined with direct immunofluorescence.

• Rabies antibody titers in serum and cerebrospinal fluid are usually measured by enzyme-linked immunosorbant assay (ELISA) or a rapid fluorescent focus inhibition test.

• Antibody usually is not detectable until late in the disease, however.

Treatment & Prophylaxis

• Clinical rabies is almost always fatal unless treated. • Once the symptoms have appeared, little other than supportive care

can be given. • Postexposure prophylaxis is the only hope for preventing overt clinical

illness in the affected person. • Although human cases of rabies are rare, appoximately 20,000 people

receive rabies prophylaxis each year in the United States alone.• Prophylaxis should be initiated for anyone exposed by bite or by

contamination of an open wound or mucous membrane to the saliva or brain tissue of an animal suspected to be infected with the virus, unless the animal is tested and shown not to be rabid.

• The first protective measure is local treatment of the wound. • The wound should be washed immediately with soap and water or

another substance that inactivates the virus. • The World Health Organization Expert Committee on Rabies also

recommends the instillation of antirabies serum around the wound.• Subsequently, immunization with vaccine in combination with

administration of one dose of human rabies immunoglobulin (HRIG) or equine antirabies serum is recommended.


• Passive immunization with HRIG provides antibody until the patient produces antibody in response to the vaccine.

• A series of five immunizations is then administered over the course of a month.

• The slow course of rabies disease allows active immunity to be generated in time to afford protection.

• The rabies vaccine is a killed-virus vaccine prepared through the chemical inactivation of rabies-infected tissue culture human diploid cells (HDCV) or fetal rhesus lung cells.

• These vaccines cause fewer negative reactions than the older vaccines (Semple and Fermi), which were prepared in the brains of adult or suckling animals.

• The HDCV is administered intramuscularly on the day of exposure and then on days 3, 7, 14, and 28 or intradermally with a lower dose of vaccine to multiple sites on days 0, 3, 7, 28, and 90.

• Preexposure vaccination should be performed on animal workers, laboratory workers who handle potentially infected tissue, and people traveling to areas where rabies is endemic.

• HDCV administered intramuscularly or intradermally in three doses is recommended and provides 2 years of protection.


• Ultimately, the prevention of human rabies hinges on the effective control of rabies in domestic and wild animals.

• Its control in domestic animals depends on the removal of stray and unwanted animals and the vaccination of all dogs and cats.

• A variety of attenuated oral vaccines have also been used successfully to immunize foxes.

• A live recombinant vaccinia virus vaccine expressing the rabies virus G protein is in use in the United States.

• This vaccine, which is injected into bait and parachuted into the forest, successfully immunizes raccoons, foxes, and other animals.


• Types of Vaccines All vaccines for human use contain only inactivated rabies virus. Three vaccines are available in the United States, though a number

of others are in use in other countries. All three US rabies vaccines are equally safe and efficacious.

• Human Diploid Cell Vaccine (HDCV) To obtain a rabies virus suspension free from nervous system and

foreign proteins, rabies virus was adapted to growth in the WI-38 human normal fibroblast cell line.

The rabies virus preparation is concentrated by ultrafiltration and inactivated with -propiolactone.

No serious anaphylactic or encephalitic reactions have been reported.

This vaccine has been used in the United States since 1980.

Treatment & Prophylaxis• Rabies Vaccine, Adsorbed (RVA)

A vaccine made in a diploid cell line derived from fetal rhesus monkey lung cells was licensed in the United States in 1988.

This vaccine virus is inactivated with -propiolactone and concentrated by adsorption to aluminum phosphate.

• Purified Chick Embryo Cell Vaccine (PCEC) This vaccine is prepared from the fixed rabies virus strain Flury LEP

grown in chicken fibroblasts. It is inactivated with -propiolactone and further purified by zonal

centrifugation. It became available in the United States in 1997.• Nerve Tissue Vaccine

This is made from infected sheep, goat, or mouse brains and is used in many parts of the world including Asia, Africa, and South America.

It has a low potency per dose, and a complete treatment involves up to 23 painful injections.

It causes sensitization to nerve tissue and results in postvaccinal encephalitis (an allergic disease) with substantial frequency (0.05%).

Estimates of its efficacy in persons bitten by rabid animals vary from 5% to 50%.


• Duck Embryo Vaccine Duck embryo vaccine was developed to minimize the problem of

postvaccinal encephalitis. The rabies virus is grown in embryonated duck eggs. Anaphylactic

reactions are infrequent but the antigenicity of the vaccine is low, so that many (16–25) doses have to be given to obtain a satisfactory postexposure antibody response.

It is no longer manufactured.• Live Attenuated Viruses

Live attenuated viruses adapted to growth in chick embryos (eg, Flury strain) are used for animals but not for humans.

Occasionally, such vaccines can cause death from rabies in injected cats or dogs.

Rabies viruses grown in various animal cell cultures have also been used as vaccines for domestic animals.

A recombinant viral vaccine consisting of vaccinia virus carrying the rabies surface glycoprotein gene has successfully immunized animals following oral administration.

This vaccine may prove valuable in the immunization of both wildlife reservoir species and domestic animals.


• Types of Rabies Antibody• Rabies Immune Globulin, Human (HRIG)

HRIG is a gamma globulin prepared by cold ethanol fractionation from the plasma of hyperimmunized humans.

There are fewer adverse reactions to human rabies immune globulin than to equine antirabies serum.

• Antirabies Serum, Equine This is concentrated serum from horses hyperimmunized with

rabies virus. It has been used in countries where HRIG is not available.


• Preexposure Prophylaxis This is indicated for persons at high risk of contact with rabies virus

(research and diagnostic laboratory workers, spelunkers) or with rabid animals (veterinarians, animal control and wildlife workers).

The goal is to attain an antibody level presumed to be protective by means of vaccine administration prior to any exposure.

It is recommended that antibody titers of vaccinated individuals be monitored periodically and that boosters be given when required.


• Postexposure Prophylaxis Although few (0–5) cases of human rabies occur in the United

States per year, more than 20,000 persons receive some treatment every year for possible bite wound exposure.

The decision to administer rabies antibody, rabies vaccine—or both—depends on several factors: (1) the nature of the biting animal (species, state of health, domestic or wild) and its vaccination status; (2) the availability of the animal for laboratory examination (all bites by wild animals and bats require rabies immune globulin and vaccine); (3) the existence of rabies in the area; (4) the manner of attack (provoked or unprovoked); (5) the severity of the bite and contamination by saliva of the animal; and (6) advice from local public health officials (Table 42–3).

Schedules for postexposure prophylaxis involving the administration of rabies immune globulin and vaccine are available from the Centers for Disease Control and Prevention and state public health offices.

family rhabdoviridae

Technology

viral g protein

n protein

rabies virus travels

protein associates

viral attachment protein

rnadependent rna polymerase

viral genomic rna

viral proteins