Rabies

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Rabies virus belongs to the family: Rhabdoviridae. (Greek: Rhabdos: rod). They can infect a variety of animals and plants

Worldwide, it is estimated that approximately 55 000 persons die of rabies each year.

Structure of rhabdoviruses

Rhabdoviruses are negative strand RNA viruses; that is they have a single strand of RNA that is anti-sense to the messenger RNA needed to code for viral proteins. This means that the RNA cannot code directly for protein synthesis and must be copied to positive strand mRNA. As a result, the virus must carry its own RNA-dependent RNA polymerase.

As their name suggests these viruses are rod shaped. They have one end that is rounded and are often referred to as bullet-shaped. Each virus particle is up to 100nm diameter and 400 nm long but this is very variable. They have an envelope derived from the host cell plasma membrane. The virus has only five proteins.

G (Surface) Protein. This is the surface glycoprotein spike and exists as trimers. There are about 1200 G proteins (400 trimers) per virus particle. It is a transmembrane protein with an N-terminal signal sequence. The G protein binds to cellular receptors and is the target of neutralizing antibodies. There are three sugar chains that are N-glycosidically attached. Penetration of the virus into the cytoplasm takes place in the endocytic pathway and not at the plasma membrane. This is because the G protein trimer undergoes a change in conformation at pH 6.1 which stabilizes the trimer and probably allows a hydrophobic region of the molecule to become exposed and to embed in the membrane of the cell to be infected.

M (matrix) protein. This is a peripheral membrane protein (originally M stood for membrane) that appears to line the inner surface of the viral membrane, though this remains somewhat controversial. It may act as a bridge between the membrane or G protein and the nucleocapsid.

Nucleocapsid. This is the infectious ribonucleoprotein core of the virus. It is a helical structure that lies within the membrane. In negative stain electron micrographs, the nucleocapsid has a striated appearance.

N (Nucleoprotein) protein. This is the major structural protein and covers the RNA genome. It protects the genome from nucleases and holds it in a conformation that allows transcription

L (Large) protein and NS (nonstructural, otherwise known as P (phospho)) protein together form the RNA-dependent RNA polymerase or transcriptase. The L protein has a molecular weight of 240 kiloDaltons and its gene takes up 60% of the genome

Replication

Binding

The receptors for rhabdoviruses have yet to be definitively identified but some experiments point to phospholipids, particularly phosphatidyl serine, as the cell surface receptor molecule.

Penetration

After endocytosis, pH-dependent fusion with the membrane of the endocytic vesicle occurs. The nucleocapsid enters the cytoplasm. All subsequent stages take place here with no involvement of the nucleus of the cell.

Transcription

First, the polymerase, which is carried in the entering virus, makes five individual mRNAs, one for each viral protein. Note, the RNA must be made before any viral protein synthesis and so the infecting virus must supply the polymerase enzyme. (As might be expected, this primary transcription process takes place in the presence of protein synthesis inhibitors). The mRNAs are capped, methylated and polyadenylated. The sequence of transcription is N, NS(P), M, G and L with synthesis of the mRNAs being attenuated at each gene junction. This means that less of the L mRNA is made than any of the others.

Replication

In addition, the polymerase transcribes the negative-sense genomic RNA into a positive sense strand. This serves as a template for the transcriptase to transcribe new negative sense genomic RNA molecules. This replicative phase does require protein synthesis and the same polymerase is involved. In the replicative phase, this enzyme must ignore signals that define the individual mRNA species and make one single RNA molecule. The switch between transcription of mRNAs and replication of genomic RNAs seems to be controlled by the level of N protein

Assembly

The G protein mRNA is translated in association with the endoplasmic reticulum and transported via the Golgi body to the cell surface. Here, it forms patches with which the M protein associates. The genomic length negative strand RNA molecules associate with N, L and NS (P) proteins forming the core nucleocapsids. This, in turn, associates with the M protein at the inner surface of the plasma membrane or perhaps in the cytoplasm. The interaction between nucleocapsid and M protein causes the former to change configuration so that it appears more condensed. The nucleocapsid then buds through the membrane.

Pathogenesis

Vesicular Stomatitis Virus (VSV)

VSV infects cattle in Carribean and occasionally in US. It is also found in horses and pigs but rarely humans

Rabies

Rabid animals become aggressive and harbor the virus in saliva and thus transmission is frequently via animal bites, although there is some evidence for spread via aerosols such as in bat urine in caves. The virus has also been transmitted by transplantation of corneas from infected individuals.

The virus binds to nerve or muscle cells at the site of the inoculation via nicotinic acetylcholine receptors. Here the virus can remain for a prolonged period of time (up to several months). The virus can replicate in muscle cells at the site of the bite with no obvious symptoms. This is the incubation phase.

The virus then moves along the nerve axons to the central nervous system using retrograde transport. The virus arrives at the dorsal root ganglia and the spinal cord. From here, spread to the brain occurs. A variety of cells in the brain can be infected including in the cerebellum, the Purkinje=s cells and also cells of the hippocampus and pontine nuclei. This is the prodromal phase. Infection of the brain leads to encephalitis and neural degeneration although elsewhere the virus seems to cause little in the way of a cytopathic effect. Involvement of the brain leads to coma and death. This is the neurological phase and during this period, the virus can spread from the central nervous system, via neurons, to the skin, eye and various other sites (adrenals, kidneys, pancreatic acinar cells) and the salivary glands.

There are various factors that determine the timing of the onset of symptomatic rabies but most important are the number of virus particles in the infection and how close the bite is to the brain. The immunological status of the patient is also important. It should be noted that the immune response to naturally acquired virus is slow and a good neutralizing response is not seen until the virus has reached the brain which is too late for survival. Cell-mediated immunity plays little role in a rabies infection. Rabies is almost always fatal and only three survivors of symptomatic rabies have been documented. Nevertheless, a good immune response that eliminates the infection, can be achieved using a vaccine even after infection because of the long incubation phase.

Epidemiology

Rabies is spread, usually by bites from animals, to other animals and to man. It is thus a zoonotic infection. Other modes of transmission may include contact of mucous membranes with saliva of a rabid animal (but this is very rare) and organ transplantation (cornea, artery). It has been suggested that inhalation of urine in caves frequented by rabid bats might be a possible mode of transmission but this is questionable since spelunkers also often become grazed on rocks and the virus may be transmitted via the wound.

The most important reservoir, as far as humans are concerned, is the dog but other animal reservoirs of importance include, in the United States, racoons, bats, skunks and foxes. Rabies is found in most continental countries of the world but not in some island nations such as the United Kingdom and Australia

Major animal reservoirs of rabies

North America Skunks, raccoons, bats, foxes
South America Rabid dogs, vampire bats
Europe Badgers, foxes

In many western countries where rabies is endemic, vaccination of animals has reduced the rate of human disease and in the United States there is approximately one case of human rabies per year. In countries such as the United Kingdom, where there is no rabies in the wild animal population, vaccination is not used. In some other countries, rabies is much more of a problem. For example, India records about 25,000 cases of human rabies per year, mainly from dog bites. In South America, rabies transmission by vampire bats is a major problem for the cattle industry.

Symptoms

Vaccination, even after exposure, is extremely effective at preventing disease. Without such treatment, rabies is almost invariably fatal (although, see the case report at left). During the incubation/prodromal period, symptoms include: pain or itching at the site of the wound, fever, headache and gastrointestinal problems. After this period (usually of up to two weeks), CNS infection is apparent. In up to half of patients, hydrophobia is seen. This fear of water is the result of the pain associated with drinking. There are also seizures and hallucinations. In some patients paralysis is the only symptom and this may lead to respiratory failure. Following the neurological phase, the patient becomes comatose. Because of the neurological problems including respiratory paralysis, death ensues.

Diagnosis

Overt symptoms clearly define symptomatic rabies in people who suffer animal bites but by this time, therapeutic intervention is too late. After a bite, laboratory tests can determine whether an animal is indeed rabid. The presence of rabies virus in an animal or an infected person can be determined by serology and immunofluorescence antigen determination using biopsy skin, brain or corneal specimens. Histologically very characteristic is the presence of Negri bodies. These are intracytoplasmic inclusions formed by aggregates of nucleocapsids in neurons of about 90% of infected humans. Other tests include the growing of virus in the brains of mice or in culture, after which antigen tests are used to determine the presence of virus. Also anti-rabies antibodies can be detected BUT only very late in the disease. Polymerase chain reaction (PCR) can also be used to detect virus

Therapy

Post-exposure prophylaxis is the major form of treatment when it is suspected that the patient may have come in contact with a rabid animal. Such contact is most often a bite but could also include contact with tissue from a rabid animal. In the United States up to 20,000 rabies inoculations are given to humans every year. After a bite, it is recommended to wash the wound and to instill anti-rabies serum into the wound. After this, the vaccine is given, accompanied by either horse anti-rabies serum or human rabies immune globulin. This suffices until the patient develops an immune response to the virus. Four more vaccinations are then given over a period of a month (days 3, 7, 14 and 28 after exposure to the virus). Pre-exposure vaccination is recommended for persons who may come in contact with tissue of rabid animals and seems effective for up to two years.

Vaccination

Vaccination against rabies has a long history, the first vaccine having been developed by Louis Pasteur. His vaccine was an attenuated form of the virus produced by inoculation of rabbit spinal cord. The present (HDCV) rabies vaccine is a chemically-inactivated one that is produced in human diploid tissue culture cells. Previous vaccines were produced in the brains of animals but these gave more side effects.