Introduction and History of the Disease:
Rabies is a viral disease which is usually transmitted to human beings through a bite from a domesticated or wild animal. The virus affects the central nervous system, causing multiple neurological afflictions and virtually no hope of survival after the onset of symptoms. It is an ancient disease which has affected humans for many centuries. In fact, references to rabies date as far back to the third century B.C. It wasn't until the late nineteenth century, however, that its biological basis started to become apparent. A strong scientific grasp on the transmission and progress of the disease has allowed many nations to initiate public health campaigns that have nearly wiped out the incidence of human rabies throughout the developed world.
Since the 1880's, there have been several significant advances in the diagnosis, treatment, and laboratory study of rabies. Beginning in 1881, Pasteur performed several experiments which showed that the disease was caused by a viral agent, and that this agent multiplied primarily in the tissues of the central nervous system. This lead to the development and testing of the first human rabies vaccine in 1885. In 1921, this vaccine was adapted for use in domesticated dogs as part of a rabies control regime in Japan. In the 1940's the United States initiated programs for mass vaccination of dogs, and later for cats as well. Meanwhile, advances in rabies diagnosis have included the 1903 discovery of the Negri body, a characteristic dense matter seen via microscopy in the brain cells of about 75% of infected individuals. An improvement on this method came with the use of the more sensitive fluorescent antibody test for rabies diagnosis in 1959. Another noteworthy achievement in the laboratory was the development of cell culture techniques for maintenance of rabies-infected cells, allowing investigators to characterize the virus and study its ability to infect. These medical and technological advances have helped lead to our current understanding of the pathology of rabies and to its dramatic decline in developed countries during the 1940's and 1950's.
Host Range and Transmission:
The rabies virus infects a wide range of hosts, including raccoons, skunks, foxes, coyotes, bats, domesticated dogs and cats, and human beings. While infection can occur in any warm-blooded animal, some (such as foxes, coyotes and wolves) are highly susceptible, while others (such as opossums) are much less susceptible. Rabies is most commonly reported among domesticated dogs in undeveloped nations, especially in parts Africa and Asia. However, in nations where canine rabies has been controlled through mass vaccination protocols, wildlife rabies accounts for the vast majority of reported cases. In 1995, only 8% of sighted cases in the United States were in domesticated animals, while more than 50% were in raccoons, 22% in skunks, 10% in bats, and 6% in foxes.
Transmission of the virus to a human host necessitates an exposure to the virus, which nearly always occurs through a bite from a rabid animal. However, exposure does not always lead to contraction of the disease. The type of infected animal, as well as the location and severity of the bite, all appear to influence the chance of infection. For example, if left untreated, severe wolf bites on the face result in 80% to 100% mortality, while a more superficial dog bite on the leg only results in a 3% mortality rate. In general, the proximity of the bite to the head determines the risk, presumably because the virus faces a more direct route to the central nervous system. The dependence of risk on the species of the attacker is most likely due to differences in the amount of virus present in the saliva.
Despite the fact that rabid dogs only present a modest risk when compared to wolves or even cats, the vast majority of human rabies deaths worldwide (99%) result from dog bites. This is due to the imbalance of cases which occur in countries where canine rabies has not been controlled and humans and dogs live in very close contact. On the other hand, in developed nations where canine vaccinations are well established, rabies cases are dramatically reduced in number, and reported bites are almost always from wild animals such as raccoons and skunks.
While bites account for the vast majority (about 99.8%) of rabies cases, other forms of transmission have been reported, including contamination of mucous membranes, faulty vaccines, corneal transplants, and aerosol transmission. There have been a total of twenty-nine reported cases caused by non-bite exposures worldwide. Eighteen of these were due to a faulty rabies vaccine lot in 1960. Six were transmitted in the form of corneal transplants from undiagnosed patients. A total of four cases of rabies have been blamed on aerosol transmission. Two of these were in lab technicians conducting research on rabies, and the others were in individuals who had spent time in caves inhabited with large numbers of infected bats.
Symptoms and Disease Progression:
The first step in the course of rabies progression (see Figure 1) is transmission of the disease following exposure (as discussed above). Once the disease is transmitted to the individual, infection ensues. The viral infection initially remains in the muscle tissue surrounding the bite site. Several lines of evidence show that the virus remains at this site during the period of time after initial infection known as the incubation period. First, amputation of the infected limb of mice injected sub-cutaneously with the rabies virus prevents disease progression and mortality. Second, tissue and organ samples from sites other than the bite site show no detectable levels of the virus throughout the entire incubation period.
The length of the incubation period varies greatly and (like the risk of developing the disease) is primarily dependent on the location and severity of the bite, and on the amount of virus originally present at the site of infection. Typically, the incubation period lasts for one to three months, though it has been known to range from 5 days to over a year. No symptoms are present, and the precise activity and location of the virus during this period have been somewhat elusive.
The incubation period ends when the virus begins to spread from the muscle at the bite site into the surrounding peripheral nerves. The neuromuscular junctions are thought to be very important during this transition, as their physical and chemical properties appear to direct the virus to infect the nerve cells. Once it has invaded the peripheral nervous system the infection enters the prodromal period which is characterized by the onset of symptoms and the rapid and irreversible progression of the disease. The virus travels up the peripheral nerve axons to the spinal ganglia which form the junction between the peripheral and central nervous systems. Once it has infected the nerves of the spinal cord, the virus travels up the axons via retrograde transport mechanisms to the brain. The affinity of the rabies virus for neurons and their close proximity throughout the central nervous system provide an ideal environment for rapid viral spread.
The prodromal period typically lasts between two and ten days, and is characterized by nonspecific symptoms which can be mistaken for a common cold or flu. These include headache, fever, fatigue, sore throat, cough, and gastrointestinal discomfort. Occasionally the individual experiences pain or numbness at the site of the bite. However, diagnosis of rabies at this stage is nearly impossible without using invasive measures (biopsy to look for Negri bodies or viral antigen).
Diagnosis based on symptoms becomes easier as the disease progresses into the next stage, the acute neurologic period. By this point the virus has spread throughout the entire central nervous system and is beginning its centrifugal spread to other areas of the body. It is carried via more nerves to almost every organ in the body, most notably the salivary glands, which cause the animal or individual to become contagious through a bite or other exchange of mucous fluids. This stage also marks the onset of the bizarre symptoms which are typically associated with rabies.
The symptoms of the acute neurologic period may manifest themselves in one of two ways. The first, experienced by about 80% of patients, is furious rabies. This form of disease is characterized by periods of extreme anxiety, violent behavior, seizures, and hallucinations alternating with periods of calm and normal behavior, as well as periods of depression. Many of these patients exhibit the classic behavior of hydrophobia, or fear of water, which results in spasms of the throat upon drinking or even the sight or mention of liquids. A similar type of response is given if the patient feels a breeze of air through the room. The reaction in thought to be an inflated version of a natural response to protect the respiratory tract. This, combined with common paralysis of the jaw, leads to the classic foaming and drooling at the mouth of a rabid animal. The acute neurologic period lasts from two to seven days in the case of furious rabies. Sometimes the patient dies suddenly from respiratory or cardiac failure, while in most cases the violence begins to subside as disorientation and paralysis set in, resulting in coma and death.
The second manifestation of symptoms during the acute neurologic period is paralytic rabies, and is experienced by about 20% of patients. This form is particularly common in those who contracted the disease from bats. Paralytic rabies generally lacks the violent symptoms of furious rabies, though occasionally some of the same symptoms occur, such as depression and hydrophobia. This form is characterized by the slow paralysis of the patient, usually starting as a numbness or weakness at the bite site and spreading to the rest of the body over time. Patients generally live longer than those suffering from furious rabies, with progression lasting up to thirty days before paralysis of the respiratory system results in death unless life support systems are put in place. Even then, the patient will experience other complications and die eventually.
Structure and Molecular Mechanisms of the Rabies Virus:
The rabies virus has been classified as a member of the genus Lyssavirus (from Greek lyssa meaning "frenzy") within the Rhabdoviridae family (from Greek rhabdo meaning "rod-shaped"). Each virus is bullet-shaped and approximately 180 nm long and 75 nm wide. The lipid bilayer membrane is coated with numerous 10 nm spikes composed of glycoprotein. Inside the membrane is a lining of matrix protein, and a core containing the ribonucleoprotein complexes (see Figure 2).
The genome of the rabies virus is made up of approximately 12,000 nucleotides of single-stranded RNA. This contains five genes which code for each of the five viral proteins. The largest of these proteins is the L protein, or large (244 kDa) polymerase. It is a multifunctional protein, but its main activity is as a polymerase which allows the genomic RNA to be copied during virus multiplication within the cell. Between 30 and 60 copies of the L protein form the ribonucleoprotein core with the RNA genome and two other proteins N (approximately 1,800 copies) and NS (approximately 900 copies). The N protein is a 55 kDa protein which is the main structural component of the ribonucleoprotein core and binds tightly to the RNA allowing it to be packed in an orderly helical structure. The NS protein is a highly phosphorylated protein whose role has not yet been fully determined but is suspected to be involved in viral RNA transcription and/or replication. The other two proteins encoded for by the rabies genome are the M (or matrix) protein, which has been shown to be crucial in the ability of a newly formed virus to bud from the host cell membrane, and the transmembrane G protein, which is involved in host cell attachment and invasion. Also, the G protein is the main target for antibody therapy against rabies, as it is the only exposed protein on the virus and binding of antibody to it can block its function in host cell infection.
The mechanism by which the rabies virus infects a cell is similar to that of many other viruses (see Figure 3). The infection is started when G protein projections on the virus interact with the membrane of the host cell. One thing that is extraordinary about the rabies virus is its high affinity for nervous tissue. The precise property of nerve cells which the virus exploits during the infection process is not not known. However, there is some evidence that the rabies G protein can bind to the acetylcholine receptor (a neurotransmitter receptor which is expressed in high levels on some nerve cells), which suggests the virus may attach to (and subsequently infect) nerve cells more frequently than other cells.
After host cell attachment via the viral G protein, the virus is adsorbed into the cell via engulfment with the plasma membrane. Once inside the cell, the viruses congregate inside endosomes which quickly drop in pH. As the pH changes, the conformation of the G protein changes such that it causes the viral membrane to fuse with the endosomal membrane. This leads to the expulsion of viral proteins and RNA into the cytoplasm. Once in the cytoplasm, the viral L protein transcribes five mRNAs from the RNA genome using free nucleotides from the host cell cytoplasm. These mRNAs are 5'-capped and poly-adenylated, allowing them to be translated into their corresponding five viral proteins using the cell's translation machinery. These proteins also undergo post-translational modification within the host cell, including the glycosylation of the G protein and phosphorylation of the NS protein. The viral RNA genome is replicated using a complex made up of the L and NS proteins. All the viral components congregate in one location of the cytoplasm, which leads to the characteristic staining seen in the diagnostic test which looks for Negri bodies. At this site the ribonucleoprotein complex is formed and it, along with the M and G proteins, are recruited to the host cell membrane. At the membrane, M protein is implicated in the bundling of the virus into a particle and initiating the budding off process which releases new virus, ready to infect a nearby cell. Inside the neuron virus spread can occur very fast, as viral particles within the cell can simply travel the length of the axon before budding off, therefore being able to cover a large distance in less time and with fewer replication cycles. This may be one reason why the disease can spread so quickly from the peripheral to central nervous system.
Treatment and Prevention:
Official diagnosis of rabies is impossible until the onset of symptoms in the prodromal stage. The oldest method of diagnosis, the Negri bodies, do not appear until the virus has infiltrated the brain, and the same is true for the fluorescent antibody test which replaced it. A measurable human immune response to the disease is not evident until at least a week after disease onset (when only slightly more than half of all patients are still surviving). By the time a conclusive diagnosis can be made, the patient's prognosis is extremely dim. Despite exhaustive attempts with different anti rabies therapies, there have only been two recorded cases of recovery and one of partial recovery worldwide. This makes traditional procedures which use diagnosis and subsequential treatment more or less useless. Therefore, the best way to decrease mortality from rabies is to prevent the disease from infecting humans to begin with.
The most significant weapon in the fight to prevent rabies was the development of the rabies vaccine. As mentioned in the introduction, this vaccine was first made and used by Pasteur in 1885. Since then a series of rabies vaccines have been released, mostly made from strains kept in tissue culture cells. As dog bites account for an astronomical proportion of rabies cases, control of canine rabies through large scale application of one of these vaccines, as well as the capture of stray animals, is the first step in prevention of the disease. As mentioned previously, these procedures have met with extraordinary success in those countries who have implemented them. However, not all countries have the money or other resources to support such programs, so canine rabies (and the subsequent high risk for humans) remains a big problem in much of the third world.
The other key component of rabies control is the use of preexposure and postexposure prophylaxis to prevent infection and disease onset in individuals who are at high risk of contracting the virus. The concept behind these therapies is to provide an individual with an initial immune response to the virus, as the normal human response does not reach an adequate level until the disease has progressed to a lethal state. Preexposure prophylaxis involves the vaccination of individuals who are frequently at risk of exposure to rabies, including veterinarians and park rangers, or who are planning on spending time in an area with limited access to postexposure prophylaxis, such as certain areas of developing countries.
Postexposure prophylaxis is used fairly commonly (an estimated 40,000 times per year in the United States) to protect individuals after they suspect an exposure with a rabid animal. The individual should receive treatment within 24 to 48 hours of the exposure, and follow all three important components to postexposure prophylaxis. The first is thorough cleaning of the wounded area, which can decrease risk by a surprising amount. The second is the injection with human rabies immune globulin (or in some cases horse anti rabies serum) to provide an initial neutralizing agent against the virus. The third component is a series of injections of rabies vaccine, which serve to speed up the individual's natural immune response to the virus. It is not known whether the treatment destroys the virus before its initial infection, or blocks the infection early on and prevents it from spreading out of the muscle tissue. Whichever route, postexposure prophylaxis has been extremely effective, with no reported cases of disease onset among individuals who have received the treatment.
Despite the success of rabies vaccines in protection of both humans and domestic animals, one must always be aware that strains of the virus in the wild can diverge evolutionarily from the vaccine strain and render the vaccine somewhat less effective. This appears to be the case with some viral strains in West Africa where failed vaccines have been reported, and also with a strain that infects some European bats which is completely divergent in the N and G proteins (those which are most important in immunity). Therefore, it is obvious that wild strains of rabies virus must be monitored for major mutational changes, and that new vaccines must constantly be developed and tested to match these mutations.
Conclusion:
The sudden and sometimes unexpected onset of disease, along with the violent and bestial actions which are manifested in the course of its progression, and the dismal prognosis after onset of symptoms, all make rabies a terrifying disease for humankind. Therefore, it is a disease that we should make efforts to prevent or even eliminate. An understanding of the biological basis for its transmission and pathological implications have allowed for the development of appropriate control and treatment programs. Strict vaccination procedures for domestic animals and postexposure prophylaxis for humans have lead to a drastic decrease in rabies cases in many developed countries, demonstrating that prevention (and possibly even elimination), is possible.
Continued research on different strains of the rabies virus, as well as the human immune response (or lack thereof), could lead to the development of more powerful or efficient vaccines. There are also several mysteries of rabies still left unsolved, such as the incidents taking place during the long incubation period, and the molecular mechanism by which the virus specifically attacks the nervous system. Insight into either of these topics might lead to a method of earlier diagnosis or a brighter hope for recovery for those patients who have already started to show symptoms.
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