The Rabies Virus

Amanda K. Marr

Biochem 316

Due: 5/21/99

The Rabies virus, which is most commonly associated with routine pet vaccinations, has caused turmoil all over the world since at least the times of Homer. It wasn’t until the late 19^th century, during the time of Pasteur, that it really began to be understood. Since then, vaccine prevention has been available for humans and animals but a cure for the disease that can kill in a matter of weeks has not yet been found. This work provides specifics about the virus, its diagnosis, how the prevention works, and information on the locations where it is most prevalent.

Rabies—The Virus

In the beginning of time, Homer’s Iliad refers to rabies when he mentions Sirius, the Dog Star of Orion, exerting a malignant influence on the health of mankind. Sirius was associated with mad dogs throughout Egypt, Rome and the eastern Mediterranean. The Greeks had Aristaeus, son of Apollo, to counteract the effect of rabies. Artemis is also represented as an early healer of rabies. (Baer, Volume 1, 1975) These thoughts and ideas predated Christ showing that this virus has had quite an affect on people for thousands of years.

The word “rabies” comes from the Sanskrit word rabhas which means, “to do violence.” The Greeks called the disease Lyssa or Lytta, meaning madness. It was described as causing hydrophobia in a sick person whom has at the same time a thirst and fear of water. Cardanus, a Roman writer, described its infectivity of the saliva of rabid dogs. It was these early writers that described the infectious material as a poison--virus in Latin. (Baer, Volume 1, 1975)

A virus particle—virion--is a nucleic acid molecule that is encased by a protein capsid. The capsid may or may not be covered by a glycoprotein-containing envelope derived from the host cell membrane. Helical viruses are constructed by the coat protein subunits forming helical tubes and spherical viruses have polyhedral shells.

The rhabdovirus, of the Lyssaviruses, is a bullet-shaped virus. Rabies virus particles are helical viruses best described as cylindrical with one round or conical end and one planar or concave end. From the surface inward, they have glycoprotein (G) surface projections that extend through the lipid membrane envelope and a helical ribonucleoprotein capsid that contains linear, single-stranded RNA genome of negative sense. The surface spike projections are 6 to 8nm long. This layer does not cover the planar end of particles. Each projection has a knoblike distal end and is placed on the virus surface at 4 to 5nm intervals. They may be randomly spaced or arrayed in rows to give a honeycomb appearance, which may reflect symmetry beneath. (Baer, Volume 1, 1975) The virus envelope’s glycoproteins have a mass of 80,000 daltons that are associated with the projections. The protein is 40,000 daltons and the glycolipid is 25,000 daltons. (Velleca, 1981) The picture below is diagram of a rabies particle. (Baer, Volume 1, 1975 p36)

rabies particle picture

The rabies virus has 30-35 coils of a strand of ribonucleoprotein. These coils form a cylinder 50nm wide and approximately 165nm long. This helical core is immediately beneath the membrane envelope. When the virus is disrupted with detergents, the single-strand wavy ribbon has a length of4.2um, thickness of 2x6.5nm, and periodicity of 7.5nm. (Baer, Volume 1, 1975)

The mode of morphogenesis involves budding upon host cell membrane with a concomitant coiling of the nucleocapsid from a less organized strand in the cytoplasm. The mature virus particle pinches off after the coiling is complete. The surface projections occur simultaneously with the budding and never extend further than what will be the envelope. (Baer, Volume 1, 1975) These glycoprotein projections are the only proteins that are capable of reaction with virus-neutralizing antibody (VNA), which has lead to evidence that this protein can be used for protection in animals against rabies (Wiktor, 1984).

The invasion of the rabies virus was studied by Iwasaki in 1973. Within 5-30 minutes after addition of the virus to cell cultures, the particles approached cell surfaces and some were engulfed by phagocytic vacuoles. The viral envelope fusion occurred with plasma membrane and vacuolar membranes. Fusion begins with the planar end of the particles. No morphogenetic events occur until five hours after the infection. The budding of the plasma membrane occurs after six hours. (Baer, Volume 1, 1975)

The virus infects the cell by viropexis, an enveloped dissolution of the envelope resulting in plasma membrane fusion. During morphogenesis, a large amount of ribonucleocpasid is produced. It forms unbounded collections of nucleocapsid called inclusions within the cytoplasm. These inclusions can be seen as matrices in the cytoplasm under the electron microscope or when stained by histological techniques as acidophilic Negri bodies containing basophilic granules under a light microscope. These are probably some cytoplasmic organelles trapped within the inclusion. (Velleca, 1981)

The envelopment of the nucleocapsid to form an intact virus particle takes place on the endoplasmic reticulum and plasma membrane of cell. However, central nervous system (CNS) envelopment occurs along the intracytoplasmic membranes. Studies in hamsters and skunks injected with virus in the hind leg indicates that the virus first replicates in muscle tissue at site of exposure and next infects the neuromuscular and neurotendinal spindles near the area of exposure. The virus later reaches the peripheral nerves and dorsal root ganglia where it ascends the spinal cord to the brain. The brain neurons become infected and in all cases, infection in the CNS is neural, involving ganglion cells but not satellite cells and axons but not Schwann cells. Virus antigen can be found in cytoplasm of the neurons. After reaching the brain, virus spreads centrifugally along neural pathways to many tissues and organs of the body. (Velleca, 1981)

There are several stages to the infection. If a rabid animal bites another, an exposure occurs. If the exposure results in inoculation of rabies virus into the animal, an infection occurs. Then, in a certain number of infected individuals, clinical symptoms or sickness will develop. The infection only occurs if the saliva of a biting animal contains the virus. (Baer, Volume 2, 1975)

Susceptibility varies with species. The most at risk are foxes, coyotes, jackals, wolves, and certain rodents. Those with a high risk are skunks, raccoons, bats, rabbits, cattle, and some members of the Felidae and Viverridae families. At moderate risk are domestic dogs, sheep, goats, horses, and nonhuman primates. At the lowest risk are all birds and primitive mammals including the opossum. Cats are more resistant to infection of the naturally occurring virus but are more prone to infection if bitten. Typically, younger animals are more susceptible than older. There has also been some evidence of virus transmission from exhaled or excreted sources. This has been suggested in large colonies of cave-dwelling bats and in a lab outbreak in 1972. There is also risk in getting the virus through the ingestion of infected tissue or transplacental. (Greene, 1990)

Clinical rabies has five stages. The incubation period is about 9 days to 19 years, typically 20-60 days. It can be shorter if the bite was on the head or in a child. People who have received postexposure treatment rather than no treatment also have a shorter incubation period. During this time, the patient is entirely well except for symptoms related to local wound healing or postexposure treatment. The second stage is called Prodrome. It shows the first symptoms including malaise, anorexia, fatigue, headache, and fever. The third stage, Acute Neurologic Phase, involves the development of objective signs of nervous system. Symptoms such as hyperactivity, disorientation, hallucinations, seizures, bizarre behavior, nuchal stiffness, and paralysis are evident. The hyperactivity includes periods of agitation, thrashing, running, biting or bizzare behavior up to five minutes. Between the outbursts the patient is relaxed and cooperative but anxious. Many patients exhibit hydrophobia. Unless the patient dies abruptly, paralysis comes on and brings on coma in stage four. This could mean hours to months during which time death is usually the result of complications suffered at this time. Complications such as cerebral swelling, hormonal imbalances, oxygen deficiency, respiratory arrest, bacterial pneumonia, irregular heartbeat, congestive heart failure, abnormally low blood pressure, blood clots, low body temperature, exceptionally high temperature, secondary bacterial infections, and gastrointestinal bleeding may lead to death. The fifth stage is recovery of which only a few have been documented. (Baer, Volume 2, 1975)

Human diagnosis can be found from the virus being isolated from antemortem human saliva on 4^th, 5^th, 6^th, 10^th 12^th, and 24^th day of illness, from cerebrospinal fluid on 7^th and 14^th day and from urine sediment on the 6^th day. Isolated postmortem specimens, in which death occurred early, might be gone after 10-14 days due to lack of neutralizing antibodies. (Baer, Volume 2, 1975) Postexposure treatment involves administration of rabies immune serum to provide antibodies during the first week of host’s immune system relating to infection.

Rabies—The Detection

The virus will remain “alive” in a body for less than 24 hours if kept at 20^oC. It can survive days if the body is refrigerated. Virus survival will increase if it is unrefridgerated and stored in 50% glycerol at room temperature, or it can be stored in pure glycerol at 4^oC. The survival rate is enhanced if a 20% suspension of infected tissue or virus culture is made with solution high in protein or amino acids. Storage for long periods of time (years) should be at ultra-low temperatures (-30^o- -80^oC) as untreated fresh-frozen tissue. (Greene, 1990) This, however, is not important for the proper diagnosis of the virus. The most popular technique for diagnosis used is immunofluorescence testing and does not depend on presence of viable viral particles.

According to Velleca & Forrester (1981), “any animal suspected of having rabies should be sacrificed immediately and sent to the laboratory for examination.” This is still true today for all wild and stray animals, but most domestic animals are allowed to have a ten day observation and quarantine period during which time the virus may appear. If it does not, the animal is free to go on with its life. If symptoms do develop the animal must be sacrificed and tested. (Texas Department of Health Zoonosis Control Division, 2/1/99) These laws vary from state to state.

A wild animal should be killed and decapitated in the field. It should be shot through the heart, not the head. The head needs to be immediately refrigerated and submitted to the lab. The rest of body should be incinerated. The entire body of a small animal is to be sent to the lab. Specimens should be placed on wet ice and delivered by messenger. (Velleca & Forrester, 1981) Most veterinary practices will give a lethal injection, do the decapitation, and submit the proper sample to a lab if desired.

At the lab, the brain gets removed and/or submaxillary salivary glands. Detection of the virus is by direct immunofluorescence (IF) technique developed in the 1950s. It is based on an antibody in gamma globulin fraction of a serum being labeled with fluorescent dye and its ability to react with the specific antigen. When fluorescent-labeled rabies antibody comes in contact with rabies antigen on a slide, an antigen-antibody reaction occurs. The sites of the specific reaction can be detected by fluorescence of the dye coupled to the antigen-antibody complex. No fluorescence is present in a negative reaction. The antibody that is responsible for staining in the IF test is the one that is directed against the nucleocapsid antigen of the virus. (Velleca & Forrester, 1981)

In immunofluorescence testing, an immunofluorescence antigen is located. It will be detectable in the brain at any stage of the disease during which the virus can be transmitted. For this test, the portions of the brain that are used are the medulla, cerebellum, and hippocampus. An impression is made on microscope slides by pressing the slide firmly against the cut surface of the brainsection. This impression is allowed to dry for thirty minutes at room temperature before the specimen is fixed. Acetone fixation increases the permeability of the cell and makes rabies antigen available for staining. Preparations that are not fixed properly may miss the antigen. Fixation involves the slides setting in acetone at –20^oC for 4 hours. A longer period of time is not harmful. (The acetone used should then be considered infectious.) Impressions are to be ringed with heavy ink or nail polish to contain the conjugate. These impressions are covered with absorbed conjugate, and the slides are placed in a humidified chamber. They are incubated at 37^oC for thirty minutes, and then rinsed of conjugate in phosphate buffered saline (PBS), pH 7.7. They should be soaked in another PBS bath for 10 min, and then rinsed with distilled water. Slide mounting involves buffered glycerol mounting medium, pH 8.5, and covering it with a coverslip. (Velleca & Forrester, 1981)

Another diagnostic test is the mouse inoculation test. It is the most sensitive method. Three to four week old female Swiss mice are most preferable especially those that are free of other viruses. Pieces of tissue from the sample brain stem, hippocampus, and cerebellum are taken from both sides of the brain. A total of about 3-4g are blended with a mortar and pestle to a paste. The suspended material is placed in a phosphate buffered saline, pH 7.2 that contains antibiotics, and a final concentration of 0.75% bovalbumin fraction V, or 2% inactivated horse serum, guinea pig serum, or hamster serum to stabilize the virus. The serum should be at 4^oC before added. The suspension must be kept cold until inoculated. Following preparation, the mice are anesthetized with ether and inoculated intracerebrally with 0.3ml of suspension. The mice are observed daily for signs of the disease, or are sacrificed and their brains are immunofluorescence tested. (Velleca & Forrester, 1981)

A histological examination for rabies-specific Negri bodies can also be done. Negri bodies are small intracellular inclusions (0.25-27um) that appear in nerve cells of animals infected with rabies. They may appear as an intracytoplasmic inclusion but may appear outside the cytoplasm in impressions or smears of material when a cell has been mechanically disrupted. They may be round, ovular, ameboid, triangular, or oblong and are found mainly in the pyramidal cells of Ammon’s horn, in the Purkinge cells of the cerebellum and in the cells of the medulla and various ganglia. They are acidophilic in staining reaction and turn a pink or purplish-pink in differential stains that use basic fuchsian or eosin with methylene blue as their base. Sellers’s staining technique is simplest, fastest, and most economical technique available. Negri bodies turn magenta red and have small (0.2-0.5um) dark-blue interior basophilic granules. Nonspecific inclusions found in dogs, foxes, cats and white lab mice are acidophilic and may be mistaken for Negri bodies but have following distinctions.

1. Negri bodies contain basophilic granules, whereas non-rabies inclusions have

no internal structures.

2. Negri bodies have a heterogeneous matrix; non-rabies inclusions have an

homogenous matrix.

3. Negri bodies have a magenta (heliotrope) tinge; non-rabies inclusions are

pinker (more acidophilic).

(Velleca & Forrester, 1981)

Rabies—The Prevention

Cures and preventions were tried for many hundreds of years to control this disease. It was difficult because no one was sure what it was. A cure that was tried in ancient medical times involved the attachment of the tongue (lingual frenulum) being cut and a fold removed in which “the worm” (the lyssa) was thought to be. It was this worm that was thought to cause the disease. This was a widely accepted idea until Pasteur proved differently. (Baer, Volume , 1975)

Pasteur developed the first rabies vaccine in 1885. He used several doses of a suspension of desiccated rabbit marrow that had been extracted from an animal infected with the rabies virus. This vaccine enabled Pasteur to cure a young boy who had been bitten by a rabid dog. (Perez, 1997) Since then a variety of vaccines have been produced and have successfully limited the spread of the disease among domestic animals and people.

The information that is valuable to the vaccine industry involves the following. The glycoprotein G-surface antigen elicits production of serum neutralizing antibody to afford protection against the disease. Slight changes in amino acid substitution in glycoprotein cause the virulent virus to be nonpathogenic and produce milder CNS lesions due to loss of neutralizing activity by antibody to this protein. As with any enveloped virus, the rabies virus can be destroyed by various concentrations of formalin, phenol, halogens, mercurials, mineral acids, and other disinfectants. It is also extremely labile when exposed to UV light and heat. (Greene, 1990)

There are several types of vaccines depending on how they are made. First generation vaccines are made using animal substrate to produce a viral mass. An example of this is one that is made with adult animal nerve tissues, embryos, or a suckling animal’s brain. Inactivation of the virus is done using phenol, ether-phenol, β-propiolactone, or ultra-violet light. Using animal nervous tissue is risky because it may still contain residual live virus, which is due to deficient inactivation with phenol. There may also be post-vaccinal encephalomyelitic reactions that are caused by the encephalitogenic factor--a basic protein associated with myelin. The reactions range from slight temporary parenthesis to permanent neurological injury to death. There have also been problems with low antigen content per dose and have stability for only six months. These vaccines are used in humans and come in a liquid form. (Perez, 1997)

Another example is vaccines made from embryos. Those of ducks produce much safer vaccines for humans. They have a low incidence of neurological reaction but may produce a local reaction. Chicken embryos are used to produce vaccines for dogs, cats, and cows. Vaccines that are produced using suckling animal brain are used most commonly in Latin America and some parts of Africa for pre- and post-treatment of animals and humans. They are made by inoculating one-day-old mice and inactivating the virus with ultraviolet radiation. (Perez, 1997)

Second generation vaccines are those produced in cell cultures. The first group uses primary mammalian cell cultures such as hamster kidney, dog kidney, fetal calf kidney, chick embryos, or quail embryos. The second group uses diploid cells of human or monkey origin. The third group uses heteroploid cell cultures. These are widely used for veterinary prevention. General steps for the production of these vaccines follows.

1. Cell culture and amplification using spinner flasks or roller bottles.

2. Infection and viral multiplication using polyions and optimizing extrinsic factors.

3. Concentration and purification using ultrafiltration in a continuous density gradient.

4. Inactivation using β-propiolactone, or ultra-violet light.

5. Stabilization and lyophilization.

(Perez 1997)

An example of a cell culture vaccine is Rabguard-TC® by Phizer Animal Health. It is prepared by growing a cell-culture adapted rabies virus in an established porcine cell line. It is chemically inactivated and combined with an adjuvant. It is packaged as liquid and used for the vaccination of healthy dogs, cats, cattle, horses, and sheep. A single dose is given at three months or older and repeated one year later. Dogs and cats get revaccinated every three years and cattle, horses, and sheep annually. Auto-immune reactions may occur with repeated doses.

Another vaccine that is used in dogs and cats 3 months of age or older is Phizer’s Defensor ® 1. It is a cell-culture-grown and chemically inactivated rabies virus. The seed virus is a highly immunogenic, fixed strain of the rabies virus which originated from Pasteur’s original isolate in 1882. The inactivated virus is also formulated with a highly purified adjuvant and is packaged in liquid form. A protective immune response may not be elicited if animal is incubating an infectious disease, is malnourished, parastitized or is stressed due to travel or environmental conditions. Also a response will be lacking if the animal is immuno-compromised or the vaccine is not administered in accordance with label directions. These rules are usually in affect for all vaccines and medicine.

Human Diploid Vaccine Cultures (HDVC) are relatively new examples of the cell culture vaccines. They have proven valuable because they elicit a good antibody response, multiple injections induce high antibody levels, and a single booster inoculation given to a previously vaccinated subject results in the rapid formation of high-titered antibodies. (Cox & Schneider, 1976) These are the qualities that every vaccine should have. Cox and Schneider did research on the quality of this type of vaccine and found that regardless of the primary schedule of inoculations and the antibody titer present at the time of booster inoculation, a single 0.2ml intradermal injection is sufficient to stimulate the development of high-titered virus neutralizing antibodies. In case of acutal exposure, a single booster inoculation would seem to be sufficient for the postexposure treatment of man. This evidence supports recommendations of the World Health Organization Expert Committee on Rabies(1973).

Research has been done with recombinant glycoprotein genes by Wiktor in 1984. The inoculation of rabbits and mice with a vaccinia-rabies glycoprotein recombinant (V-RG) virus resulted in rapid induction of high concentrations of rabies virus-neutralizing antibodies and protection from severe intracerebral challenge with several strains of rabies virus. Effective immunization by V-RG depended on expression of rabies glycoprotein that registered proline rather than leucine as 8^th amino acid from its NH₂-terminus (V-Rgpro8). The research showed that effective immunization of mice was 10⁴ plaque-forming units of V-Rgpro8 virus. β-propiolactone inactivated the preparations of V-RGpro8 which also induced high levels of rabies virus-neutralizing antibody and protected mice against intracerebral challenge with street rabies. It was also shown to be highly effective in priming mice to generate a secondary rabies–specific cytotoxic T-lymphocyte (CTL) response following culture of lymphocytes or PM strains. V-Rgpro8 did not prove to have ability to induce CTL memory specific for rabies glycoprotein but did induce primary vaccinia specific CTL response. (The CTL response is one of the most important defense mechanisms the body has because it can stimulate an immune response upon recognition of the virus.) This may be evidence of some form of immunodominance, but the mechanisms are unclear.

According to Baer (Volume II, 1975), postexposure treatment uses the equivalent of 2ml of a 5% brain-tissue vaccine or a dose recommended by the producer given daily for 14 consecutive days. Additional boosters are given at 10, 20 and 90 days after the last dose in all cases to ensure production and maintenance of high levels of serum-neutralizing antibodies. At this time, the combined serum-vaccine is the best specific treatment available for postexposure prophylaxis of rabies in man. The vaccine alone is sufficient for minor exposure. The serum should be a single dose of 40IU per kg of body weight for heterologous serum and 20IU per kg of body weight for human antirabies immunoglubulin. The first dose of vaccine is given at the same time as the serum but at another site. Treatment should be started as early as possible after exposure and should not be denied regardless of whatever time interval has elapsed.

Additionally, wound treatment should include immediate washing and flushing with soap and water, detergent, or water alone. This should be followed by a rinse with 40-70% alcohol, tincture or aqueous solutions of iodine, or 0.1% quaternary ammonium compounds.

There is some evidence that the rabies vaccine can cause a type of cancer—a sarcoma—in a small percentage of cats. No similar reaction has been noted in dogs. The numbers are one in every 2,500-10,000 cats will get cancer. It is thought that the vaccine induces a form of inflammation that can turn on an oncogene to cause the cancer. Although this is a risk, the rabies vaccine is still highly recommended because the risk of getting rabies is much greater unless the cat is completely isolated from all outdoor animals. (Gustafson, 2/1/99)

Rabies—The Worldwide Distribution

The epizootiology, the study of the spread of a disease in animal populations, of rabies is a very popular research topic. Overall, there seems to be a pattern because it occurs in waves throughout history. The current epidemic in Europe began south of Gdansk in Poland in 1939 and has since spread 1,000 miles across the northwest. The map below gives an indication of the European spread between 1940 and 1979. (MacDonald, 1980)

European map 1940-1979

Following is a table of cases observed in West Germany between 1965 and 1972. And the countries that were then infected. (MacDonald, 1980 p13)

Country	1965	1966	1967	1968	1969	1970	1971	1972
West Germany	3910	3661	4373	4353	3917	2036	2214	2524
Denmark	52	1	0	1	71	83	0	0
Austria	0	8	79	173	93	116	213	78
Belgium	0	40	326	453	161	20	4	7
Luxembourg	0	49	294	31	12	11	0	0
Switzerland	0	0	193	713	393	295	353	499
France	0	0	0	63	334	513	896	1027

According to Schering-Plough more than 30,000 people in the United States undergoes treatment every year for possible exposure from domestic animals. In 1990, 4,881 cases of animal rabies were reported to the Center for Disease Control by all 50 states, District of Columbia, and Puerto Rico.

Meltzer & Rupprecht compiled some statistics on the occurrence of rabies worldwide and found that in the 1950s, the incidence of rabies was 0.2 to 2.5/1000 dogs (0.22 to 3.8/1000 of unvaccinated dogs) in the United States. In the 1960s, the incidence of rabies in the Philippines was 0.4 to 1.0/1000 dogs and of Ecuador was 1.5/1000 dogs. (Meltzer & Rupprecht, 1998)

Developing countries have a low vaccination rate. For example, a 1990s survey of Turkey showed at least 40% of owned dogs had not been vaccinated. These countries are also known for numerous unowned dogs. However, a 1980 survey in Yuba County of California found that only 20% of dogs and cats had up-to-date vaccinations. So it is not just those who don’t have the means who don’t get the treatment. The World Health Organization (WHO) recommends a minimum of 70% of the dog population to be effectively vaccinated to block transmission. (Meltzer & Rupprecht, 1998)

Dog populations in North America and Europe are about 9-16% of that of the human population. Africa, Asia, and Latin America has approximately 12.5% of the human population. Studies from Tunisia and Turkey found 60-80% of all dogs roam freely, but only 15% of these were ownerless. Malawi has up to 95% of the dogs roam free. There is no evidence that the removal of dogs has ever had a significant impact on dog population densities or the spread of rabies. (Meltzer & Rupprecht, 1998) This was once thought of as a possible control method.

In 1980, California had one rabid dog cause a local health department to locate and/or destroy 300 unclaimed cats and dogs and provide a clinic to vaccinate 2,000 dogs. Clinic costs totaled $4,190 and additional costs were $8,950. This came to a cost per pet for 2,300 animals of $5.70. As of 1993, the average veterinary cost for a rabies vaccine in the United States is $0.50 per dose. (Meltzer & Rupprecht, 1998)

In 1995, about 50% of all wildlife rabies cases in the United States were associated with raccoons. Europe has the most problem with Red foxes which had 5,800 confirmed cases in 1994. These have been documented to spread at a pace of 20-60km per year. Vampire bats in Latin America cause the most serious rabies problem in livestock. Latin America has suffered losses of $30 million from 100,000 rabies-related deaths. For this wild animal control, there has been the use of oral vaccines for fox and raccoon rabies but there is no economic data to support its cost effectiveness. (Meltzer & Rupprecht, 1998)

Some of the areas free from rabies are the United Kingdom and Hawaii which have mandatory quarantine periods for all incoming cats and dogs regardless of vaccine status. France has cattle vaccinated routinely against rabies because of the risk of exposure from bats. (Meltzer & Rupprecht, 1998) The following page is a worldwide map of the animals that are the principal vectors in major regions of the world as of 1980. Australia, British Isles, Antarctica, and most of Scandinavia are free of rabies. (Greene, 1980)

world map

According to Rupprecht, the source of the disease has changed from domesticated animals to wildlife, principally raccoons, skunks, foxes and bats. Human deaths have declined to average about 1-2 per year.

Rupprecht’s theory of rabies migration involves pathogen migration during the exchanges of fauna and human populations over the Bering Strait 50,000 years ago. He gives some folklore evidence of a rabies-like sickness among native people through Pacific Northwest. Records at the time of the Spanish conquest in Middle America associate vampire bats with human illness. However, the first indication of terrestrial rabies did not surface until 1703 in the California area. Then, a dog and fox outbreak was reported in the mid-Atlantic colonies throughout the late 1700s and was probably due to the introduction of dogs and red foxes. (These were imported for British-style fox hunting throughout New England in the 1800s.) Fox rabies epizootic ensued and spread to the eastern United States by the 1940s –1950s. Skunk were also frequent vectors throughout the western states in the 19^th century. The highest record of human deaths from rabies is 143 in 1890. However, it wasn’t until 1938 that rabies in humans and other animals became a nationally reportable disease. At that time the total rabies cases were 9,412 per year and caused 47 human deaths. These are probably underestimated because surveillance was limited and sensitive diagnostic tests for human and animal rabies were not developed until the mid-1950s. (Rupprecht, 1998)

In the 1920s, the United States prevention began with vaccination programs, stray animal removal, and leash and muzzle ordinances. In 1971, rabies was reported for the first time as affecting all 48 states and Alaska. The rabies reservoir was now found worldwide. (Rupprecht, 1998)

Following is a table of human rabies cases in United States reported by exposure Category from 1946-1995(through Oct.) (Rupprecht, 1998)

Exposure Source

Years	Domestic	Wildlife	Other	Unknown (5)	Case total
1946-1955	86	8	0	26(22)	120
1956-1965	21	7	0	10(26)	38
1966-1975	6	7	1	2(13)	16
1976-1985	6	1	2	11(55)	20
1986-1995	2	2	0	14(78)	18

Rupprecht estimates the cost of prevention to be $230 million to $1 billion per year. The cost of postexposure treatment which consists of rabies immunoglobulin and five doses of vaccine given over a 4-week period exceeds $1,000 per case.

Currently, the United States is trying to control raccoon and skunk rabies. Greenwood, Newton, Pearson, and Schamber did surveillance work on the movement patterns of healthy and rabid skunks. They observed a total of 102 striped skunks (Mephitis mephitis) from March to July of 1991 and 1992 in Stutsman County, North Dakota. They detected no differences in 1992 between healthy and rabid skunks in average rate of travel, distance traveled, or home range size during half month periods. Among rabid skunks, the rate of travel tended to decrease from about 298 m/hr during the 14 days preceding the clinical period of rabies to about 172m/hr during clinical period. The average distance traveled at night also decreased from about 2,318m pre-clinical to about 1,297m clinical. The average home range size of males (2.8m) was greater than of females (1.2m) during the pre-clinical period but the clinical period home range sizes were similar (1.8m). They detected no correlation between locations of animal found dead of rabies and dates of death.

Pobojewski of the University of Michigan has observed the raccoon migration into northeast Ohio and assumes it will move into Michigan. Raccoon rabies first appeared in Florida in the 1950s and has been moving north through the mid-Atlantic states and New England ever since. According to Segelken, the virus made a leap north in the late 1970s when 3,500 raccoons were transported from Florida to Virginia. According to Pbojewski, before 1991, no rabid animals had been identified in Connecticut for more than a decade, but over the next four years, Connecticut officials reported 2,612 rabies-positive animals. Most of these were raccoons of which 80%+ were found in property owner’s yards. During the same period, 939 people were exposed to virus by handling a pet that had just fought with a rabies-positive animal. The first documented case in northeast Ohio occurred in April 1997, according to Ohio Department of Health. Since then, 75 additional cases have been recorded. Officials are trying an oral vaccine program to create a firewall, but problems arise when people trap and release animals several miles away or when raccoons stowaway in garbage trucks.

Segelken states that a northward spread of raccoon rabies can be halted by these vaccination barrier zones. This is according to veterinarians and wildlife biologists at Cornell University College of Veterinary Medicine. The oral rabies vaccine, Raboral, is a capsule that is concealed in flavored baits and dropped from aircraft or distributed by hand in populated areas. The same vaccine has been shown to control rabies in coyotes and red and grey foxes. The scientists plan to take a regional approach of New York and Vermont first, then the northeastern and southern states. The Appalachian Mountains should serve as a natural barrier. To be cost effective, the team uses the smallest possible number of baits per square mile and vaccinates in the fall instead of biannually. They are also testing to find a favorite raccoon bait.

Krebs, Strine, and Childs also did some United States surveillance work. They found one strain of the virus in raccoons, two strains in skunks—one in North Central state and California and the other in South Central states—and 3 in foxes. There are 5 antigenic variants or strains of rabies that are currently recognized for terrestrial animals in the 10 epizootics in US. There is also a distinct strain for bats vs. terrestrial carnivores.

Following are several graphs indicating the United States current problems with rabies. The first shows the increase in cases of animal rabies from 1955-1992. The next two break it into wild animal cases to show the increase and domestic animal cases to show the decrease. (Krebs, Strine & Childs, 1992)

three graphs-Rabies 1955-1992

Wild Animal Cases

Domestic Animal Cases

Finally, this is a more focused map of the United States and the locations of the current epidemics among the dominant species. (Krebs, Strine & Childs, 1992)

United States Map

Current research on this virus involves the development of new and better vaccines for post-exposure treatment. One such vaccine is the DNA vaccine that is made by injecting genetic material into the body to produce proteins which would in turn prompt an appropriate immune response. It passed the tests on monkeys with all of the vaccinated monkeys developing anti-rabies antibodies; when exposed to the disease six months later, they developed no signs of the disease. (Manning, 1/14/99) This vaccine is given high hopes of providing a blueprint for vaccines against HIV, hepatitis, and other illnesses. (Verrengia, 1998)

Other research being performed by Thomas Lentz of Yale University is on the receptors. Specifically, he is looking at the nicotinic acetylcholine receptor which appears to be the host cell receptor for the rabies virus. He is looking at a way of blocking these receptors to prevent infection.

In conclusion, the rabies virus is still a worldwide problem. Its solutions seem to come from its prevention because death from the infection is almost inevitable. Perhaps the virus will someday have more that just vaccines to fight it off and maybe even a cure.

Bibliography

Aiello, Susan E. ed. The Merck Veterinary Manual 8^th ed. Merck & Co., Inc. Whitehouse Station, NJ: 1998.

Baer, George M. The Natural History of Rabies. Vol. 1. New York: Academic Press. 1975.

Baer, George M. ed. The Natural History of Rabies. Volume 2. New York: Academic Press. 1975.

Cox, James H. and Lothar G. Schneider. “Prophylactic Immunization of Humans Against Rabies by Intradermal Inoculation of Human Diploid Cell Culture Vaccine.” Journal of Clinical Microbiology. Feb. 1976: 96-101.

Greene, Craig E. Infectious Diseases of the Dog and Cat. W.B. Saunders Company. Philadelphia. 1990.

Greenwood, Raymond J., Wesley E. Newton, Gary L. Pearson, & George J. Schamber. Population and Movement Characteristics of Radio-Collared Striped Skunks in North Dakota During an Epizootic of Rabies. <http://www.npwrc.usgs.gov/resource/othrdata/skunks/skunks.htm> Located: 2/1/99.

Gustafson, Blair. Vaccine-Induced Cancer In Cats. The Country Internet Vet: Dr. Blair Gustafson. <http://wavecom.net/inetvet/vaccinecat.htm> Located: 2/1/99.

Krebs, John W., Tara W. Strine, James E. Childs. “Rabies surveillance in the United Stated during 1992.” Journal of the American Veterinary Medical Association. Vol.203. No. 12. Dec. 15, 1993. p1718-1731

Lentz, Thomas L. “Cell Biology at Yale. Faculty: T.L. Lentz” <http://info.med.yale.edu/cellbio/Lentz.html> Published Sept. 8, 1997. Located: 2/1/99.

MacDonald, David W. Rabies and Wildlife: a biologist’s perspective. New York: Oxford University Press. 1980.

Manning, Anita. “DNA-based rabies vaccine passes tests.” USA Today Health. <wysiwyg://90/http://www.usatoday.com/life/health/general/lhgen013.htm> Published: 1/14/99. Located: 2/1/99.

Meltzer, Martin I. and Charles E. Rupprecht. “A Review of the Economics of the Prevention and Control of Rabies. Part 2: Rabies in Dogs, Livestock and Wildlife.” Pharmacoeconomics. Nov. 14, 1998: 481-498.

Perez, O. and CC Paolazzi. “Production Methods for Rabies Vaccine.” Journal of Industrial Microbiology and Biotechnology. Vol. 18 No. 5. 1997: 340-347.

Phizer. Rabies Vaccine-Killed Virus: Defensor ® 1

Phizer Animal Health. Rabguard-TC® <http://www.pfizer.com/ah/level3/tref/merch/prgd107.html> Located 2/1/99.

Pobojewski, Sally. Raccoon rabies headed toward Michigan. <http://www.umich.edu/~newsinfo/U_Record/Issues98/May06_98/raccoon.htm> Located:2/1/99.

Rupprecht, Charles E. et.al. The Ascension of Wildlife Rabies: A Cause for Public Health Concern or Intervention? <http://www.columbia.net/phys/rabies.html> Located: 2/1/99.

Schering-Plough Animal Health Corp., Rabies Vaccine—Killed Virus. 1998

Rabdomun®1—use in dogs and cats only

Segelken, Roger. Wildlife Rabies Vaccination Barrier Zones May Be Working. <http://www.cornell.edu/News/extension_news/en199710/en19971024.html> Located: 2/1/99.

Texas Department of Health Zoonosis Control Division. Animal Bites. 9/94. <http://www.ipt.com/bas/bites.htm> Located: 2/1/99.

Wiktor, T.J. et.al. “Protection from rabies by a vaccinia virus recombinant containing the rabies virus glycoprotein gene.” Proc. National Academy of Science. Nov. 1984: 7194-7198.

Velleca, Wallis M., & Francis T. Forrester. Laboratory Methods for Detecting Rabies. Atlanta, Georgia: U.S. Department of Health and Human Services, Public Health Services, Center for Disease Control. 1981.

Verrengia, Joseph B. “New Rabies Vaccine: New DNA Rabies Vaccine Passes Tests.” Acme Pet-DNA Rabies Vaccine. <http://www.acmepet.com/content/health/rabies.html> 1998. Located:2/1/99

Voet, Donald & Judith G. Voet. Biochemistry 2^nd ed. New York: John Wiley & Sons, Inc. 1995.