Rhabdoviruses: Rabies Virus
Charles E. Rupprecht
Rabies virus causes acute infection of the central nervous system. Five general stages are recognized in humans: incubation, prodrome, acute neurologic period, coma, and death. The incubation period is exceptionally variable, ranging from fewer than 10 days to longer than 2 years, but is usually 1-3 months.
Rabies virus is a rod- or bullet-shaped, single-stranded, negative-sense, unsegmented, enveloped RNA virus. The virus genome encodes five proteins.
Classification and Antigenic Types
Placement within the family is based on the distinctive morphology of the virus particle. Cross- reactive nucleoprotein antigens or comparative genomic sequences determine inclusion in the genus Lyssavirus, which includes rabies virus and at least five other pathogenic rabies-like viruses.
The viral RNA uncoats in the cytoplasm of infected cells. The genome is transcribed by a virion-associated RNA-dependent RNA polymerase. Viral RNA is then translated into individual viral proteins. Replication occurs with synthesis of positive-stranded RNA templates for the production of progeny negative-stranded RNA.
After inoculation, rabies virus may enter the peripheral nervous system directly and migrates to the brain or may replicate in muscle tissue, remaining sequestered at or near the entry site during incubation, prior to central nervous system invasion and replication. It then spreads centrifugally to numerous other organs. The case:fatality ratio approaches unity, but exact pathogenic mechanisms are not fully understood.
Susceptibility to lethal infection is related to the animal species, viral variant, inoculum concentration, location and severity of exposure, and host immune status. Both virus-neutralizing antibodies and cell-mediated immunity are important in host defense.
Rabies occurs in nearly all countries. Disease in humans is almost always due to a bite by an infected mammal. Nonbite exposures (e.g., mucosal contact) rarely cause rabies in humans.
Early diagnosis is difficult. Rabies should be suspected in human cases of unexplained viral encephalitis with a history of animal bite. Unvaccinated persons are often negative for virus-neutralizing antibodies until late in the course of disease. Virus isolation from saliva, positive immunofluorescent skin biopsies or virus neutralizing antibody (from cerebrospinal fluid, or serum of a non-vaccinated patient), establish a diagnosis.
Vaccination of susceptible animal species, particularly dogs and cats, will control this zoonotic disease.
The family Rhabdoviridae consists of more than 100 single-stranded, negative-sense, nonsegmented viruses that infect a wide variety of hosts, including vertebrates, invertebrates, and plants. Common to all members of the family is a distinctive rod- or bullet-shaped morphology. Human pathogens of medical importance are found in the genera Lyssavirus and Vesiculovirus. Only rabies virus, medically the most significant member of the genus Lyssavirus, is reviewed in this chapter.
Five general stages of rabies are recognized in humans: incubation, prodrome, acute neurologic period, coma, and death (or, very rarely, recovery) (Fig. 61-1). No specific antirabies agents are useful once clinical signs or symptoms develop. The incubation period in rabies, usually 30 to 90 days but ranging from as few as 5 days to longer than 2 years after initial exposure, is more variable than in any other acute infection. Incubation periods may be somewhat shorter in children and in individuals bitten close to the central nervous system (e.g., the head). Clinical symptoms are first noted during the prodromal period, which usually lasts from 2 to 10 days. These symptoms are often nonspecific (general malaise, fever, and fatigue) or suggest involvement of the respiratory system (sore throat, cough, and dyspnea), gastrointestinal system (anorexia, dysphagia, nausea, vomiting, abdominal pain, and diarrhea), or central nervous systems (headache, vertigo, anxiety, apprehension, irritability, and nervousness). More remarkable abnormalities (agitation, photophobia, priapism, increased libido, insomnia, nightmares, and depression) may also occur, suggesting encephalitis, psychiatric disturbances, or brain conditions. Pain or paresthesia at the site of virus inoculation, combined with a history of recent animal bite, should suggest a consideration of rabies.
Figure 61-1 Pathogenesis of rabies.
The acute neurologic period begins with objective signs of central nervous system dysfunction. The disease may be classified as furious rabies if hyperactivity (i.e., hydrophobia) predominates and as dumb rabies if paralysis dominates the clinical picture. Fever, paresthesia, nuchal rigidity, muscle fasciculations, focal and generalized convulsions, hyperventilation, and hypersalivation may occur in both forms of the disease.
At the end of the acute neurologic phase, periods of rapid, irregular breathing may begin; paralysis and coma soon follow. Respiratory arrest may occur thereafter, unless the patient is receiving ventilatory assistance, which may prolong survival for days, weeks, or longer, with death due to other complications.
Although life support measures can prolong the clinical course of rabies, rarely will they affect the outcome of disease. The possibility of recovery, however, must be recognized, and when resources permit, every effort should be made to support the patient. At least seven cases of human "recovery" have been documented.
The rabies virus is a negative-sense, non-segmented, single-stranded RNA virus measuring approximately 60 nm x 180 nm. It is composed of an internal protein core or nucleocapsid, containing the nucleic acid, and an outer envelope, a lipid-containing bilayer covered with transmembrane glycoprotein spikes (Fig. 61-2).
Figure 61-2 Virion structure of rabies virus.
The virus genome encodes five proteins associated with either the ribonucleoprotein (RNP) complex or the viral envelope (Fig. 61-3). The L (transcriptase), N (nucleoprotein), and NS (transcriptase-associated) proteins comprise the RNP complex, together with the viral RNA. These aggregate in the cytoplasm of virus-infected neurons and compose Negri bodies, the characteristic histopathologic finding of rabies virus infection. The M (matrix) and G (glycoprotein) proteins are associated with the lipid envelope. The G protein forms the protrusions that cover the outer surface of the virion envelope and is the only rabies virus protein known to induce virus-neutralizing antibody.
Figure 61-3 Genome of rabies virus (ERA strain). This contains single-stranded RNA (12 kilobases); N, NS, M, G, and L genes; a leader sequence at the 3' end; and four intergenic regions.
Classification and Antigenic Types
The genus Lyssavirus includes rabies virus and the antigenically- and genetically-related rabies- like viruses: Lagos bat, Mokola, and Duvenhage viruses, and two suggested subtypes of European bat lyssaviruses. Cross-protection studies suggest that animals immunized with traditional rabies vaccines may not be fully protected if challenged with other lyssaviruses.
Rabies viruses may be categorized as either fixed (adapted by passage in animals or cell culture) or street (wild type). The use of monoclonal antibodies and genetic sequencing to differentiate street rabies viruses has been helpful in identifying viral variants originating in major host reservoirs throughout the world and suggesting the likely sources of human exposure when a history of definitive animal bite was otherwise missing from a patient's case history.
The replication of rabies virus is believed to be similar to that of other negative-stranded RNA viruses. The virus attaches to the host cell membranes via the G protein, penetrates the cytoplasm by fusion or pinocytosis, and is uncoated to RNP. The core initiates primary transcription of the five complementary monocistronic messenger RNAs by using the virion-associated RNA-dependent RNA polymerase. Each RNA is then translated into an individual viral protein. After viral proteins have been synthesized, replication of the genomic RNA continues with the synthesis of full length, positive-stranded RNA, which acts as a template for the production of progeny negative-stranded RNA.
Rabies virus is most commonly transmitted through the bite of an infected mammal, all of which may be susceptible, but to greatly varying degrees. The virus may enter the peripheral nervous system directly, or may replicate in muscle tissue after entering the host, remaining at or near the site of introduction for most of the incubation period. However, the precise sites of viral sequestration remain unknown, since neither antigen nor virus can usually be found in any organ during this phase.
Virus may enter the peripheral nervous system via the neuromuscular junctions, and moves rapidly centripetally to the central nervous system for replication; symptoms may develop shortly thereafter. The virus then begins to pass centrifugally to many tissues and organs, such as the salivary glands.
In general, gross examination of the brain shows mild congestion of the meningeal vessels; microscopic examination usually demonstrates slight perivascular cuffing, limited tissue necrosis, acidophilic intracytoplasmic neuronal inclusions, and rarely, neuronophagia.
The host animal species, viral variant, inoculum concentration, body location and severity of exposure, and host immune status have been associated with overt susceptibility to infection and with different incubation periods. The association of virus-neutralizing antibody, principally IgG, and protective immunity is well known. Production of cytokine, such as interferon, induced during rabies virus infection or vaccination, has been reported to abort the disease if it occurs shortly after viral infection. In one clinical trial, however, all subjects died despite experimental treatment with high doses of alpha interferon.
Recently it has been demonstrated that animals immunized with purified RNP complexes or recombinant nucleoprotein vaccines resisted lethal challenge with rabies virus, although the role of N protein in protection, illness, or recovery is unclear.
Rabies has been recognized for over 4,000 years. Today it is found in most countries, with the exception of those regions from which it has not been naturally reported, including many Australian islands, or areas achieving secondary elimination, such as the United Kingdom. Almost all human rabies is caused by the bite of a rabid animal (Fig 61-4). The risk of rabies is highest in countries with hyperendemic canine rabies, including most of Asia, Africa, and Latin America. In the United States and Europe, domestic animal rabies was largely controlled during the 1940-50s and now represents less than 10% of all animal rabies recorded. Wildlife rabies in the United States occurs primarily among wild terrestrial carnivores, such as raccoons, skunks, foxes, and coyotes, and in insectivorous bats.
Figure 61-4 Life cycle of rabies.
Human rabies is almost always attributable to a bite (any penetration of the skin by the teeth). Nonbite exposures (contamination of an open wound or a mucous membrane via scratches, licks, and inhalation of aerosol) rarely cause rabies in humans. In the United States, nonbite exposures were reported as the source of infection for only 5 (3 %) of the 154 cases reported from 1950 through 1980. Of these five human cases, four were apparently attributable via exposure to aerosols containing highly concentrated live rabies virus: two in spelunkers (cave explorers) and two in rabies research laboratory workers. The fifth case occurred in the recipient of a cornea transplanted from a patient dying of unsuspected rabies encephalitis. An increasing proportion of current human rabies patients in the United States have had no known exposure to the virus; since 1980, 19 of 25 (90%) of the rabies patients have had no definitive rabid animal exposure. This may be attributable either to an inability of the patient to recognize actual rabies exposure at the time, or a failure to properly question the patient concerning potential animal contact. Although it is now a rare human disease in the United States, its actual incidence may be higher than generally believed. The initial suspicion of rabies only occurred at postmortem examination in five reported human cases in the United States since 1985.
The diagnosis of human rabies is usually suggested by epidemiologic and clinical findings and confirmed in the laboratory. The diagnosis is not difficult if there is a history of animal bite exposure and if a full spectrum of symptoms and signs has appeared. Otherwise, careful but rapid assessment of the epidemiologic and clinical features of less typical cases is essential before special laboratory tests are performed. Every patient with neurologic signs or symptoms or unexplained encephalitis should be questioned about the possibility of animal exposure in a rabies-endemic area inside or outside the country of residence. The failure to suspect rabies in several of the recent human deaths in the United States may have occurred because no thorough exposure history had been sought.
Early in the course of illness, rabies can mimic numerous infectious and noninfectious diseases. Many other encephalitides, such as those caused by herpesviruses and arboviruses, resemble rabies. Other infectious diseases also may resemble rabies, such as tetanus, cerebral malaria, rickettsial diseases, and typhoid. Paralytic infectious illnesses that may be confused with rabies include poliomyelitis, botulism, and simian herpes type B encephalitis.
Noninfectious diseases that may be confused with rabies encompass a number of neurologic syndromes, especially acute inflammatory polyneuropathy (Guillain-Barre syndrome), as well as allergic postvaccinal encephalomyelitis secondary to vaccination with nervous-tissue rabies vaccines, intoxication with poisons or drugs, withdrawal from alcohol, acute porphyria, and rabies hysteria. Guillain-Barre syndrome may be mistaken for the paralytic form of rabies, and vice versa.
The detection of rabies antigen, antibody, viral RNA, or the isolation of virus establishes a diagnosis of rabies. Because any individual test may not be positive in a patient with rabies, serial serum specimens for detection of rabies antibodies, saliva specimens for culture of virus, and skin biopsies for direct immunofluorescence testing for virus antigen are sometimes necessary, especially when rabies is strongly suspected.
One of the most rapid methods to diagnose rabies antemortem in humans is to perform a direct immunofluorescence test on a skin biopsy from the nape of the neck for evidence of rabies antigen. The direct immunofluorescence test is the most sensitive and specific method of detecting rabies antigen in skin and other fresh tissue (e.g., brain biopsy), although the results may occasionally be negative in early stages of the disease. If fresh tissue is unavailable, enzyme digestion of fixed tissues may enhance the reactivity of the immunofluorescence test; however, sensitivity may be unacceptably low.
The diagnosis can also be established if virus is isolated from saliva after inoculation of neuroblastoma cells or laboratory rodents; this is generally most successful during the first 2 to 3 weeks of illness. The detection of rabies virus-neutralizing antibody, as typically performed by the rapid fluorescent focus inhibition test (RFFIT), in the serum of unvaccinated individuals is also diagnostic. The presence of antibody in the cerebrospinal fluid confirms the diagnosis, but it may appear 2 to 3 days later than serum antibody and may there fore be less useful early in the disease. Whereas the serological response after vaccination cannot be generally differentiated from that due to disease, vaccination does not typically produce cerebrospinal fluid antibody.
Only seven "recoveries" from rabies, all in the past 25 years, have been well documented. Although rabies virus was not isolated in any of the patients, the high rabies-neutralizing antibody titer in serum samples and the presence of neutralizing antibodies in cerebrospinal fluid strongly supported the diagnoses.
Animal rabies is prevented by vaccinating susceptible species, particularly dogs and cats. Mass dog vaccination programs in the United States and Europe were largely responsible for a dramatic reduction in canine and human rabies during the 1940's and 1950s. In these countries, the number of reported cases in wildlife is currently about 10-fold greater than that in domestic animals; wildlife therefore constitute the greatest risk to human beings. Oral vaccination of wildlife with attenuated and recombinant rabies vaccines by the use of vaccine-containing bait offers hope of controlling the disease in susceptible wild free-ranging animal populations.
Human rabies is best prevented by avoiding exposures to the disease. When an exposure is suspected, the patient's physician and local health department authorities should determine whether an exposure actually occurred and whether a risk of rabies exists in the geographic area (Table 61-1). If treatment (postexposure prophylaxis) is necessary, it should be initiated promptly. Postexposure prophylaxis consists of the combination of local wound cleansing, human rabies immune globulin (HRIG) and rabies vaccine. Two cell culture products currently licensed in the United States include the human diploid cell vaccine (HDCV) and rabies vaccine adsorbed (RVA). Postexposure treatment will abort the infection (Table 61-2), but there is no cure for clinical disease.
Preexposure immunization may be offered to persons at high risk (Table 61-3), such as veterinarians, animal handlers, certain laboratory workers, and persons spending time (e.g., 1 month or more) in foreign countries where rabies is a constant threat. Persons, such as spelunkers, whose vocational or recreational pursuits bring them into frequent contact with potentially rabid animals should also be considered for preexposure prophylaxis. The schedules for preexposure prophylaxis are given in Table 61-4.
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