Lyssaviruses and Rabies Vaccines

HISTORICAL PERSPECTIVE

Tens of thousands of highly relevant scientific papers exist on the diagnosis, epidemiology, immunology, molecular biology, pathogenesis, prevention, treatment, and control of this malady from the late 19th through early 21st centuries, of which only a fraction are cited in this chapter. Moreover, considering that rabies is one of the oldest infectious diseases, the following summary cannot do justice to the rich history accumulated over the past four millennia. Interested readers are directed to G.M. Baer’s Rabbis and Rabies: A Pictorial History of Rabies Throughout the Ages (Tlaxcala, Mexico: Laboratorios Baer; 1996) for a much more engaging and erudite introduction to this fascinating and overlooked topic.

Ancient texts suggest that rabies may have existed before 2000 BCE, largely because of a description in the Mesopotamian Laws of Eshnunna, related to deaths from dog bites. Other historical writings from China, Egypt, India, Persia, and the Levant also seem to contain allusions to this zoonosis. Clearer references appear in Greek contributions. The Homeric poems hint of the disease. Democritus and Aristotle referred to lyssa (related to a mythological Greek goddess of rage and fury) in their writings. During the 1st century CE, the Roman scholar Aulus Cornelius Celsus provided an accurate description and a wide range of susceptible species. Rabies is mentioned frequently in Arabic and Western writings during the Middle Ages. In the 15th century CE, the Italian savant Girolamo Fracastoro conceptualized the “incurable wound,” in which the disease was always considered lethal and not treatable. Similarly, the Talmud mentions that those who say they were bitten by a rabid animal, yet survived, should not be believed.

By the 1500s CE, shortly after European colonization of the Americas, the first Bishop of Oceania described “small animals” that bit the toes of Spanish soldiers during the night. The soldiers died of a disease that may have been rabies. Other sources over the subsequent centuries describe a role for bats that even now have a major role in rabies perpetuation. However, virologic data suggest that canine rabies may have been absent in the New World until introduced within the 16th century CE, as part of the Columbian exchange. ^,

Early in the 19th century, one school of thought held that rabies was purely a psychiatric illness. Nevertheless, the dramatic symptoms and clinical signs, and nearly 100% fatality rate of rabies attracted the scientific curiosity of many of the first modern microbiologists. The German scientist, Gottfried Zinke, demonstrated experimental transmission of rabies virus in 1804 by inoculation of human saliva into animals. During 1879, in Lyon, France, Pierre-Victor Galtier transmitted rabies virus from a dog to a rabbit and from rabbit to rabbit and used intravenous injections of infected material to immunize sheep and goats. Nevertheless, Galtier’s work was overshadowed by that of his famous contemporary, Louis Pasteur, celebrated today in art, script, and screen. Much of the credit for applied rabies research belongs to Pasteur, but one must not overlook the contributions of his collaborators, Roux, Chamberland, and Thuillier. These investigators conducted much of his critical laboratory manipulations because Pasteur was partially disabled by a stroke.

During 1881, the Pasteur group established that the central nervous system (CNS) was the principal site of rabies virus replication (even though their concept of the virus as a “slimy liquid” was applied only generically at the time). They used submeningeal inoculation of infected neural material into rabbits and were able to maintain the resulting “fixed” (i.e., laboratory-adapted) rabies virus in this laboratory host (deemed much safer than the routine use of rabid dogs!) for more than 100 passages. Roux noticed that the virulence of infected rabbit spinal cords decreased rapidly when they were suspended in dry air and was extinguished almost completely in 15 days. From this observation, Pasteur developed an experimental method of vaccination. Dogs injected subcutaneously with serial suspensions of fragments of infected spinal cords, beginning with cord dried long enough to be avirulent and using successively less-dried cords, resisted a lethal rabies virus infection when they were injected intracerebrally with a virulent “street” (i.e., wild-type) virus.

Thereafter, at least 50 dogs were protected by Pasteur’s protocol. Although experiments performed to demonstrate that animals could be made refractory to rabies by vaccination after bites by “mad” dogs were somewhat inconclusive, successful inoculation of one person, Joseph Meister, was attempted on July 6, 1885 (after other unsuccessful and less publicized attempts at human vaccination). Meister, who had been bitten multiple times by a rabid dog some 60 hours previously, received a subcutaneous inoculation of spinal cord suspension derived from rabid rabbits, preserved in a flask of potassium hydroxide for 15 days. Twelve successive inoculations were made with cords of increasing virulence, for a total of 13 inoculations during an approximately 10-day period. The boy not only resisted the original animal source of rabies virus infection but also survived the large quantities of highly virulent virus that were contained in the final doses of vaccine.

This Pasteurian method, as the first generation of rabies vaccination, aroused great interest (and suspicion) in medical circles and, despite some major disagreements, was accepted relatively rapidly. Thereafter, the Pasteur Institute of Paris was founded during 1888. Within a decade, there were Pasteur Institutes throughout the globe (including the United States), providing human rabies vaccination by this method and setting a precedent for prophylaxis well into the 20th century.

Criticism, however, was forthcoming. Occasional failures raised questions about the safety of the vaccine, especially because virulent material was inoculated into patients by the end of the vaccination series. Moreover, there were no controls for comparison to confirm the efficacy of the method. Much later, one tragic accident in Fortaleza, Brazil, in which children died after vaccination, showed that the so-called “fixed” virus could retain virulence if not prepared properly, which led to a century of focus upon the improvement of rabies vaccine purity, potency, safety and efficacy, which prevails to this day.

Understandably, few diseases have carried the cachet of loathing and superstition attributed to this zoonosis, bridging the chasm from fact to folklore, including potential credence to the legends of vampires and werewolves (and even zombies!). In a curious twist on the macabre, some even believe Edgar Allan Poe may have died of rabies in Baltimore, which is not at all surprising given the variety of ways the disease may present clinically.

CLINICAL PRESENTATION

Rabies vaccination is not successful once a patient presents with signs of encephalitis. Prompt recognition of the signs and symptoms is necessary for confirmatory diagnosis, grief counseling, utilization of proper PPE and practices by healthcare staff, national reporting requirements, epidemiological investigations, and institution of palliative care or consideration for extreme therapeutic intervention. Comprehensive clinical descriptions of human rabies have been published by many authors for centuries.

The specific disease may be considered a continuum because of a diversity of variables. In general, the clinical syndrome in humans consists of the following overlapping stages: incubation period, prodrome, acute neurologic phase, coma, and death. Very rarely does recovery occur, even after heroic measures. Once clinical signs occur, death is nearly inevitable.

During the incubation period, there are no symptoms and no means of diagnosis. After exposure, the time to onset is highly variable in humans, ranging in extreme cases from approximately 5–6 days to several years. Typically, humans develop symptoms after 20–60 days. The length of the incubation period is in part influenced by the site of exposure and tends to be shorter when the virus enters locations closer to the brain. Exceptionally long incubation periods more than several years have been reported. ^{, ,} Initially, these were viewed with doubt. Nevertheless, such lengthy periods have been substantiated by genetic analysis of the infecting virus, which may pinpoint where and how the infection was acquired. For example, immigrants or travelers may become infected originally with a viral variant from a region they had not visited for many months to years and such variants did not occur in the region where the patient succumbed. ^{, ,} During such long incubation periods, the specific anatomical location where lyssaviruses remain in vivo is unclear. Opinions differ if virions remain locally in the periphery, close to the site of infection, or become “dormant” after entry to the nervous system. After breaching the skin or mucosae, rabies virus may exist in a stable form within a cellular compartment, relatively shielded from proteolytic enzymes or detection by the immune system. Prophylaxis success after neural entry remains highly uncertain.

Most human rabies cases are caused by dog bites. ^{, , ,} Approximately 37–57% of unvaccinated individuals with known exposure to a rabid dog develop rabies. The risk of disease is linked presumably to the amount of virus present in saliva as well as the location and the severity of exposure. The latter is supported by an increased (∼80%) risk of disease, following extreme attacks by rabid wolves. The incidence of symptomatic rabies in unvaccinated persons after exposure to other rabid animals, such as bats, is unknown, but may be lower, as suggested by documentation of viral seropositivity in healthy humans residing in areas of Amazonia, where vampire bat rabies is common.

The initial prodromal symptoms of rabies consist of general malaise, anorexia, fatigue, headache, vomiting, sore throat, fever, and in 50–80% of cases, pain or paresthesia close to the bite site, potentially indicative of neuronal damage. Some patients display psychological symptoms, such as anxiety, agitation, irritability, nervousness, insomnia, or depression. The prodromal phase, lasting for 2–10 days, is followed by neurological symptoms of rabies, which in humans can present as a furious or paralytic form. The “furious” form, generally associated with dog bites, is more common and afflicts approximately 65–70% of patients. Furious rabies is characterized by periods of hyperactivity, aggressive behavior, agitation, hallucinations, delusions, hypersalivation, muscle fasciculations, seizures, fever, excessive sweating, and priapism. Patients develop difficulties swallowing and hydrophobia (literally, fear of water) because of painful spasms of their throat muscles, which can be triggered by the mere sight, sound, or even mention of water. Patients may also develop fear of light (photophobia) and air currents (aerophobia). Hyperactive periods in humans last approximately 1–5 minutes. These episodes may occur spontaneously or are triggered by a variety of stimuli. Between hyperactive periods, patients are alert and able to communicate, although over time their mental faculties deteriorate.

Paralytic, or so-called “dumb,” rabies may be under reported and seems more common after exposure to rabid bats. ^, Patients develop ascending muscle weakness, loss of sensation, and paralysis starting in the extremities and spreading centrally. Eventually, with either form of rabies, patients fall into a coma and die of cardiac or respiratory arrest. Without supportive care, death occurs within approximately 5 days of furious rabies and approximately 13 days of paralytic rabies. Intensive care can prolong life for a few days or even months, but in general does not prevent death. ^,

In countries where canine rabies is enzootic, this zoonosis is somewhat easier to diagnose, with the onset of classical symptoms in an encephalitic patient with a history of a suspicious animal bite. However, rabies may not be suspected or easily diagnosed, especially in developed countries where canine rabies is eliminated. ^{, ,} This, in turn, is one explanation for the occurrence of human transplantation of tissues and organs, from individuals who succumbed to an undiagnosed illness, to recipients who later developed rabies, and thereby inadvertently allowing for a postmortem diagnosis from the donors, as to the cause of death. ^,

Rabies may be confused with encephalitis caused by other infectious agents, such as herpes simplex virus, enteroviruses, arboviruses, toxoplasma, malaria, or prions, or toxins, such as tetanus toxin. Rabies may mimic atropine poisoning, intracranial malignancies, acute cerebral injuries (e.g., a stroke), or transverse myelitis caused by infections with measles virus or mycoplasma. Some individuals may exhibit symptoms suggestive of a hysterical reaction to an animal bite. For a thorough diagnosis of rabies, careful history about contacts with animals, unprovoked animal attacks, availability of the animal for testing, vaccination status of the animal, camping with potential exposure to bats, foreign travel, and the like is essential. Physical examination should reveal autonomic instability in furious rabies and symmetric paralysis that may be mistaken for Guillain–Barré syndrome (GBS) in paralytic rabies. Laboratory testing can be used to rule out other infectious diseases. Magnetic resonance imaging (MRI) and computed tomography (CT) scanning of the brain may show some abnormalities, but neither is specific for a diagnosis of rabies. ^,

Electroencephalography may show sudden drops in amplitude because of vasospasms of cerebral vessels, but, again, those findings are not specific to rabies. ^, Cerebrospinal fluid (CSF) commonly shows lymphocytic pleocytosis, but few to no changes in glucose or protein content. Positive antemortem diagnosis requires several tests, performed by a qualified reference laboratory ( Table 51.1 ). Skin biopsy specimens can be tested with fluorescent-labeled antibodies for detection of viral antigens present in the cutaneous nerves at the base of hair follicles. Impressions of corneal epithelia can be analyzed using the same method. Harvesting of either type of sample should be conducted with topical anesthesia by a dermatologist or an ophthalmologist. Saliva, CSF, urine sediment, and pulmonary secretions may allow for isolation of virus within approximately 2 weeks after onset of symptoms or such samples can be tested for viral transcripts by reverse transcription and amplification by polymerase chain reaction (PCR) and other molecular tests. Antibodies to rabies virus can be detected from serum or CSF via neutralization by 1–2 weeks after onset of symptoms.

As in humans, clinical signs of rabies in animals are compatible with an acute, progressive encephalitis. An initial prodromal phase, often characterized by changes in behavior and loss of appetite, is followed by a phase where the animal becomes aggressive and may bite without provocation. Other animals may become somnolent. Animals may develop paralysis and die quickly thereafter. As a rule, rabies in animals is diagnosed postmortem from brain impressions using the direct fluorescent antibody test. Microscopic examination of stained sections demonstrates inclusion bodies in the cytoplasm of neurons, especially in the brainstem and Ammon’s horn of the hippocampus. Brain impressions can be tested by a fluorescent-conjugated antibody for detection of viral antigens. Alternative molecular methods, or microscopic techniques based on biotinylated virus-specific antibodies that allow for easier and faster onsite diagnosis by routine light microscopy, rather than immunofluorescence, have also been developed. ^,

Historically, isolation of rabies virus had been reported from as much as 0.5% of so-called “normal” dogs in Ethiopia and elsewhere, presumably in the late incubation stage or early prodromal phase, but the actual significance of this finding to human rabies occurrence is unknown. Moreover, animals may excrete virus in their saliva for days to weeks before overt illness. The ultimate documentation of a true “carrier state” in rabies for any species awaits further rigorous, objective corroboration, given several alternative explanations.

APPLIED VIROLOGY

An introduction to applied virology yields important insights as to the basic immune mechanisms that are triggered by rabies prophylaxis. Rabies virus belongs to the family Rhabdoviridae, genus Lyssavirus , consisting of genetically related enveloped viruses with a single, nonsegmented, negative-stranded RNA. The virion is shaped like a bullet, approximately 200 nm long and 75 nm wide. The virus contains multiple copies of five structural proteins: the virion transcriptase (L), glycoprotein (G), nucleoprotein (N), phosphoprotein (P), and matrix protein (M). The L, N, and P proteins are bound noncovalently to the virion RNA, and the resulting ribonucleoprotein (RNP) complex forms a helically coiled nucleocapsid structure within the virion. The crystal structure of the RNP has been resolved. The N protein encapsidates the RNA, shielding it from the intracellular environment. This RNP complex is surrounded by a lipoprotein envelope consisting of the M protein and the surface projections of the trimeric G protein extend to the exterior of the virion.

Like other RNA viruses, rabies viruses have been proposed by some to exist as quasispecies. ^, Compatible with its purported ancient origins, phylogenetic analyses suggest rabies evolved in bats. Transfer of certain carnivore rabies viruses from the Old to the New World may have occurred at the time of colonization of the Americas, where it was likely already established in bats, and subsequently in mammalian carnivores, but the evidence is incomplete. ^{, ,}

The rabies virus G protein, which is a trimer of approximately 67 kDa, is the major antigen responsible for inducing production of VNAs and for conferring immunity against lethal infection with rabies virus. ^, The G protein also contains determinants of virulence. ^, The G gene was the first rabies virus gene to be cloned and sequenced. From the nucleotide sequence, a polypeptide 524 amino acids long was deduced, which included a signal sequence of 19 amino acids. An arginine at position 333 seems important as one virulence factor, that increases neuroinvasiveness and spread through the nervous system. ^,

Despite the importance of the G gene to virulence and attenuation, critical features related to viral pathogenesis are diverse. For example, the P protein interferes with host interferon production. ^, The development of a reverse genetics system for application to rabies virus offers a more detailed analysis of virulence factors. Attenuation may be appreciated as a mirror image to virulence and includes additional elements, such as inhibition of apoptosis of the infected neuron. The G and M proteins are responsible largely for blocking apoptosis after infection by virulent viruses, which is, in effect, a protective mechanism for the host, although it also allows the virus to finish its replication cycle. Highly attenuated strains have alterations in those proteins, rendering them proapoptotic. Rabies virus genomic RNA is also protected from generating innate immune responses by its tight physical association with the N protein. The immunogenic activity of purified native and smaller fragments of G protein, both naturally occurring and derived by chemical cleavage of the rabies virus G protein, has been compared in a variety of ways to determine the structural basis of VNA production after immunization. ^{, , ,} Although the definitive role of the humoral immune response to rabies may be debated, evidence for the importance of VNA in prevention of viral infection is convincing in humans and other mammals. Regarding a cellular response, T-helper cells are necessary for antibody induction, whereas cytolytic T cells directed against the N protein induced by vaccination may be important for clearing infected extraneural cells before entry of virus into the CNS. Interestingly, the cytolytic T-cell response may be suppressed in natural infection.

Routine sequence analysis of rabies virus variants has not revealed substantive differences accounting for the clinical appearance of furious or paralytic rabies, as opposed to host-specific difference in viral transport. ^,

PATHOGENESIS AS IT RELATES TO PREVENTION

If lyssaviruses are quintessential neurotropic agents and the CNS is considered under immune privilege, how does prophylaxis act? Facets related to inoculum dose, route, severity, and timing are key to successful intervention. Clearly, appreciation of viral pathobiology is critical as background to understanding how, when, and why PEP may be used appropriately to prevent this fatal zoonosis. After a bite and salivary inoculation of thousands to millions of particles in a wound, rabies virus may take days, weeks or longer to reach the CNS. This biological revelation, at least in part, buys precious time for intervention and allows medical engagement. During this early phase, rabies virus is more susceptible to the combined impact of clinical action (such as first aid and mechanical removal of pathogens by thorough washing and wound debridement) with innate, passive and adaptive immunity. There is some evidence that replication may occur in peripheral tissues, such as in extrafusal muscle fibers or fibrocytes surrounding the wound, providing an amplification of the original inoculum, yet experimental laboratory animal data show that neural entry can occur without any prior replication in other tissues. ^, Besides muscle cells, another site proposed for possible harborage of rabies virus before entry into the nervous system includes macrophages, from which virus could in theory reactivate to cause disease. However, the importance of replication in nonneural cells to the overall pathogenesis of rabies virus remains highly controversial. ^,

Rabies virus G protein has sequences similar to certain neurotoxins, and there are several posited viral receptors, including the α subunit of the nicotinic acetylcholine receptors, the neural cell adhesion molecule, the neurotrophin receptor, the low affinity p75 neurotrophin receptor, the metabotropic glutamate receptor subtype 2, and perhaps certain lipoproteins on cellular membranes. For nonbite exposure, such as via the airborne route, access to the CNS may occur thru the nasal mucosa as a portal of entry, albeit much more rarely than by bite transmission. After reception at membranes, viral intracellular penetration depends on clathrin-mediated endocytosis.

Upon entry to the neuronal cytosol, replication occurs, and the virus spreads centrifugally to the CNS. ^{, , ,} While VNA was thought to act largely on extracellular virus, the action of rabies VNA may not be exerted solely outside the cell. In one animal model, the effectiveness of antibody was associated with its entry into the cell by endocytosis and inhibition of viral transcription. Mechanisms of viral neutralization appear to be multiple and whether antibody may act directly or indirectly by signal transduction to inhibit viral protein synthesis is unclear.

Once in the nervous system, rabies virus travels rapidly within motor (and potentially sensory) axons, at a rate of approximately 8–20 mm/day in rodents, probably faster in humans (15–100 mm/day). Experimental studies showed that rabies virus can reach the CNS in 3–5 days in retrograde fashion from the periphery, wherein it causes a widespread encephalitis.

After viral establishment in the neurons of the CNS, centrifugal spread occurs in the opposite, anterograde direction, down the axons to replicate in peripheral tissues, most notably in the nerve plexus and acinar cells of the salivary glands, from which excretion in saliva permits viral transmission by bite to maintain the circuit of infection. ^{, ,} However, at the end stage of infection, neurons in several other extraneural tissues are also affected, including the cornea, hair follicles, lungs, heart, liver, kidneys, pancreas, adrenal glands, and gastrointestinal tract. ^{, , ,} The finding of widespread dissemination at a diversity of sites throughout the body explains the cases in patients who acquired rabies from virus-infected tissue and organ transplants (i.e., cornea, liver, lung, kidney, etc.) and raises questions about the possibility of altered pathogenesis through such unusual routes. Presence of virus in nonneuronal cells might also explain cases with long incubation periods.

The pathophysiology of neural dysfunction is not completely understood. Although encephalitis is widespread, neuronal destruction is not. Death may result from the involvement and dysfunction of brain centers controlling the cardiovascular and respiratory systems. In general, the histologic presence of inclusions (i.e., Negri bodies) parallels that of rabies virus antigens, although many infected cells do not have these inclusions. Rabies virus antigens are most prevalent in the periaqueductal gray matter and the Purkinje cells of the cerebellum, but the quantity of rabies virus does not correlate with severity of symptoms. ^, Highly virulent rabies virus variants may be more capable of evading host innate immunity and of destroying neuronal processes. ^, The production of nitric oxide within the brain and general downregulation of host genes have been advanced as possible explanations for brain dysfunction. Although the fatality rate in rabies is extremely elevated, recovery has been documented in some species.

Clinically, the neural mechanisms that distinguish furious from paralytic rabies are not completely defined, but electrophysiologic studies suggest that denervation and anterior horn cell dysfunction are prominent in the former, whereas in paralytic rabies, there is inflammation and demyelination of peripheral nerves. ^, The mechanisms causing demyelination could involve autoimmunity or a bystander effect of immune response to virus in the axons. Hemachudha and colleagues emphasized the role of cellular immunity in the course of rabies. They correlated strong T-cell responses and cytokine secretion (in particular, interleukin-6) with early death and the encephalitic form of the disease, and weak T-cell responses with paralysis and longer survival. Whereas it is certain that lack of an immune response to rabies virus, particularly VNA, leads to fatal disease, there is thus some evidence that, on the contrary, some cellular responses are responsible for neural pathology. ^,

Evidence of a serologic response to rabies viral antigens can be demonstrated by a variety of laboratory techniques, including historical mouse neutralization, fluorescent focus inhibition, indirect fluorescent antibody, plaque neutralization, immunolysis of rabies virus–infected cells, and binding techniques using radioimmunoassay or enzyme-linked immunosorbent assay procedures, to help better understand the dynamics of infection to immunity, from the periphery to the brain. ^, Serum antibodies develop relatively late after natural infection in humans. In persons without a history of vaccination, serum antibodies may be first detected on or about the 6th to 10th day of illness, and thereafter can rise rapidly to high levels. Antibodies are also present in CSF later in the clinical course. Antibody titers in CSF are higher than might be expected from seepage into CSF from circulating blood, suggesting local production. Because vaccination does not ordinarily induce CSF antibodies, the presence of CSF antibody titers supports the diagnosis of clinical rabies. ^{, ,}

The absence of most detectable serum antibodies until around the second week of illness (if at all) and of CSF antibodies until approximately the third week of illness (when significant systemic and neurologic problems occur) raises the possibility that some of the clinical symptoms result from the interaction of host antibodies with rabies virus–infected cells. In laboratory animal experiments, neutralizing antibodies and infiltration of cellular inflammatory cells were necessary to clear infection with an attenuated rabies from the CNS, but this process occurs relatively late in the typical clinical situation.

Most persons who die of rabies may develop specific antibodies to the virus, particularly if they received some form of PEP. However, that response does not protect against a lethal outcome once illness is present and might even contribute to the disease. Experimental findings suggest that the inflammatory response is associated with opening the blood–brain barrier, delivering antibody-producing B cells to the CNS. Nevertheless, apparently this response usually occurs too late to be therapeutic in most cases, regardless of detectable VNA titers, and is one reason to place doubt upon the subjective nature of any arbitrary or absolute “seroprotective” level. ^{, , , ,}

DIAGNOSIS

The history of an associated bite from a known or suspected rabid animal, coupled with the striking clinical manifestations, should provide a reasonably simple diagnosis of rabies. However, such straightforward attributes are not always present. Especially in the absence of a documented exposure source, clinical diagnosis of rabies requires differentiation from a wide variety of diseases that can cause neurologic symptoms. Because laboratory diagnosis may not be obtained during the first week of illness, a presumptive diagnosis based on clinical symptoms is important.

The definitive diagnosis of rabies virus infection of humans and suspected animal vectors depends on the detection and identification of rabies virus antigens or intracytoplasmic neuronal inclusions (Negri bodies) in infected brain tissue; viral nucleic acids by reverse transcription–polymerase chain reaction (RT-PCR); the presence of rabies virus–specific antibodies in the CSF, or in the serum of unvaccinated patients; and on the isolation and identification of the virus from brain tissue, saliva, or other infected tissues. ^, The standard diagnostic technique is to search for rabies virus antigens in brain tissue by fluorescent antibody staining. The brainstem, cerebellum, and thalamus provide some of the best samples for diagnostic testing. Identification of viral nucleic acids such as by primary or real-time RT-PCR is useful, particularly if specimens are in poor condition. Demonstration of Negri bodies has a variable sensitivity and is of only historical interest as a primary method, notwithstanding their detection microscopically upon postmortem examination of brain tissue, particularly when rabies is not suspected. Virus isolation is a procedure used for confirmation of other positive test results. Isolation can be accomplished in mouse neuroblastoma cell culture or by intracerebral inoculation of suckling mice. Although diagnostic procedures generally are initiated in tissue specimens obtained postmortem, rabies virus infection also can be identified during the extended course of the disease. In addition to its usefulness for brain biopsy specimens, the fluorescent antibody staining technique enables detection of viral antigens in impressions of infected cornea or in frozen sections of skin biopsy samples from the hairline of the neck, where antigens can be detected in the nerves surrounding the hair follicles.

In addition, as shown in Table 51.1 , molecular tests conducted on saliva provide a rapid and sensitive method for rabies as early as 5 days after onset of symptoms. ^, Improved sensitivity was obtained when an RNA polymerase was included in the reaction and electrochemiluminescence was used for detection of product. This technique, called nucleic acid-based sequence amplification, detected the rabies virus genome in saliva, concentrated urine sediment, and CSF as early as 2 or 3 days after symptoms. ^, Serial tests should be performed to increase sensitivity. One study in Thailand showed high sensitivity (91%) of rabies virus genome detection on the first day of hospitalization, particularly in saliva. Both CSF and concentrated urine sediment specimens may also be useful. However, viral nucleic acids were not detected in all patients before death. A latex agglutination test for viral antigen in dog saliva also has been developed. One report found rabies virus–specific immune complexes in the CSF of 77% of patients with rabies 5–7 days after onset of disease.

As an example of human rabies diagnosis in a developed country, between 1980 and 2020, more than 95 cases of human rabies were reported in the United States, but not all were diagnosed before death, probably because only a minority had a known history of exposure to potentially rabid animals. This problem arises in large part because several patients had been infected with rabies virus variants from bats, possibly with undocumented, or recognized but ignored, bat bites.

EPIDEMIOLOGY

A thorough understanding of rabies as a fundamental disease of nature, affecting many other species, is essential, to properly conduct risk assessments for both human and domestic animal PrEP and PEP applications.

Animal Reservoirs and Vectors

Rabies is a disease of domestic and wild mammals, particularly dogs and related canid species, as well as raccoons, mongooses, skunks, and bats. Although birds are susceptible under experimental conditions, natural cases are very rare. In areas where domestic animal control programs are not extensively developed, dogs and cats account for most of the rabid animals reported and cause the majority (>90%) of human rabies virus exposures and deaths. After effective domestic animal rabies control programs in these areas, the numbers of rabid dogs and cats markedly decrease, as illustrated in the United States from the 1940s to the 1960s ( Fig. 51.1 ). With a substantial burden of disease removed, multiple species of wildlife may then be recognized and appreciated as the main reservoirs of rabies virus. For example, in the United States since 1960, most cases of animal rabies have been in wildlife, and most human rabies cases have been secondary to bites by rabid wildlife, such bats. ^, Fortunately, insectivorous bats do not often transmit rabies virus to dogs or cats.

Illustrating the relationship among different viral variants and hosts, Fig. 51.2 is a composite map of the United States showing the major carnivore reservoirs in each region. ^, The salient features show that skunks are the important vectors for rabies in the western and central United States, whereas raccoon rabies dominates in the east. The original focus of raccoon rabies was in the southeast, but a second front developed in the mid-Atlantic and northeast owing to translocation of infected animals, and now raccoon rabies is contiguous from Maine to Florida and west to Ohio. ^, Foci of red fox rabies are evident in Canada and Alaska. Rabies in Puerto Rico is caused by the mongoose, which was introduced for pest control in sugar cane fields but emerged as an invasive species and viral reservoir. Clearly, even after canine rabies elimination, management remains costly: in New York State alone, the cost of human rabies prevention was calculated to be more than $2.3 million per year.

Table 51.2 delineates rabies cases by species for the United States during 2019. By comparison, Table 51.3 lists the principal animal vectors of rabies throughout the world. In Western Europe, before oral wildlife vaccination programs, foxes accounted for up to 80% of rabid animals. Infection in raccoon dogs is becoming more common in eastern Europe. ^, Molecular analysis of European viruses suggests that rabies there originated in dogs, but now affects red foxes and raccoon dogs. Infection in red foxes and raccoon dogs has gradually spread from east to west. Canine rabies is still widespread in Asia, Africa, and parts of Latin America. Between 1993 and 2009, there was a significant drop in canine and human cases in Latin America. However, dogs remained the predominant vector for humans (65%), followed by bats (15%) and cats (3%). Vaccination of pet dogs is an effective strategy for protection of humans and has eliminated carnivore rabies in Great Britain, Iceland, Japan, and many other islands. ^, An immunization coverage of 60–70% is estimated to prevent canine rabies outbreaks.

TABLE 51.2

Cases of Rabies in the United States, 2019 ^a

	Number	Percentage
Wildlife
Bats	1387	29.6
Raccoons	1545	32.9
Skunks	915	19.5
Foxes	361	7.7
Rodents/lagomorphs	40	0.9
Other	57	1.2
Subtotal	4305	91.8
Domestic Animals
Cats	245	5.2
Cattle	39	0.8
Dogs	66	1.4
Other	35	0.7
Subtotal	385	8 . 2
Humans b	0
Total	4690	100%

a During 2019, more than 97,000 samples were submitted for consideration in laboratory diagnosis.

b During 2021, at least two human rabies cases were reported, including one with a history of PEP. Data from: Ma X, Monroe BP, Wallace RM, et al. Rabies surveillance in the United States during 2019. J Am Vet Med Assoc. 2021;258:1205–1220 .

TABLE 51.3

Principal Global Animal Reservoirs/Vectors of Lyssaviruses ^a

Location	Reservoirs/Vectors
North America	Bats, coyotes, foxes, mongooses, raccoons, and skunks
Western Europe	Bats
Eastern Europe	Bats, cats, dogs, foxes, raccoon dogs, and wolves
Latin America	Bats, cats, coati (?), dogs, foxes, marmosets, and skunks
Caribbean	Bats, dogs, and mongooses
Australia	Bats
Africa	Bats, cats, dogs, jackals, foxes, and mongooses
Asia	Bats, cats, dogs, foxes, ferret badgers, mongooses, monkeys, and mongooses

a None listed in Antarctica, Oceania, and a few other, mostly insular locations, reported as presumably “rabies-free” (even though laboratory-based surveillance may be entirely lacking or inadequate by current global epidemiological standards). As described in: Rupprecht CE, Bannazadeh Baghi H, Del Rio Vilas VJ, et al. Historical, current and expected future occurrence of rabies in enzootic regions. Rev Sci Tech. 2018;37:729-739 .

In support of historical and epidemiologic observations in developed countries, one Thai study confirmed that rabid dogs and cats uniformly die within 10 days of the onset of illness, similar to classical laboratory observations.

Foxes are important vectors in Canada, Alaska, and Russia. Just as mongooses imported into the Caribbean Islands now form a reservoir for rabies, several mongoose species play a similar role in southern Africa and parts of Asia. The common vampire bat is a major threat to livestock in Latin America and has been involved in many biting incidents in humans. Rabid cattle also may excrete rabies virus in saliva. Like all mammals, rodents are susceptible to infection but are infrequently rabid, and human transmission of disease by these animals has not occurred. In Thailand, 95% of the animals involved in biting incidents are dogs, often younger than 6 months, with cats accounting for another 3%. The remaining 2% include monkeys, civets, tigers, and other animals, which testifies to the wide host range of rabies virus. Dogs randomly captured in Thailand developed rabies within 1 month in 3% to 4% of cases, and, curiously, there was serologic evidence suggestive of prior rabies virus exposure or prior vaccination in approximately 15–20% of dogs.

Rabies in animals (excluding bats) is absent in many islands such as the United Kingdom, Australia, and Japan. Much of Western Europe is also now considered “rabies-free” in carnivores, although rabies still occurs in Central and Eastern Europe. For example, from 2000 to 2005, there were 45 nonimported cases of human rabies in Europe, but 44 occurred in Eastern Europe, and the remaining case was caused by a bat lyssavirus. Many of the larger Asian countries (such as China), most of Africa, Russia and the republics from the former Soviet Union, and some parts of South America still report considerable rabies in domestic animals.

Travelers are at risk, as confirmed by the report of more than 18 human cases of rabies diagnosed in the United States but acquired abroad between 1990 and 2020. The risk of rabies exposure in Nepal was calculated to be 5.7 per 1000 person-years for expatriates and 1.9 per 1000 person-years for tourists. The epidemiology of rabies has been revolutionized by viral typing, initially with monoclonal antibodies since the late 1970s. Panels of these antibodies, directed against epitopes specific to isolates from different animal species and from different geographic locations, were used to identify viruses. Thus, it is now possible to identify the source of an isolate from humans or animals and to demonstrate that the infection was transmitted far away in time and place. Significantly, genetic sequencing of the viral genome after RT-PCR amplification has supplemented greatly our knowledge of viral variability. Laboratory “fixed virus” strains differ in sequence from wild “street viruses” by as much as 10–15% of nucleotides. Sequence data suggest that many viruses in the Western Hemisphere and in South Africa, and elsewhere, were imported from Europe during colonization. ^,

NONBITE TRANSMISSION

Most rabies cases occur after animal bite transmission. Nonbite transmission is rare but causes a great deal of anxiety among persons in contact with suspect animals and a substantial outlay of investigational public health resources. As reviewed by Gibbons, at least 27 cases of transmission by means other than bites were well-documented, and 17 other less-well-documented cases. Of 44 suspected cases, 18 were caused by improperly inactivated vaccine in Brazil, 8 were due to corneal transplants, 8 from contamination of skin whose integrity had been impaired, 4 by aerosols created in the laboratory or bat-infested caves, and 6 from human-to-human transmission. Among the alleged historical causes of human-to-human transmission were transplacental passage, lactation, kissing, intercourse, and providing healthcare to a rabid patient. Gibbons also found three reports of transmission by human bite. One recent case of nonbite transmission of rabies virus was reported in China, concerning a well-intentioned but unvaccinated father who succumbed after sucking a dog bite lesion of his child, who received PEP and survived.

Transmission to transplant recipients from organ donors is particularly worrisome when rabies is not suspected. In the first reported series from the United States, transmission occurred to four patients who received kidneys, liver, and an arterial segment from a donor with unsuspected rabies after a bat exposure. A similar scenario was reported for German transplant recipients during 2005, and additional cases were reported again in the United States, from a donor unknowingly infected with raccoon rabies virus. Harkening back to pathobiology, these cases demonstrate that rabies virus is disseminated extensively throughout the body, at least late during the disease, and that direct implantation of infected tissues from a donor may transmit rabies virus to human organ recipients. Deaths of suspected encephalitis should be investigated for rabies or other etiologic agents before tissues from the donor are used.

Infection by aerosol is unusual but has been suspected in caves inhabited by millions of bats and implicated under certain laboratory conditions. ^{, ,} Besides rabies virus, in theory, other lyssaviruses could also be transmitted by aerosols. Despite the frequency of rabies virus perpetuation in nature, close human engagement with their companion animals, intimate contacts among rabies patients, family members and healthcare workers, etc., documentation of nonbite transmission remains extremely uncommon.

HUMAN RABIES

No one knows the actual burden of human rabies, because surveillance is much less than ideal, reflective in part of existing health disparities. Generally, the epidemiology of human rabies follows closely the epizootiology of animal rabies as regards exposures and case causality. The dog is the major global reservoir of rabies, and hence the focus of the “Zero by Thirty” GEHRD program. Yet, even with the elimination of dog-to-dog transmission, spillover infections from wildlife will occur, and in the United States alone, more than 1 million dog bites are reported each year. The situation is much worse in many other parts of the world. Human rabies has been reported from all continents except Antarctica, but most cases occur in countries where canine rabies is not well controlled. The World Health Organization (WHO) estimate for humans vaccinated due to exposure to rabies exceeds 15 million annually. The WHO infers tens of thousands of unconfirmed human rabies cases occur, most of them in canine-enzootic countries of Africa and Asia. Recent global estimates suggest that 70,000 human rabies cases or more may occur annually.

The annual incidence of rabies deaths per 100,000 population has been calculated as approximately 2 in India, 0.01–0.2 in Latin America, and an uncertain 0.0001–13 in Africa. ^, Human rabies is common in persons younger than 18 years of age, with approximately 40% of cases found in children 5–14 years of age. However, all age groups are susceptible. Most rabies victims are male. In one study in the United States, the highest incidence of human rabies PEP occurred among rural boys, primarily during the summer months.

In the United States, bats have emerged as the leading transmitter of rabies virus to humans. Excluding transplant recipients infected by a common donor, after 1990 bats were associated in most diagnosed human rabies cases of presumed domestic origin. ^, There were also other cases associated with nonindigenous canine rabies viruses during the same period. No definitive contact with bats could be elicited in the majority of cases, and only a handful of patients had a documented history of a bite reported before diagnosis, suggesting that memory lapses, neural impairment, and, rarely, ignored or unperceived bites in some persons may have been responsible. A similar situation exists in other parts of North America, and this phenomenon should be operative in any areas in which bats serve as lyssavirus reservoirs. Avoiding suspect animal contacts, and appropriate prophylaxis after exposures, prevent human cases.

VACCINATION

CELL CULTURE RABIES VACCINES

The solution to the problem of safety of rabies vaccines lay in the development of vaccines prepared from rabies virus grown in tissue culture free of neuronal tissue. The first attempts to develop a tissue culture vaccine were made by Kissling during 1958 and by Fenje during 1960. Both investigators used primary hamster kidney (PHK) cells for rabies virus production.

Typically, laboratory strains of rabies virus are produced in embryonated eggs or tissue culture, concentrated, purified, inactivated, formulated, and lyophilized, with a shelf life >2 years. All modern CCVs contain rabies virus antigens with a potency of at least 2.5 IU per intramuscular dose. However, a meta-analysis of immunogenicity data revealed that higher concentrations of antigen did not translate into higher immune responses. Historically used fixed rabies viruses adapted for vaccine production include the Pasteur, Pitman-Moore (PM), challenge virus standard (CVS), Flury low egg passage (LEP), Flury high egg passage (HEP), Kelev, and ERA/Street Alabama Dufferin (SAD) strains.