Читать книгу To Catch a Virus - John Booss - Страница 20

Extensive experimental work on what would turn out to be the second human disease shown to be caused by a filterable virus, rabies, had been under way for virtually a century. The medical historian Lise Wilkinson credits Georg Gottfried Zinke’s 1804 publication as being “. . . the first description of experiments specifically intended to follow the transmission of the unknown agent of rabies” (69). Zinke reported successful transmission of rabies to several species, including dogs, cats, rabbits, and fowl (72). While certain characteristics, such as incubation time and clinical characteristics, differed from those in later reports, Zinke’s published work was an important landmark. Studies of diseases in animals in the 18th and 19th centuries involved agents of agricultural import—cattle plague (rinderpest), foot-and-mouth disease, bovine pleuropneumonia, and glanders—and established the basis for comparative medicine, the study of human diseases in animals (68). Rabies straddled human and animal medicine.

Rabies is horrific in all aspects: in the savage bites by crazed wolves or dogs to implant infection, in the anxiety and fear in anticipation of whether the disease will develop, in the torturing expression of the acute disease, and in the knowledge that once expressed, rabies is an essentially fatal disease. The fear induced by the approach of a rabid dog was used to good effect in Harper Lee’s To Kill a Mockingbird: “. . . motivated by an invisible force that was inclining him toward us. We could see him shiver like a horse shedding flies; his jaw opened and shut; he was alist, but he was being pulled gradually toward us” (35) (Fig. 1). Accounts of terrifying attacks by rabid wolves in country villages have played a role in the development of treatment. It has often been cited that one of the motivations for Pasteur’s study of rabies was a childhood memory of a rabid wolf attack on the village of Villers-Farlay and the town of Arbois (21) (Fig. 2). It was reported that Pasteur was 8 when he listened in horror to the screams of victims who had come to a blacksmith’s shop for “treatment”—cauterization. In 1886, soon after Pasteur’s publicity spread worldwide concerning postexposure prophylaxis treatment of rabies, 19 Russians came to Paris seeking treatment. In the cases of two victims of a rabid wolf’s attack, one had a lip and cheek bitten off, and the other’s face had been ripped off. Of the 19, 3 died during treatment and 16 ultimately returned to Russia (11). In 1955, the WHO, reporting on the use of hyperimmune serum and vaccine, described the effects of a rabid wolf’s attack on an Iranian village (2). Twenty-nine persons were bitten, 18 with severe head wounds, including a 6-year-old boy in whom a skull bone was crushed and the dura mater covering of the brain slashed. Remarkably, 25 of 29 wounded survived, including the young boy, who received intensive treatment with hyperimmune serum and vaccine.

Figure 1 Mad Dog. The fear of rabid dogs has been portrayed throughout history. This caricature by T. L. Busby was published in London in 1826. (Courtesy Yale University, Harvey Cushing/John Whitney Medical Library.)

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Figure 2 Louis Pasteur. Pasteur was one of the principal founders of germ theory. He disproved the theory of spontaneous generation; linked agricultural, animal, and human diseases to specific infections; and developed vaccinations, including that to prevent rabies. (Courtesy of the National Library of Medicine.)

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Rabies in humans may start after an incubation period of weeks or months with itching at the site of the bite and growing apprehension. Thereafter, aerophobia and hydrophobia may develop. The description of hydrophobia by D. A. Warrell et al. serves well: “The patient picks up a cup to drink but, even before the liquid has reached his lips, his arm begins to shake, he takes a rapid succession of inspiratory gasps, his neck muscles are seen to contract forcibly, and the spasm ends with throwing away his cup and falling back with neck extended” (64). Death ensues after periods of marked agitation and fearfulness alternating with lucidity. It is no wonder that rabies attracted attention out of proportion to its incidence.

In studies of rabies transmission to animals, several experimental aspects were considered and refined, many by Pasteur and his colleagues on the road to producing a postexposure vaccine. One was the type of inoculum, infected central nervous system (CNS) tissue. Other crucial considerations were the animal species used and the route of inoculation.

In 1821, F. Magendie reported the transmission of rabies from a sick person to a dog by injecting saliva beneath the skin (39). Magendie reported that the dog became rabid in about a month. This appears to be the first acknowledged transmission of rabies from a human to an experimental animal (69). Use of the dog as the test animal, while logical as the original species tested, is dangerous because the “furious” type of rabies is expressed. The indiscriminate and aggressive biting presented very significant risks to experimenters. In contrast, a more tractable model, expressing “dumb” or paralytic rabies, would have much to recommend it. Another distinct weakness of the model as first employed was the long and variable period of incubation before disease expression. Pasteur and his colleagues would later address that issue, modifying the nature of the inoculum and route of delivery in a different host.

Great progress in the development of an experimental rabbit model was achieved by M. Galtier, who held the chair of pathology in the veterinary school at Lyons, France (68). In 1879, he reported that rabies in the dog could be transmitted to the rabbit and could be serially transmitted from rabbit to rabbit (20). Significantly, he found that the predominant symptoms in the rabbit were paralysis and convulsions, hence of much less risk to the experimenter than the furious biting behavior of the infected dog. Galtier reported that once the disease declared itself, the rabbit was able to live for 3 or 4 days, thereby facilitating further observations and transmission studies as well as an accurate demonstration of positive transmission. Of great importance as a model, the rabbit revealed a shorter incubation period than other species: the peak onset of illness in 25 cases occurred between 13 and 23 days. Thus, the rabbit model had been well characterized in Galtier’s hands by 1879, before Pasteur and his associates launched their epic work. From an examination of Pasteur’s laboratory notebooks, Gerald L. Geison confirmed that Pasteur’s work on rabies began at the end of the following year, 10 December 1880, to be precise (21). Pasteur’s laboratory notebooks, numbering over 100, had become available to scholars at the Bibliothèque nationale (National Library of France) in the 1970s, so Galtier’s priority in establishing and refining the rabbit model could be verified (21). Patrice Debré, a biographer of Pasteur, suggested that Galtier had been considered for the Nobel Prize in 1908, but he died before the award decision, precluding the honor (11).

Galtier had also attempted, without success, transmission of rabies by CNS tissue. However, Henri Duboué, a Paris physician, found otherwise. According to Debré, Duboué wrote to Pasteur in January 1881 that the “‘morbid agent’” traveled to the medulla oblongata from the site of the bite (11). Vallery-Radot’s biography of Pasteur confirmed the theory of Duboué but indicated that it was unsupported by experimental evidence (63). It remained for Pasteur and his colleagues, notably Emile Roux, to exploit the CNS aspects of the model. The goal was to shorten the period of incubation and to achieve full success in experimental transmission of rabies in a convenient animal model (63).

The motivation to select a particular experimental problem by a scientist is usually complex and may reflect the less tangible facts of the scientist’s life. The distinguished Pasteur biographer René J. Dubos, an eminent 20th-century biologist, noted that “There is something odd in the selection by Pasteur of rabies as the next subject for his experimental studies” (12). Perhaps Pasteur’s horrifying experience as a young boy witnessing the effects of a rabid wolf’s attack was a motivation. More pressing than the relatively low prevalence of rabies was the nature of the disease itself. As well summarized by Gieson, “. . . rabies embodied the ultimate in agony and degradation, stripping its victims of their sanity and reducing them to quivering convulsive shadows of their former selves” (21).

Pasteur refined the animal model. Vallery-Radot noted that “Evidently the saliva was not a sure agent for experiments, and if more information was to be obtained, some other means had to be found of obtaining it” (63). With the growing realization of a seat of infection in the brain, Pasteur and his collaborators—“actually it was Emile Roux” as noted by Geison—developed an intracranial method of inoculation (2). The skull was trephinated, and infected cerebral matter from the rabid animal was deposited under the dura mater on the surface of the brain. This method achieved success: in the words of Vallery-Radot, “. . . rabies was contracted surely and swiftly” (63), and according to Geison, these collaborators “. . . had at last developed a uniformly successful method of transmitting the disease from animal-to-animal” (21). The next step was development of a brief and reproducible incubation period. Subdural transmission in rabbits resulted in a progressively shortened, “fixed” incubation period, as brief as 7 days (63).

What followed in the Pasteur story of rabies postexposure prophylaxis is the stuff of legend. Briefly told, a method of attenuating the infectivity of the rabid rabbit CNS by desiccation was devised, and the material could protect dogs from virulent infection. Circumstances led to the historic postexposure treatment in 1885 of Joseph Meister, a 9-year-old boy brought to Pasteur from Alsace (Fig. 3). Because the boy was suffering severe bites from a rabid dog, his case was thought likely to be fatal (69). The dog was killed and its stomach was found to contain hay, straw, and wood fragments, apparently leaving “no doubt that the dog was rabid” (34). Such was the state of diagnostic virology! After a series of inoculations of progressively more virulent rabies spinal cord tissue, Meister survived. The floodgates of postexposure treatment opened, with hundreds arriving from all parts of the globe. Gieson, in his review of Pasteur’s laboratory notebooks, observed “some remarkable discrepancies between the public and private versions of this celebrated story” (21). According to the notebooks, Joseph Meister was not the first person in whom the antirabies treatment was attempted. Of importance, however, is the train of events leading to the demonstration of rabies as a filterable virus.

Figure 3 Rabies prevention. The recognition that the Pasteurian treatment could prevent rabies after the bite of a rabid animal resulted in dramatic public acceptance. In this print from 1885, Pasteur is depicted observing the inoculation of a boy to prevent hydrophobia (rabies in humans). (Courtesy of the National Library of Medicine.)

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In 1884, “. . . Pasteur did concede that he and his collaborators had still not managed to isolate and cultivate a rabies microbe in artificial media” (21). While rabies virus was incapable of growth on artificial media, one of the criteria that defined a viral agent, rabies was transmissible to susceptible animal hosts. The stage was set for the demonstration that rabies was a filterable, “ultravisible” agent. That task would prove to be a technical challenge. However, in 1903, Paul Remlinger, the Director of the Imperial Institute of Bacteriology at Constantinople, reported “Le passage du virus rabique à travers les filtres” (51). His success depended on the techniques of filtration and inoculation and on the number of experimental animals tested. As summarized by a contemporary, John McFadyean, a veterinarian, the most porous of the Berkefeld filters would allow passage of the virus, whereas more finely porous filters would hold the agent back. The filtrates were deposited under the dura, and several rabbits were inoculated with each filtrate (41). The success in filtration was confirmed by others (41). Remlinger was gracious, attributing to Pasteur the idea of rabies as ultramicroscopic (51). It was a fitting tribute to Pasteur.

Coincidentally, in 1903, A. Negri described inclusions in the neurons of the CNS of rabid animals and humans (44). While the inclusions were misinterpreted as parasites, they became in the following years “an extremely important method of diagnosis” (28). Thus, 1903 was a seminal year in the study of rabies. Using the animal model developed by Galtier and refined by Pasteur and his colleagues, Remlinger demonstrated the filterable, that is, viral, nature of the agent of rabies. Negri developed a histopathological hallmark for rabies which, when positive, was a “rapid viral diagnostic technique.”