Читать книгу To Catch a Virus - John Booss - Страница 16

By 1900, several pieces of the puzzle were in place that would help to explain the spread of yellow fever. Of principal interest was the mode of transmission; the precedent had been set in 1897 with malaria when mosquitoes were identified as vectors. Walter Reed acknowledged “. . . the splendid work of Ross, Bignami, and others with regard to the propagation of malarial fever. . .” (30). Reed also acknowledged the work of J. C. Nott in 1848 in suggesting “. . . that the spread of yellow fever could not be assumed by the assumption of a diffusible miasm in the atmosphere but required the presence of an intermediate host. . . .” The specific mosquito, then called Stegomyia fasciata, later called Culex fasciatus and finally Aedes aegypti (19), had been identified by Carlos Finlay in 1886. However, Finlay had failed to convince his colleagues that this mosquito was responsible for disease spread. One of the principal reasons for that failure was ignorance of an extrinsic incubation period, a time during which the virus matures in the mosquito.

Reed recognized the careful work of Henry Rose Carter (8) (Fig. 5) in two small towns in Mississippi in 1898, “‘demonstrating the interval between the infecting and secondary cases of yellow fever.’” Reed was gracious in declaring, “To Dr. Carlos J. Finlay, of Havana, must be given, however, full credit for the theory of the propagation of yellow fever by means of the mosquito” (30).

Figure 5 Henry Rose Carter, 1909. As a member of the Marine Hospital Service, he was able to deduce a delay between primary and secondary cases of yellow fever. This extrinsic incubation period implied the need for another, nonhuman, host, later shown to be the mosquito. He was assigned to the Panama Canal Zone in 1904 to work on yellow fever. (Courtesy of Historical Collections & Services, Claude Moore Health Sciences Library, University of Virginia.)

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Sill missing was a crucial piece of the puzzle: isolation of an agreed-upon etiological agent of yellow fever. Although this was a time of exciting discoveries in medical bacteriology, credit in disproving putative bacterial causes must go to George Miller Sternberg, a pioneer American bacteriologist. An author of early American textbooks of bacteriology in the 1890s, he spent most of his career in the U.S. Army and was largely self-taught in bacteriology. In 1890, he published Report on the Etiology and Prevention of Yellow Fever, in which he thoroughly disposed of the several candidate bacteria as the cause of yellow fever (36). In 1897, Sternberg appointed Reed and James Carroll to investigate yet another candidate, Bacillus icteroides (Sanarelli) along with his own candidate, Bacillus X. Reed and Carroll, who were joined by Aristedes Agramonte in Cuba in 1898, demonstrated that Bacillus icteroides “bore no relation to the disease” (7).

In 1900, by-then Surgeon General Sternberg appointed Reed, Carroll, Agramonte, and Jesse W. Lazear to a board of army medical officers to investigate yellow fever in Cuba (7). The board first met 25 June 1900 (2). Astonishingly, within 4 months the board was able to report at the Annual Meeting of the American Public Health Association in October 1900 that Culex fasciatus served as the intermediate host for yellow fever (29). In clearing the field of bacterial contenders and in appointing the U.S. Army Yellow Fever Commission in 1900, Sternberg can be credited as the catalyst of these findings on the transmission and etiology of yellow fever (Fig. 6).

Figure 6 George Miller Sternberg. Known as America’s first bacteriologist, he produced the first textbook of bacteriology in the United States. He was Surgeon General of the Army from 1893 to 1902, during which time he appointed the Yellow Fever Commission. (Courtesy of the Historical Collections & Services, Claude Moore Health Services Library, University of Virginia.)

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Without a bacterial agent identified in culture, the need remained to do studies in human subjects. The use of experimental animals was to come later (37, 38, 39). Finlay had already used human subjects in his earlier studies (14). Still, the investigators recognized the ethical implications of the studies in humans and offered themselves first. In James Carroll’s words, “Then arose the question of the tremendous responsibility involved in the use of human beings for experimental purposes. It was concluded that the results, if positive, would be sufficient justification of the undertaking. It was suggested that we subject ourselves to the same risk, and this suggestion was accepted by Dr. Reed and Dr. Lazear” (7). Carroll became the first experimental subject, accepting the risks ahead of other volunteers.

The circumstances to further study yellow fever were propitious. Following the Spanish-American War, yellow fever appeared yet again in Cuba, placing the populace and American troops at risk. Soon after arrival in Cuba, Reed and his Commission colleague, Agramonte (Fig. 7), visited an army barracks at Pinar del Rio where an outbreak was occurring. Observations made on that visit “. . . did not tend to strengthen one’s belief in the theory of the propagation of yellow fever by fomites” (30). A curious story was told of that visit. Only one of nine prisoners, well guarded in jail, had come down with yellow fever. Speculation was raised that an insect such as a mosquito had bitten the one prisoner. That speculation was buttressed by the observations of Carter of the interval between infecting and secondary cases (9, 21). It was decided to test Finlay’s theory of mosquito transmission of yellow fever.

Figure 7 The Yellow Fever Commission consisted of (upper left) Walter Reed, who led the Commission; (lower left) James Carroll, who performed the filtration experiment; (upper right) Aristides Agramonte; and (lower right) Jesse W. Lazear, who became infected in the course of the experiments and died. In a remarkably brief period of time at the turn of the 20th century, the Commission under Reed demonstrated that the disease was transmitted by mosquitoes and that it could be transmitted by filtered blood and thus was caused by a virus. (Courtesy of the Historical Collections & Services, Claude Moore Health Sciences Library, University of Virginia, except for the image of Walter Reed, courtesy of The National Library of Medicine.)

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In Reed’s words, “. . . the search for the specific agent of yellow fever while not abandoned, should be given secondary consideration, until we had first definitely learned something about the way or ways in which the disease was propagated from the sick to the well” (30). In preliminary experiments by Lazear, mosquito eggs were supplied by Finlay, and mosquitoes were raised in the laboratory, allowed to feed on yellow fever patients, and allowed to bite human subjects. First among the subjects was Carroll, who fell ill and almost perished (21). Lazear, apparently bitten by a stray mosquito in 1900, was a victim of their research efforts: he contracted yellow fever and died. The results of the experiments showed that 2 of 11 experimentally infected subjects developed yellow fever. It was concluded that “The mosquito acts as the intermediate host for the parasite of yellow fever, and it is highly probable that the disease is only propagated through the bite of this insect” (italics in the original) (29).

There followed the construction of two small buildings in an open field to compare the transmission of yellow fever by fomites with transmission by the bites of infected mosquitoes or inoculation of infected blood. The “Infected Mosquito Building” was well ventilated and divided into two compartments by a screen. The “Infected Clothing and Bedding Building” was purposely not well ventilated so as to retain any noxious effects of bed clothing, pajamas, and other items from previously infected cases. After some early discouraging results, John R. Kissinger, a soldier who Reed praised for having volunteered “solely in the interest of humanity and the cause of science” and who would accept no payment, came down with experimental yellow fever from the bites of infected mosquitoes (30). In these experiments, six of seven “non-immunes” bitten by infected mosquitoes in the Infected Mosquito Building became ill with yellow fever (32). None of the seven subjects in the Infected Clothing and Bedding Building exposed to fomites from cases of yellow fever became ill, nor did subjects become ill who had remained behind the screen, not bitten by mosquitoes.

The clarity of the design of comparison groups and the results were decisive: 85.71% infected by mosquitoes versus 0% by fomites. In the definitive publication in JAMA, “The Etiology of Yellow Fever: an Additional Note,” Reed, Carroll, and Agramonte ended with several major conclusions. In addition to confirming that “C. fasciatus serves as the intermediate host,” they determined that 12 days or more was required after contamination for the mosquito to transmit the infection. Thus, they determined experimentally what Carter had observed epidemiologically. They found that yellow fever could be transmitted by blood subcutaneously inoculated when taken from a patient on the first 2 days of the illness. They concluded that yellow fever resulting from a mosquito bite “confers immunity” against attempted reinfection with infected blood (32).

In memory of Lazear, the experimental station established by Reed, where the crucial studies were conducted demonstrating the transmission of yellow fever by mosquitoes and not by fomites, was christened Camp Lazear. Ironically, although Carroll recovered from acute yellow fever infection, he tragically died 7 years later of myocarditis attributed to that attack of yellow fever.

An important piece of the puzzle still remained to fall in place. Walter Reed and his colleagues’ final conclusion of their JAMA report was that “. . . the specific cause of this disease remains to be discovered” (32). Having turned away from that goal in their transmission studies, Carroll returned to the project. Initially confronted with local objections to further experimentation, Carroll resumed his studies in September 1901 in Cuba on the nature of the infecting agent (7). In the crucial experiment, six individuals were exposed to the bites of infected mosquitoes (33). Four did not develop yellow fever, but two did. Blood was taken from patients I and II for further transmission study, but due to an accident to the vacuum pump, the blood from patient I could not be used. The blood from patient II was divided into three aliquots of partially defibrinated and diluted serum. The first aliquot, a positive control, was left untreated and successfully transmitted yellow fever to patient III. The second aliquot was heated to 55°C for 10 minutes and failed to transmit disease to patients IV, V, and VI. Based on previous work on heat stability with toxins, Reed and Carroll argued against a toxin. The third aliquot was “slowly filtered through a new Berkefeld laboratory-filter” and the filtrate was inoculated into patients VII, VIII, and IX. Patients VII and VIII developed “unmistakable” attacks of yellow fever; patient IX remained well. The scientific data were presented at the annual meeting of the Society of American Bacteriologists, 31 December 1901 and 1 January 1902. Thus, clinical virology can be said to have started in the first years of the 20th century.

Presciently, they noted that the most effective means of controlling the spread of yellow fever was through destruction of mosquito breeding areas and prevention of mosquitoes biting the sick (31). This strategy was employed with extraordinary success by William Crawford Gorgas of the U.S. Army, also present in Havana at that time. A brilliant story unto himself, Gorgas cleared Havana of yellow fever. An interesting connection can be noted here between Gorgas and J. C. Nott, mentioned above, who had suggested that yellow fever transmission required an intermediate host. Nott, coincidentally, was the doctor who delivered the infant Gorgas, whose success in the story of yellow fever eradication was based on Nott’s theory. A few years later, Gorgas also cleared the Canal Zone of yellow fever and malaria, allowing the successful construction of the Panama Canal (17, 18, 25). Thus, the yellow fever-mosquito story was intimately wound into America’s expanding international role (26).

It was clear from the classic studies of the Yellow Fever Commission that transmission experiments had to be performed in human subjects. However, that presented significant ethical issues, not only for potentially lethal viral infections such as yellow fever but also for permanently disabling anterior poliomyelitis.

Although Carroll reported in 1904 that others had also shown that the agent of yellow fever was filterable (7), attempts to identify a bacterial cause continued (2). It was not until a successful experimental animal host, the rhesus monkey, was demonstrated in 1928 (37) and then the successful use of intracerebral inoculation of white mice (38, 39) that large-scale studies of the yellow fever virus could be undertaken and the bacterial candidates dismissed.