href="#ulink_4aa6aa9e-5d86-5b90-b89e-5eef745bb5ae">57). The article described work that was fastidious in the care to exclude exogenous infection and elegant in the clarity of its results. It also demonstrated the experimental serendipity of susceptibility to various viral infections by different species of animals, so-called “species specificity.” Although the possibility of influenza being a viral disease had been raised in about 1914 (31), ambiguous results had been obtained in humans, and unsuccessful attempts were made in other species. Smith et al. commented that “The filtrates, proved to be bacteriologically sterile, were used in attempts to infect many different species. All such attempts were entirely unsuccessful until the ferret was used . . .” (57).
The report in 1933, which the investigators termed “a preliminary communication,” detailed a number of critical parameters for experimentation. These included the source and nature of the inoculum, throat washings from people sick with influenza. Experimental manipulation and important clinical observations in ferrets included intranasal instillation, the biphasic clinical course, the nasal histopathology in infected ferrets, and serial passage. Finally, the characteristics of the agent were documented, including filterability, the absence of bacterial growth, and the neutralization of the virus by serum taken from people who had recovered from clinical influenza and from ferrets that had recovered from experimental infection. Smith and colleagues also studied the relationship to the virus of swine influenza received from Richard Shope; they found “a close antigenic relationship” (57). However, “[i]n striking contrast to swine influenza,” there was no synergistic role for H. influenzae suis in the production of experimental disease in ferrets.
The Mill Hill investigators’ report on the successful use of the ferret as an animal model received prompt confirmation from investigators on other continents. For example, T. Francis, at the Rockefeller Institute in New York City, working with sputum obtained from patients in a 1934 influenza epidemic in Puerto Rico, transmitted the disease to ferrets (18). F. M. Burnet reported the experimental transmission of influenza to ferrets from a 1935 epidemic in Melbourne, Australia (4).
The successful isolation of human influenza virus in the ferret in several laboratories triggered the exploration of other biological systems. Andrewes et al. reported the successful transmission of ferret-passed virus to mice (1), which was also reported in the following month by Francis (18). Reports of the successful cultivation of the virus in minced chicken embryo soon emerged (19, 58). Smith concluded that the egg membrane technique was “unsuitable for the study of this virus” (58). However, further studies would show that this biological system, the embryonated egg of chickens, was remarkably productive for the understanding of human influenza infection.
There are parallels between the yellow fever story and the study of the cause of human influenza. Noguchi claimed to have isolated a bacterial cause of yellow fever, Leptospira icteroides, in guinea pigs. The bacterium, while causing symptoms in guinea pigs similar to those of yellow fever, turned out not to be the cause of yellow fever, and the guinea pig turned out not to be a susceptible host of yellow fever. As noted above, Pfeiffer in 1893 reported the isolation of a gram-negative bacterium as the cause of human influenza (48). Haemophilus influenzae, or Pfeiffer’s bacillus, as it came to be known, was for some time thought to be the cause of influenza (42). Richard Shope and Paul Lewis found Haemophilus influenzae suis to be associated with swine influenza (36, 54, 55). It was to be shown that the ferret was the model of choice to study human influenza and that a synergistic bacterial infection was not present (57).
There was also a tragic link to three of the diseases discussed in this chapter. Paul A. Lewis was one of the first to transmit poliovirus serially in monkeys (16) and to identify it as a filterable agent in his work with Flexner (17). He had collaborated with Shope on his studies of swine influenza (70), only to later succumb to yellow fever while studying it in Brazil.
Embryonated Eggs
“The recognition of the potentialities of the method for virus research was almost entirely due to Ernest William Goodpasture and his collaborators,” wrote W. I. B. Beveridge and F. M. Burnet in their 1946 monograph on the use of the chicken embryo for the cultivation of viruses and rickettsiae (3). Goodpasture was a Tennessee-born pathologist who early in his career had trained with William Welch, who was one of the founders of American pathology at Johns Hopkins (70). As the medical writer Greer Williams told the story, working at Vanderbilt University, Goodpasture had put an M.D. pathology trainee, Eugene Woodruff, to work on investigating the pathogenesis of fowlpox (70). His wife, Alice Woodruff, a Ph.D. physiologist, joined the effort somewhat later in an attempt to culture the virus. After their unsuccessful attempts in tissue culture, Goodpasture suggested to Alice Woodruff that they try embryonated chicken eggs for the study of fowlpox.
As later reviewed by Goodpasture, there was a very long history of the study of embryonated eggs in experimental biology (23). He noted that the first reported creation of a “window” in the egg to observe the embryo was by L. Beguelin in 1749. He also noted that the first published study of the use of embryonated eggs in infection was in 1905 by C. Levaditi working with the spirillum of fowl. The laurel for the first application of embryonated eggs to the study of viral infection goes to Peyton Rous and James B. Murphy, who in 1911 reported the successful transplantation of a transmissible sarcoma of fowl (52). The tumor was transmitted to the chick not only by finely divided tumor but also by “a filtrate free of the tumor cells,” resulting in tumors in the membranes of the embryo.
Starting with the technique of E. R. Clark for operating on chicken embryos (9), Goodpasture’s group developed and adapted the technique for virological studies. The methods to deposit the virus in a sterile fashion on the chorioallantoic membrane or in the allantoic or amniotic cavities were carefully described (22). The first report by the Goodpasture group on the use of the embryonated egg was that of Alice Woodruff and Goodpasture for infection with fowlpox virus (71). Goodpasture’s principal interest was in the pathogenesis of viral infections; however, with others he also developed the embryonated egg for vaccine production (22). That work laid the foundation for the
