The next major development was the report by Macfarlane Burnet in 1935 of the growth of influenza virus in embryonated eggs (5). Serial passage of influenza in eggs was required before egg membrane lesions became obvious. Hence, it did not appear that the technique would prove of use for primary virus isolation. Presciently, Burnet suggested that the method could serve as a means of antigen preparation for immunization.
The necessary step to bring the technique into primary diagnostic work resulted from a chance observation. George Hirst, working at the Rockefeller Foundation in New York, reported that “When the allantoic fluid from chick embryos previously infected with strains of influenza A virus was being removed, it was noted that the red cells of the infected chick, coming from ruptured vessels, agglutinated in the allantoic fluid” (26). This observation was confirmed by L. McClelland and R. Hare at the University of Toronto (40). Further expansion of this observation facilitated the development of diagnostic tests for influenza, both primary isolation and antibody development. For virus isolation, clinical specimens were inoculated into eggs, the amniotic fluids were harvested, and a dilution of fluid was mixed with red blood cells. Agglutination indicated likely influenza virus infection, which was proven by the inhibition of agglutination by virus-specific antisera. Henceforth, use of the ferret, with its attendant costs, housing, and susceptibility to exogenous infection, was superseded by use of embryonated eggs (27). The embryonated egg proved to be a boon to virological studies in general and to influenza in particular. As a measure of the importance of the technique, Nobel Laureate Macfarlane Burnet simplified his description of many years of research to say, “From 1935–1955, one can summarize my life as learning about influenza virus in chick embryos” (6).
The use of experimental animals and that of embryonated eggs were the principal means of virus isolation prior to the development of cell culture in the 1950s. Animals were crucial to the isolation and characterization of herpes viruses, among many others. In parallel, development of serological techniques to measure virus-specific antibodies was to become the major means of viral diagnosis for several decades. That story is told in the next chapter.
References
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