CULTIVATION 959 



only in the earliest stages ; in herpetic keratitis of rabbits the inclusion bodies 

 appear within the first 24 hours ; in venereal herpes they are best seen on the 

 third day, and so on. There appears to be some relationship between the presence 

 of inclusion bodies and the infectivity of the tissue ; in herpetic keratitis of rabbits, 

 for example, it is said that with the disappearance of the nuclear inclusion bodies 

 the disease can be no longer propagated. Experimentally, the formation of 

 inclusion bodies can be stimulated by the inociilation not only of infected tissue 

 extracts, but often of filtered cell-free material. They can, moreover, often be 

 demonstrated in tissue cultures (Andrewes 1929, Elvers et al. 1929). 



The earlier workers regarded these inclusion bodies as protozoa, and pictured 

 them as varying stages of an elaborate life-cycle. Subsequently they were believed 

 to represent cellular degeneration products due to nucleolar extrusion, vacuolation 

 of the cytoplasm, and other processes consequent on the attack of the virus. 

 Von Prowazek regarded them as of a dual nature, consisting of micro-organisms 

 embedded in material deposited around them as the result of a reaction of the 

 cell protoplasm ; for these bodies the term " Chlamydozoa " — literally cloak 

 animals — was proposed. It is now, however, becoming increasingly clear that 

 intracellular inclusion bodies are essentially colonies of the infecting virus. Since 

 Woodruff and Goodpasture (1929, 1930) showed that the Bollinger inclusion body 

 of fowl-pox consisted of 10,000-20,000 minute Borrel bodies, and that a singly 

 Bollinger body, washed free from surrounding virus, was capable of giving rise to 

 a typical fowl-pox lesion on skin inoculation, it has been difficult to regard inclusion 

 bodies as other than intracellular aggregations of elementary virus particles. By 

 tryptic digestion, by maceration, by surface tension, or other means it has now been 

 shown that the inclusion bodies of ectromelia (Barnard and Elford 1931), vaccinia 

 (Ledingham 1931, Paschen 1932), and psittacosis (-Bedson and Bland 1932, 1934) 

 contain masses of elementary bodies which are apparently responsible for giving 

 rise to characteristic intracellular changes. Moreover, the formation of inclusion 

 bodies from elementary bodies has now been watched experimentally in the chorio- 

 allantoic membrane and the rabbit's cornea after inoculation with vaccinia virus 

 (Herzberg 1936, Tang and Wei 1937, Himmelweit 1938, Bland and Eobinow 1939). 

 Whether the intranuclear acidophilic bodies, which are so common in infections 

 caused by neutrotropic viruses, are of the same nature as the intracellular but 

 extranuclear bodies has not yet been made clear either by morphological study 

 or by the micro-incineration technique (see Cowdry 1933). There is some evidence 

 that they result from flocculation of the nuclear colloids (Findlay 1939). What- 

 ever the structure of inclusion bodies may be, however, there is no doubt whatever 

 of their significance ; their presence in the tissue is a sure sign of infection, and 

 is made use of in the routine diagnosis of certain of the filtrable virus diseases, 

 such as rabies. (For a pictorial review of inclusion bodies see Findlay and Ludford 

 1926, and for a general account of their properties see Goodpasture 1929, 1929-30, 

 Ledingham 1935. See also Figs. 298-302.) 



Cultivation. — With the possible exception of saprophytic viruses cultivable 

 in serum broth, the filtrable viruses have proved refractory to cultivation in the 

 absence of living cells. In 1915 Noguchi succeeded in obtaining pure cultures in 

 vivo of vaccinia virus, by growing it in the testicles of rabbits and bulls. The virus, 

 obtained from skin scrapings, was first freed from bacteria by suitable means, and 

 was then inoculated intratesticularly into rabbits. Transfers were made every four 

 days. Several passages were necessary before the virus became adapted to growth 



