The eclipse phase of vaccinia virus growing in chick embryo cell monolayers and some technical procedures which affect its demonstration

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Abstract
1. When chick embryo cell monolayers were infected with ultrasonically treated elementary body suspension by a short period of adsorption some of the adsorbed virus lost infectivity in a few minutes. This was followed by a more gradual loss during incubation of the cultures. The lowest point was reached at 4–6 hr. followed by a rise in titre due to production of new virus at 7–8 hr. The quantitative assessment of these changes is discussed.2. In such cultures, titration of intact and disintegrated cells after incubation for 4–5 hr., indicated an eclipse phase by showing an absence of infective virus in cells which ultimately produced it. The process of eclipse continued for several hours. With inoculum that had not been subjected to ultrasonic treatment it was not possible to show this by infecting HeLa cell monolayers.3. Ultrasonically treated inoculum, compared with a similar, but untreated inoculum, had a three to flvefold higher titre. The virus units in it were more uniformly capable of infecting cells. After its adsorption to monolayers a rapid loss of some infective virus occurred within a few minutes which was not demonstrable with untreated inoculum. During incubation of monolayers infected with treated inoculum the loss of infectivity was faster, there was a more rapid production of new virus and a greater yield. The nature of the changes in the virus which were brought about by ultrasonic treatment have not been explained, but the use of such an inoculum markedly affected the results of experiments.4. Treatment of infected monolayers with trypsin or EDTA caused the cells to shed as much as half their virus. This virus would not be released spontaneously into the medium or removed from monolayers by washing. This applied to all periods of incubation up to 24 hr. or more when marked increase of virus had occurred.5. The disintegration of infected cells by ultrasonic treatment was efficient in releasing infective virus from them, although it was not possible to be certain all the virus in the cells was rendered detectable by this technique.6. The effect of some technical procedures on the interpretation of results is discussed.