1.3 MULTIPLICATION OF VIRUSES

Although methods of assaying viruses had been developed, there were still considerable doubts as to the nature of viruses. d’Hérelle believed that the infecting phage particle multiplied within the bacterium and that its progeny were liberated upon lysis of the host cell, whereas others believed that phage-induced dissolution of bacterial cultures was merely the consequence of a stimulation of lytic enzymes endogenous to the bacteria. Yet another school of thought was that phages could pass freely in and out of bacterial cells and that lysis of bacteria was a secondary phenomenon not necessarily concerned with the growth of a phage. It was Delbruck who ended the controversy by pointing out that two phenomena were involved, lysis from within and lysis from without. The type of lysis observed was dependent on the ratio of infecting phages to bacteria (multiplicity of infection). At a low multiplicity of infection (with the ratio of phages to bacteria no greater than 2 : 1), then the phages infect the cells, multiply, and lyse the cells from within. When the multiplicity of infection is high, i.e. many hundreds of phages per bacterium, the cells are lysed directly, and rather than an increase in phage titer there is a decrease. Lysis is due to weakening of the cell wall when large numbers of phages are attached. Convincing support for d’Hérelle’s hypothesis was provided by the one-step growth experiment of Ellis and Delbruck in 1939. A phage preparation such as bacteriophage λ (lambda) is mixed with a suspension of the bacterium Escherichia coli at a multiplicity of infection of 10 PFU per cell, ensuring that virtually all cells are infected. Then after allowing 5 minutes for the phage to attach, the culture is centrifuged to pellet the cells and attached phage. Medium containing unattached phage is discarded. The cells are then resuspended in fresh medium. Samples of medium are withdrawn at regular intervals, cells removed and assayed for infectious phage. The results obtained are shown in Fig. 1.2. After a latent period of 17 minutes in which no phage increase is detected in cell-free medium, there is a sudden rise in PFU in the medium. This “burst” size represents the average of many different bursts from individual cells, and can be calculated from the total virus yield/number of cell infected. The entire growth cycle here takes around 30 minutes, although this will vary with different viruses and cells. The amount of cell-associated virus is determined by taking the cells pelleted from the medium, disrupting them, and assaying for virus infectivity as before. The fact that virus appears inside the cells before it appears in the medium demonstrates the intracellular nature of phage replication. It can be seen also that the kinetics of appearance of intracellular phage particles are linear, not exponential. This is consistent with particles being produced by assembly from component parts, rather than by binary fission.


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