Читать книгу Principles of Virology - Jane Flint, S. Jane Flint - Страница 105

One-step growth analysis soon became adapted for studying the reproduction of animal viruses. The experiment begins with removal of the medium from the cell monolayer and addition of virus in a small volume to promote rapid adsorption. After ∼1 h, unadsorbed inoculum containing virus particles is removed, the cells are washed, and fresh medium is added. At different times after infection, samples of the cell culture supernatant are collected and the virus titer is determined. The kinetics of intracellular virus production can be monitored by removing the medium containing extracellular particles, scraping the cells into fresh medium, and lysing them. A cell extract is prepared after removal of cellular debris by centrifugation, and the virus titer in the extract is measured.

The results of a one-step growth experiment establish a number of important features about viral reproduction. In the example shown in Fig. 2.19A, the first 11 h after infection constitute the eclipse period, during which the viral nucleic acid is uncoated from its protective shell and no infectious virus can be detected inside cells. The small number of infectious particles detected during this period probably represents adsorbed virus that was not uncoated. Beginning at 12 h after adsorption, the quantity of intracellular infectious virus particles begins to increase, marking the onset of the synthetic phase, during which assembly begins. During the latent period, no extracellular virus can be detected. At 18 h after adsorption, virions are released from cells into the extracellular medium. Ultimately, virus numbers plateau as the cells become metabolically and structurally incapable of supporting additional reproduction.