Читать книгу Principles of Virology - Jane Flint, S. Jane Flint - Страница 106

Infection depends on the random collision of cells and virus particles. When susceptible cells are mixed with a suspension of virus particles, some cells are uninfected and other cells receive one, two, three, etc., particles. The distribution of virus particles per cell is best described by the Poisson distribution:

P(k) = e^–mmk/k!

In this equation, P(k) is the fraction of cells infected by k virus particles. The multiplicity of infection, m, is calculated from the proportion of uninfected cells, P(0), which can be determined experimentally. If k is made 0 in the above equation, then

P(0) = e^–m and m = −1n P(0)

The fraction of cells receiving 0, 1, and >1 virus particle in a culture of 10⁶ cells infected with an MOI of 10 can be determined as follows.

The fraction of cells that receive 0 particles is

P(0) = e–10 = 4.5 × 10^–5

and in a culture of 10⁶ cells, this equals 45 uninfected cells.

The fraction of cells that receive 1 particle is

P(1) = 10 × 4.5 × 10^–5 = 4.5 × 10^–4

and in a culture of 10⁶ cells, 450 cells receive 1 particle.

The fraction of cells that receive >1 particle is

P(>1) = 1 − e^–m(m + 1) = 0.9995

and in a culture of 10⁶ cells, 999,500 cells receive >1 particle. [The value in this equation is obtained by subtracting from 1 (the sum of all probabilities for any value of k) the probabilities P(0) and P(1).]

The fraction of cells receiving 0, 1, and >1 virus particle in a culture of 10⁶ cells infected with an MOI of 0.001 is

1 P(0) = 99.99%P(1) = 0.0999% (for 106 cells, 104 are infected)P(>1) = 10–6The MOI required to infect 99% of the cells in a cell culture dish isP(0) = 1% = 0.01m = −1n (0.01) = 4.6 PFU per cell.

The yield of infectious virus per cell can be calculated from the data collected during a one-step growth experiment. This value varies widely among different viruses and with different virus-host cell combinations. For many viruses, increasing the multiplicity of infection above a certain point does not increase the yield: cells have a finite capacity to produce new virus particles. In fact, infecting at a very high multiplicity of infection can cause premature cell lysis and decrease virus yields.

The kinetics of the one-step infectious cycle can vary dramatically among viruses. For example, enveloped viruses that mature by budding from the plasma membrane, as discussed in Chapter 13, generally become infectious only as they leave the cell, and therefore, little intracellular infectious virus can be detected (Fig. 2.19B). The curve shown in Fig. 2.19A illustrates the pattern observed for a DNA virus with the long latent and synthetic phases typical of many DNA viruses, some retroviruses, and reovirus. For small RNA viruses, the entire growth curve is complete within 6 to 8 h, and the latent and synthetic phases are correspondingly shorter.

One-step growth curve analysis can provide quantitative information about different virus-host systems. It is frequently employed to study mutant viruses to determine what parts of the infectious cycle are affected by a particular genetic lesion. It is also valuable for studying the multiplication of a new virus or viral reproduction in a new virus-host cell combination.

When cells are infected at a low multiplicity of infection, several cycles of viral reproduction may occur (Fig. 2.18B). Growth curves established under these conditions can also provide useful information. When infection is carried out at a high multiplicity, a mutation may fail to have an obvious effect on viral reproduction. The defect may only become evident following a low-multiplicity infection. Because the effect of a mutation in each cycle is multiplied, a small effect can be amplified after several cycles. Defects in the ability of viruses to spread from cell to cell may also be revealed when multiple cycles of reproduction occur.