Kinetics of virus production from single cells

Abstract

The production of virus by infected cells is an essential process for the spread and persistence of viral diseases, the effectiveness of live-viral vaccines, and the manufacture of viruses for diverse applications. Yet despite its importance, methods to precisely measure virus production from cells are lacking. Most methods test infected-cell populations, masking how individual cells behave. Here we measured the kinetics of virus production from single cells. We combined simple steps of liquid-phase infection, serial dilution, centrifugation, and harvesting, without specialized equipment, to track the production of virus particles from BHK cells infected with vesicular stomatitis virus. Remarkably, cell-to-cell differences in latent times to virus release were within a factor of two, while production rates and virus yields spanned over 300-fold, highlighting an extreme diversity in virus production for cells from the same population. These findings have fundamental and technological implications for health and disease.

Figure 1

Kinetics of virus release from single infected cells. (a) Our method combines steps of virus infection, cell immobilization by adsorption, multi-time sampling, and analysis, and (b) one-step virus release profiles, from which one can estimate parameters of single-cell virus production: latent time, rise rate, yield and rise time.

Figure 2

Virus production profiles for virus-infected cells. (a) Production rate profiles for 12 cells drawn from the same initial population of infected cells, and (b) one-step production of virus from single cells.

Figure 3

Correlations among virus-release traits from single cells. Cells that produce virus earlier (shorter latent times) tend to produce fewer virus particles at slower rates as indicated by (a) yield vs latent time and (b) rise rate vs latent time; (c) yields and rise rates are correlated owing to relatively narrow distributions among rise times, (d) larger yields require a longer period of virus production, and (e) earlier producers tend to be productive for shorter periods than later producers.

Figure 4

Virus yields from single infected cells. (a) Dependence of single-cell yields on MOI. Average single-cell yields at MOI 5 and MOI 15 did not differ (p-value 0.076, Student’s t-test, non-equal variance, 95 percent confidence interval). Error bars at each MOI reflect one standard deviation of the average yield. (b) Yield distribution from single-cells from the MOI-series experiment (MOI 5 Yield) and the kinetic experiment (MOI 5 Cumulative Yield) where the average yields did not differ (p-value 0.60, Student’s t-test, non-equal variance, 95 percent confidence interval).