Apparent cooperativity between human CMV virions introduces errors in conventional methods of calculating multiplicity of infection.

Whether infection of cells by individual virions occurs randomly, or if there is some form(s) of competition or cooperativity between individual virions, remains largely unknown for most virus-cell associations. Here, we studied cooperativity/competition for three different strains of human cytomegalovirus (HCMV) on two different cell types (fibroblasts and epithelial cells). By titrating viral inocula concentrations in small steps over several orders of magnitude, and by using flow cytometry to precisely measure the frequency of infected cells, we found that for most virus-cell associations, the frequency of cell infection increases faster than linear with an increasing inoculum concentration, indicating cooperativity between individual infecting virions. Mathematical modeling suggests that this apparent cooperativity cannot be explained by heterogeneity in either the infectivity of the individual virions or the resistance of individual cells to infection, or by simple aggregation/clumping of viral particles. Stochastic simulations of two additional alternative models that allow for (1) reduction in cell resistance to infection when exposed to multiple virions, or (2) compensation in infectivity of poorly infectious virions when coinfecting cells with more infectious virions, resulted in apparent viral cooperativity. Analysis of other published datasets suggests the presence of apparent viral cooperativity for HIV and vaccinia virus, infecting CRFK or HeLa cells, respectively, but not for tobacco mosaic virus forming plaques on plant leaves. We thus (1) propose a methodology to rigorously evaluate apparent cooperativity of viruses infecting target cells, and (2) demonstrate that knowing the degree of virus cooperativity for any given virus-cell combination is important for an accurate quantification of multiplicity of infection.