Plaques Formed by Mutagenized Viral Populations Have Elevated Coinfection Frequencies

Abstract

The plaque assay is a common technique used to measure virus concentrations and is based upon the principle that each plaque represents a single infectious unit. As such, the number of plaques is expected to correlate linearly with the virus dilution plated, and each plaque should be formed by a single founder virus. Here, we examined whether more than one virus can contribute to plaque formation. By using genetic and phenotypic assays with genetically marked polioviruses, we found that multiple parental viruses are present in 5 to 7% of plaques, even at an extremely low multiplicity of infection. We demonstrated through visual and biophysical assays that, like many viral stocks, our viral stocks contain both single particles and aggregates. These data suggest that aggregated virions are capable of inducing coinfection and chimeric plaque formation. In fact, inducing virion aggregation via exposure to low pH increased coinfection in a flow cytometry-based assay. We hypothesized that plaques generated by viruses with high mutation loads may have higher coinfection frequencies due to processes restoring fitness, such as complementation and recombination. Indeed, we found that coinfection frequency correlated with mutation load, with 17% chimeric plaque formation for heavily mutagenized viruses. Importantly, the frequency of chimeric plaques may be underestimated by up to threefold, since coinfection with the same parental virus cannot be scored in our assay. This work indicates that more than one virus can contribute to plaque formation and that coinfection may assist plaque formation in situations where the amount of genome damage is high.

Single-Cell Virology: On-Chip Investigation of Viral Infection Dynamics

“…A few years ago, we showed for PV that formation of a plaque required more than a single genome (Korboukh et al, 2014). Similar conclusions have been reached by others studying PV (Aguilera et al, 2017) and vesicular stomatitis virus (Combe et al, 2015). Together, these studies highlight the concept of an infectious unit, the number of virions or genomes required to establish infection of a cell.…”

Viral coinfection is shaped by host ecology and virus–virus interactions across diverse microbial taxa and environments

“…If this is correct, variability in potential and realized coinfection, as found in this study, suggests that the manifestation of superinfection exclusion will vary across viral groups according to their ecological context. Accordingly, there are specific viral mechanisms that promote coinfection, as found in this study with ssDNA/dsDNA coinfections and in other studies (Turner et al 1999; Dang et al 2004; Cicin-Sain et al 2005; Altan-Bonnet and Chen 2015; Aguilera et al 2017; Erez et al 2017). I found substantial variation in cross-infectivity, culture coinfection, and single-cell coinfection and, in the analyses herein, ecology was always a statistically significant and strong predictor of coinfection, suggesting that the selective pressure for coinfection is going to vary across local ecologies.…”

Limits of variation, specific infectivity, and genome packaging of massively recoded poliovirus genomes

“…Note. During review of this paper, Aguilera et al, published a study [mBio, March/April 2017 (84)] concluding that in infections with purified PV (expressing no phenotype) at low MOI (0.000001 PFU/cell) approximately 5% of the cells were infected with >1 virion. Dual infections increased (to 8%) when infections with two different PV mutants were analyzed and assayed phenotypically.…”