Plaques formed by mutagenized viral populations have elevated co-infection 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, plaque number 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 5-7% 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 co-infection and chimeric plaque formation. In fact, inducing virion aggregation via exposure to low pH increased co-infection in a flow cytometry-based assay. We hypothesized that plaques generated by viruses with high mutation loads may have higher co-infection frequencies due to fitness restoring processes such as complementation and recombination. Indeed, we found that co-infection 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 three-fold, since co-infection 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 co-infection may assist plaque formation in situations where the amount of genome damage is high.

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 (Dang et al, 2004; Cicin-Sain et al, 2005; Turner et al, 1999; Altan-Bonnet and Chen, 2015; Erez et al, 2017; Aguilera 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.…”

“…Most attention in virus-virus interactions has focused on mechanisms limiting coinfection, with the assumption that coinfection invariably reduces host fitness (Berngruber et al, 2010). However, some patterns of non-random coinfection suggest elevated coinfection (Dang et al, 2004; Cicin-Sain et al, 2005; Turner et al, 1999) and specific viral mechanisms that promote co-infection have been identified, including enhanced expression of the phage attachment site upon infection (Joseph et al, 2009), particle aggregation (Altan-Bonnet and Chen, 2015; Aguilera et al, 2017), and phage-phage communication (Erez et al, 2017). Systematic coinfection has been proposed (Roux et al, 2012) to explain findings of chimeric viruses of mixed nucleic acids in metagenome reads (Diemer and Stedman, 2012; Roux et al, 2013).…”

Enteric Viral Co-Infections: Pathogenesis and Perspective

“…Recent studies have revealed that multiple enteric eukaryotic viruses, including RV, NoV, and PV may be transmitted in aggregates or viral clusters [ 134 , 135 ]. Groups of assorted virions, often containing mutagenized genomes, are replicated and transmitted cell-to-cell in vesicle-like structures [ 135 , 136 , 137 ]. For aggregation to occur, virions must be able to accumulate and be transmitted from infected cells to neighboring cells with minimal interference.…”

“…For aggregation to occur, virions must be able to accumulate and be transmitted from infected cells to neighboring cells with minimal interference. Aggregation may offer viruses multiple advantages, including an increased cellular multiplicity of infection [ 138 ], and greater opportunity for complementation or recombination to restore fitness [ 135 ]. Gut bacteria have been shown to facilitate the aggregation of PV, and this could be a mechanism of the bacterially-mediated enhancement of infection by other enteric viruses [ 139 ].…”