Collective Viral Spread Mediated by Virion Aggregates Promotes the Evolution of Defective Interfering Particles

Abstract

A growing number of studies report that viruses can spread in groups in so-called collective infectious units. By increasing the cellular multiplicity of infection, collective dispersal may allow for social-like interactions, such as cooperation or cheating. Yet, little is known about how such interactions evolve. In previous work with vesicular stomatitis virus, we showed that virion aggregation accelerates early infection stages in most cell types, providing a short-term fitness benefit to the virus. Here, we examine the effects of virion aggregation over several infection cycles. Flow cytometry, deep sequencing, infectivity assays, reverse transcription-quantitative PCR, and electron microscopy revealed that virion aggregation rapidly promotes the emergence of defective interfering particles. Therefore, virion aggregation provides immediate fitness benefits to the virus but incurs fitness costs after a few viral generations. This suggests that an optimal strategy for the virus is to undergo virion aggregation only episodically, for instance, during interhost transmission.IMPORTANCE Recent insights have revealed that viruses use a highly diverse set of strategies to release multiple viral genomes into the same target cells, allowing the emergence of beneficial, but also detrimental, interactions among viruses inside infected cells. This has prompted interest among microbial ecologists and evolutionary biologists in studying how collective dispersal impacts the outcome of viral infections. Here, we have used vesicular stomatitis virus as a model system to study the evolutionary implications of collective dissemination mediated by viral aggregates, since this virus can spontaneously aggregate in the presence of saliva. We find that saliva-driven aggregation has a dual effect on viral fitness; whereas aggregation tends to increase infectivity in the very short term, virion aggregates are highly susceptible to invasion by noncooperative defective variants after a few viral generations.

Keywords: collective infectious units; defective interfering particles; experimental evolution; social evolution; vesicular stomatitis virus.

FIG 1

Evolution of saliva-driven coinfection rates. (A) Founder virus. Fluorescence micrographs of infection foci produced at 12 hpi in BHK-21 cells. Right, cells were inoculated with a 1:1 mix of VSV-GFP and VSV-Cherry (nonaggregated). Left, prior to inoculation, viral particles were aggregated in the presence of human saliva. Yellow foci indicate coinfection of cells with the two variants. The gray background shows noninfected cells in phase contrast. Bar = 1 mm. (B) Progressive loss of coinfection in viruses evolved under the saliva-driven aggregation regime (orange) and titer reached by these lines after each transfer (blue). The percentages of cells coinfected with VSV-GFP and VSV-mCherry were determined in cultures infected with saliva-treated virus after each evolution transfer using flow cytometry. Viral titers were quantified after each transfer by the plaque assay. Each dot represents an evolution line (one dot for the founder, three dots for evolved lines). Least-squares regression lines (dashed) are shown.

FIG 2

VSV deep sequencing. (A) Nonsynonymous mutation frequencies along the viral genome. Mutation frequencies (no. of mutated reads/no. of total base reads, excluding indels) were calculated by pooling all reads of lineages of the same treatment (aggregated versus nonaggregated). (B) Nonsynonymous polymorphisms at >0.1% in frequency found in VSV genes in the founder virus and evolved lines.

FIG 3

Virion aggregation promotes the emergence of DIPs. (A) Yield reduction assay. The titer of a reporter virus at 16 hpi is shown as a function of the fraction of tested/(reported + tested) viruses in the inoculum. The titer of the reporter virus decayed roughly proportionally to the fraction of founder virus in the inoculum (gray), as expected from direct competition (dashed line; r² = 0.884). In contrast, the titer of the reported virus decayed faster when mixed with A1 virus (red) or a virus serially transferred at high density (10 PFU/cell; blue), suggesting the presence of interfering viruses in these tested viral populations. (B) Electron micrographs of A3 viruses (left) and C2 viruses (right). Bullet-shaped virions correspond to VSV carrying complete genomes, whereas shorter, thimble-shaped viruses corresponded to DIPs. Scale bars = 200 nm. DIPs were found in all A lines but only rarely in C lines.