Generated randomly and selected functionally? The nature of enterovirus recombination

Abstract

Recombination within RNA viruses is an important evolutionary process that can significantly influence virus fitness and has been repeatedly reported to compromise vaccine effectiveness. However, its precise mechanism is poorly understood. Here, we used an established poliovirus-based in vitro assay (CRE-REP) to investigate the molecular determinants of recombination and show that neither sequence identity, nor RNA structure, have any significant effect on recombination frequency. Since the CRE-REP assay is confined by the ability to detect infectious virus progeny, we utilized deep sequencing to study the recombinant genome population that arises early in infection and before any bottleneck of selection for viable progeny. We were able to detect and analyse hundreds of recombinants containing sequence insertions or deletions, or that were of wild type genome length. While we found higher diversity in recombination events than from CRE-REP assays, the analyses demonstrate no biases towards sequence or structure, in support of the CRE-REP assay findings. The results suggest that genome functionality and fitness are of greater importance in determining the identity of recombinants. These studies provide critical information that can improve our understanding of the recombination process, and consequently allow for the production of less recombinogenic and more stable vaccines.

Imprecise recombinant viruses evolve via a fitness-driven, iterative process of polymerase template-switching events

“…We therefore investigated the latter using Illumina next generation sequencing and observed significant levels of diversity within the RNA population (Fig 5A; Fig S3). We have previously demonstrated that the Illumina library preparation methods utilised here do not generate artefactual junctions that could confound the results [13]. We demonstrated a very good overlap between precise junctions independently identified using both ONT and Illumina technologies (Fig 5A).…”

“…As recently demonstrated for recombination [13], viruses undergoing resolution generate an extensive population of diverse molecules (Fig S3), the identity of which could arise from additional in trans polymerase-mediated template switching events.…”

“…In this we were able to detect a much greater range of crossover junctions in the RNA population than were present in the resulting recombinant infectious virus population [13]. We therefore applied this method to investigate the process of resolution and to compare with results from the ONT analysis.…”

First come, first served: Superinfection exclusion in Deformed wing virus is dependent upon sequence identity and not the order of virus acquisition

“…Analysis of these sequences showed that the majority were out of frame deletions (Woodford, unpublished), and so incapable of replicating. Our studies using analogous approaches in other RNA viruses show that these types of aberrant products are not unusual and reflect the random nature of the molecular mechanism of recombination ([42, 56]).…”

“…In arthropods antiviral RNAi response acts via generation of short double stranded RNAs (siRNA) from virus RNA replication intermediates through cleavage by the enzyme Dicer. These are further used by the RNA induced silencing complex (RISC) to target the destruction of complementary sequences [55, 56]. Hence viral RNA genomes exhibiting greater identity are likely to generate higher numbers of cross-reactive siRNAs.…”