Abstract
The viral multipartite genomic architecture and packaging strategy, in which the genome is divided in multiple segments separately encapsidated, stands as an evolutionary conundrum associated with theoretical high fitness costs associated to the maintenance of genome integrity. Earlier studies within the research team showed the capacity of the faba bean necrotic stunt virus (FBNSV; Nanovirus genus, genome composed of 8 segments) to accumulate each segment in distinct host cells and to complement the system at the supra-cellular level by exchanging gene products among interconnected cells, thereby greatly reducing the theorized intra-host necessary MOI. The aim of this doctoral research was to delve into the mechanisms through which nanoviruses manage to maintain their genome integrity, with a specific focus on reassortments and on their effects on the fitness of resulting hybrid genotypes. A reassortment corresponds to the replacement of one or more segments by homologous ones from a distinct parental genotype. The primary objective was to conduct a systematic comprehensive phenotypic characterization of all 16 possible single-segment reassortants involving two distinct FBNSV isolates.The objectives were addressed through the utilization of experimental techniques such as agro-inoculation, aphid inoculation, qPCR, phenotyping of infected plants, and confocal microscopy. Our findings revealed the FBNSV capacity to transmit its distinct segments from host-to-host non-concomitantly and to eventually reconstitute a complete genome, thereby significantly diminishing the costs related to maintenance of the genomic integrity.These findings, along with the capacity of nanovirus genomic segments to effectively complement each other at the supra-cellular level, significantly expand the spatial scale at which reassortment can occur. A thorough literature review on reassortments in single-stranded DNA multipartite viruses underscored that the replication of reassorted segments appears as the primary constraint for the success of the new generated genotypesThis doctoral research has led to the production of two new infectious clones, [AZ;15] and [AZ;10_12b], each representing distinct FBNSV isolates. Together with the isolate [JKI-2000], which was already available at the start of the PhD, we now possess three infectious clones representing isolates from the three main FBNSV phylogenetic clades. These clones will facilitate additional investigations of genetic exchanges, mixed infections, and ecological dynamics.The phenotypic characterization of isolates was conducted across three host plants (faba beans, lentils, and vetches) and one aphid vector (Aphis craccivora), who appeared to be common to all three viral isolates holding significant implications on ecological dynamics of nanovirus natural populations. This analysis revealed phenotypic diversity among isolates, notably regarding their genome formula, across all host plants and aphid vectors. Finally, the systematic phenotypic characterization of single-segment reassortants between [AZ;15] and [AZ;10_12b] isolates demonstrated the viability of all 16 possible combinations across three host plants. While these findings hold potential ecological and evolutionary significance for nanoviruses, it is important to emphasize that they were obtained under controlled environment with no competition from other genotypes.The results from this doctoral research significantly expand the genetic resources and opportunities available for exploring nanovirus biology, reassortment dynamics, and the strategies employed by ssDNA multipartite viruses in general, at least nanoviruses, to mitigate the costs associated with the maintenance of genome integrity.