Reassortments in multipartite viruses – Reassort

All experiments used FBNSV as the main model species. Its genome is composed of 8 ssDNA segments (designated C, M, N, R, S, U1, U2, U4), each encoding a single gene. Transmission by vector (aphid) is assumed to follow the circulating non-propagative type, with virus particles moving from the intestine to the salivary glands of aphids without replication.

Two genotypes, [FBNSV-AZ;15] and [FBNSV-AZ;10/12b], and their respective field hosts, Vicia sativa [vetch] and Lens culinaris (lentil), were used for reassortment studies. These isolates were selected because they belong to different clades of FBNSV, and therefore their segments can be genetically distinguished, and they were isolated from localities very close geographically and on different host species. Since infectious clones of these two genotypes were not available prior to our study, they had to be created ex silico. Infectious clones can be infiltrated into beans or transmitted by aphid to vetches and lentils from infected beans. Other species of the genus Nanovirus used to study interspecific reassortments are faba bean necrotic yellows virus (FBNYV), whose field host is the faba bean, Vicia faba, and the more genetically distant pea necrotic yellows dwarf virus (PNYDV), with pea (Pisum sativum) as its field host. Both have a genomic organization similar to FBNSV. Infectious clones of both species were already available.

For studies on the involvement of nonconcomitant transmission of segments to reassortments, we used the genotype [FBNSV – JKI2000] whose field host plant is faba bean and for which we had an infectious clone before the start of this project. We systematically studied the effect of single segment reassortments between viral genotypes by infecting plants with genotypes consisting of one heterologous segment and seven genomic background segments. For each infected plant, we characterized the effects of infection on the plant over time (symptoms, height), quantified the load of each segment and their sum (viral load) by qPCR, and estimated their aphid transmission capacity. We competed segments of two genotypes in a given genomic context (e.g., infection by C segments of two isolates and the other seven segments of one of the two isolates) to observe the outcome of competition. We evolved some reassorting genotypes, and in parallel parental genotypes, for 10 successive passages by aphid to see if reassortant characteristics change over time.

We knew that the genomic formula (frequency distribution of different segments within a host) changes with the host species; here we were able to show that the genomic formulae of three FBNSV genotypes differ for a given host species. Thus, the genomic formula varies according to both the host species and the viral genotype. Systematic analysis of the effects of intraspecific reassortments of a segment of the FBNSV showed that the overall infection phenotype (host plant symptoms and height, viral load, transmission rate) is not very sensitive to reassortments. Conversely, the genomic formula is very labile, with reassortants having different genomic formulae from their parents in almost all cases. For the following experiments we had to focus on two segments for logistical reasons. We chose C and U1 because we observed large differences in genomic formula for reassortants of these segments. Competitions between segments coding for the same protein in a given genomic context showed that [FBNSV-AZ;10/12b] segments accumulated more than [FBNSV-AZ15] segments in both genomic contexts. Experimental evolution during 10 aphid passages of reassortant genotypes led to extinction during passages of almost all reassortant lineages, while parental lineages were maintained. Interspecific reassortments between FBNSV, FBNYV, and PNYDV were studied for the R, C, and U2 segments because of their low, medium, and high levels of genomic divergence among species. Over the entire genome FBNSV and FBNYV are close, whereas PNYDV is more genetically distant. The results depend on the genetic distance between parents over the whole genome, the genetic distance on the reassorting segment, and the host plant. On faba bean, the original host of FBNSV and FBNYV, no reassortment involving any of the three segments of PNYDV in a FBNSV or FBNYV background worked: for U2 and C we obtained infections but consisting only of the 7 segments of the majority parent. Since R is indispensable for infection, reassortantss for R are not viable at this genomic differentiation scale. The reassortants between FBNSV and FBNYV for R complement each other well, but for U2 and C the complementation is unidirectional: FBNSV segments complement FBNYV genomic background well, but in the reverse direction FBNYV reassorting segments are not found in infected plants, we find only the 7 FBNSV genomic background segments. Finally, on pea, the original host of PNYDV, reassortants involving a segment of this species function in all cases except U2 in an FBNYV background.

The transmission of the different segments can be done independently and delayed in time.

The discovery of the possibility of non-concomitant transmission of genomic segments opens up several possibilities. First, this discovery was made by studying the transmission of non-essential segments. It would be important to confirm that this non-concomitant transmission can also occur when key segments are involved. In particular, it will be necessary to characterize the time window during which the rescue of inoculated segments during a first transmission can be done by a second inoculation bringing the missing segments; in other words, estimate the life expectancy of latent genomic segments. In addition to better estimating the putative cost of maintaining the genomic integrity of multipartite viruses, this knowledge would allow a better characterization, and hence understanding, of the circulation of genomic segments in a landscape. The results on the phenotypic effects of intra- and interspecific reassortments show a high robustness to the former, probably mediated by the lability of the genomic formula, and an intolerance to the latter though dependent on the host plant. Thus genomic exchange and complementation seem to be easy between different genotypes of the same species, allowing viruses to explore the genotypic space but also to easily complement incomplete inoculations, bypassing the problem of maintaining genomic integrity. At the interspecific level, more stringent barriers are observed, though host-dependent exchange bridges exist. It should be borne in mind that these results and inferences are based on the exchanges of a single segment; potentially the barriers could be greater if several segments are involved, which remains to be confirmed. Thus, in combination with nonconcomitant transmission of segments, it becomes important to focus now on the circulation of genomic segments of several genotypes of several viral species in a landscape of several host plant species, more or less favourable to different genotypes and permissive of their reassortments, connected by different vector species. Such a study would make it possible to know the constraints these viruses face in natura, but also the opportunities they have to survive, transmit, invent genetic novelty and adapt to their environment. This knowledge should allow us to better characterize the risk that multipartite viruses potentially represent for their host plants, which in the case of Nanoviruses are the important for agriculture family of Legumes (https://agriculture.gouv.fr/les-legumineuses-une-famille-de-vegetaux-redecouverture).

Genetic exchanges are one of the main mechanisms generating within-population diversity that selection can act on. Even though some extremely successful genotypes may be thus produced, many deleterious genotypes are also produced; the net balance that could result from systematic experimental gene exchange between two distinct genotypes is unknown, and this is true for any organisms. This project aims at filling this gap by using as model system multipartite viruses, whose genome is divided in several segments packaged independently, each viral particle carrying only one segment and each segment encoding only one gene. Multipartite viruses can exchange entire genomic segments, an additional mechanism of genomic shuffling termed reassortment. Using the octopartite nanovirus faba bean necrotic stunt virus (FBNSV), a parasite of legume plants, we will measure several viral fitness components for intra- and inter-specific single segment (and thus single gene) reassortants. This will allow us to estimate whether reassorting is on average beneficial/detrimental and whether specific viral genes/functions are more likely to produce beneficial reassortants. Competition experiments between reassorting and parental-genotypes will show whether mechanisms favoring species/genotype genomic integrity are at play, and experimental evolution will show whether deleterious effects of reassortment may be alleviated during coevolution of reassorting segments with the rest of the genome. Finally, transmission experiments will investigate whether reassorting may facilitate the transmission of multipartite viruses by allowing for the non-concomitant transmission of different genomic segments. This project may thus provide (i) the first systematic characterization of the fitness and biological trait effects of reassortment; (ii) a potential mechanism through which the between-host transmission cost of multipartitism may be alleviated.