Etude de la structure génétique d'une population de Plum Pox virus (PPV) au sein d'un hôte pérenne et de son évolution

Equipe 2La structure génétique d’une population virale au sein d’un hôte constitue un maillon essentiel pour la compréhension de l’évolution des virus et de leur épidémiologie, cependant cet aspect reste très peu étudié. Dans le cas des phytovirus, les plantes pérennes constituent un modèle de premier choix pour une étude intra-hôte détaillée. Le travail de thèse se positionne sur cette question et propose d’étudier la structure génétique des populations de Plum pox virus (PPV) --organisme de quarantaine responsable de la maladie de la Sharka chez les arbres fruitiers à noyau du genre Prunus-- au sein d’un hôte pérenne, et d’évaluer les effets de l’alternance des saisons et de la transmission par puceron sur leur évolution. Nous avons réalisé un échantillonnage exhaustif d’un pêcher chroniquement infecté par le PPV depuis 13 ans, suite à un événement d’inoculation unique. Notre échantillonnage a pris en compte l’architecture de l’arbre. L’analyse des échantillons démontre pour la première fois qu’à l’intérieur d’un arbre, il existe des populations virales totalement isolées qui évoluent indépendamment et qui sont en expansion contiguë dans des charpentières différentes. Nous avons aussi mis en évidence un phénomène de "clonage" biologique des variants de PPV à l’intérieur des feuilles. Ainsi, la somme des variants contenus individuellement dans l’ensemble des feuilles, représentent une sorte de collection de l’immense variabilité des génomes viraux que peut contenir la partie aérienne de l’arbre. Un suivi de la variabilité des populations de PPV, au sein de plantes ligneuses inoculées par une source virale homogène, au cours d’une succession de cycles saisonniers accélérés au laboratoire, a confirmé le mécanisme biologique permettant l’évolution de PPV dans son hôte par expansion contiguë : le PPV est transmis depuis les tissus néoformées d’une saison (jeunes branches) à ceux qui les prolongent l’année suivante (nouvelles branches et feuilles associées). Enfin, le suivi de nombreuses lignées de PPV au fil d’une série de cycles de transmission, de plantes à plantes par pucerons vecteurs, suggère la présence d’une dérive génétique plus importante que celle détectée dans les cycles de simulation de saisons

Etude de la structure génétique d une population de Plum pox virus au sein d un hôte pérenne et de son évolution

MONTPELLIER-SupAgro La Gaillarde (341722306) / SudocSudocFranceF

Adaptation du CaMV à son environnement: évolution des séquences nucléotidiques

National audienc

Etude de la structure génétique intra-arbre d'une population de Plum pox virus (PPV)

National audienc

Distinct Viral Populations Differentiate and Evolve Independently in a Single Perennial Host Plant

The complex structure of virus populations has been the object of intensive study in bacteria, animals, and plants for over a decade. While it is clear that tremendous genetic diversity is rapidly generated during viral replication, the distribution of this diversity within a single host remains an obscure area in this field of science. Among animal viruses, only Human immunodeficiency virus and Hepatitis C virus populations have recently been thoroughly investigated at an intrahost level, where they are structured as metapopulations, demonstrating that the host cannot be considered simply as a “bag” containing a homogeneous or unstructured swarm of mutant viral genomes. In plants, a few reports suggested a possible heterogeneous distribution of virus variants at different locations within the host but provided no clues as to how this heterogeneity is structured. Here, we report the most exhaustive study of the structure and evolution of a virus population ever reported at the intrahost level through the analysis of a Prunus tree infected by Plum pox virus for over 13 years following a single inoculation event and by using analysis of molecular variance at different hierarchical levels combined with nested clade analysis. We demonstrate that, following systemic invasion of the host, the virus population differentiates into several distinct populations that are isolated in different branches, where they evolve independently through contiguous range expansion while colonizing newly formed organs. Moreover, we present and discuss evidence that the tree harbors a huge “bank” of viral clones, each isolated in one of the myriad leaves

Specialization of a phytovirus to its environment: experimental evidence for a cost of adaptation

National audienceStudying patterns and genetic basis of adaptation is crucial to understand viral emergence and to anticipate suitable strategies of control. In this work, we propose to detect adaptive mutations in different environments (constant and variable) and to evaluate their phenotypic expression (measured by evaluation of fitness and virulence) in different environments. We designed an experimental evolution of Cauliflower mosaic virus by transmitting viral populations from plant to plant in either two homogeneous environments (Arabidopsis thaliana or Nicotiana bigelovii) or a variable environment (alternation of both species). For each of these three treatments, 10 independent viral populations were evolved in parallel. After five and ten passages (approx. 150 and 300 viral generations, respectively), we sequenced complete genome length of consensus viral populations and look for mutations appearing at the same locus in more than two independent populations. These parallel mutations have a high probability to be a signature(s) of adaptive changes. Genomic data revealed that parallel mutations appeared differentially depending on environmental contexts: (i) most of them were accumulating in constant environments; (ii) none were common to several environments. Evaluation of phenotypic effects of viral evolution in different environment gave the evidence for a cost of adaptation and suggest a positive correlation between virulence and virus accumulation. These genomic and phenotypic results will be discussed in light of evolution of specialist/generalist strategies

Quantitative PCR analysis of the salivary gland hypertrophy virus (GpSGHV) in a laboratory colony of Glossina pallidipes

International audienceMany species of tsetse flies can be infected by a virus that causes salivary gland hypertrophy (SGH) and virus isolated from Glossina pallidipes (GpSGHV) has recently been sequenced. Flies having SGH have a reduced fecundity and fertility. To better understand the impact of this virus in a laboratory colony of G. pallidipes, where the majority of flies are infected but asymptomatic, and to follow the development of SGH in symptomatic flies in relation to virus copy number, a quantitative PCR (qPCR) method was developed. The qPCR analyses revealed that in asymptomatic flies virus copy number averaged 1.68E+5, 2.05E+5 and 1.07E+7 log10 in DNA from an excised leg, salivary glands and a whole fly, respectively. In symptomatic flies the virus copy number in the same organs averaged 1.34E+7, 1.42E+10 and 1.5E+9, respectively. Despite these statistically significant differences (p much less-than 0.0001) in virus copy number between asymptomatic and symptomatic flies, there was no correlation between age and virus copy number for either sets in adult flies. A clear correlation between virus copy number in pupae and their mothers was observed. Reverse transcription quantitative PCR (RT-qPCR) of the viral messenger RNA encoding ODV-E66, an envelope protein, revealed a clear correlation between virus copy number and the level of gene expression with values of 2.77 log10 in asymptomatic males and 6.10 log10 in symptomatic males. Taken together these results confirm the close relationship between virus copy number and SGH syndrome. They demonstrate the vertical transmission of GpSGHV from mother to progeny, and suggest that the development of SGH may be correlated to the virus copy number acquired by the larva during its intra-uterine development

VAPA, an Innovative ‘‘Virus-Acquisition Phenotyping Assay’ ’ Opens New Horizons in Research into the Vector- Transmission of Plant Viruses

Host-to-host transmission—a key step in plant virus infection cycles—is ensured predominantly by vectors, especially aphids and related insects. A deeper understanding of the mechanisms of virus acquisition, which is critical to vectortransmission, might help to design future virus control strategies, because any newly discovered molecular or cellular process is a potential target for hampering viral spread within host populations. With this aim in mind, an aphid membranefeeding assay was developed where aphids transmitted two non-circulative viruses [cauliflower mosaic virus (CaMV) and turnip mosaic virus] from infected protoplasts. In this assay, virus acquisition occurs exclusively from living cells. Most interestingly, we also show that CaMV is less efficiently transmitted by aphids in the presence of oryzalin—a microtubuledepolymerising drug. The example presented here demonstrates that our technically simple ‘‘virus-acquisition phenotyping assay’ ’ (VAPA) provides a first opportunity to implement correlative studies relating the physiological state of infected plan

Protoplast transmission of TuMV.

<p><b>A.</b> Effect of different acquisition times on TuMV transmission. After 60 min of starving, aphids were allowed different protoplast acquisition periods before being placed on test plants for inoculation. No effect of different acquisition time was observed (<i>P</i> = 0.942; df = 2; Kruskal-Wallis test). The graph combines data from 24 test plants per assay with 6 assays for acquisition times from 15 to 60 min (<90 min), 6 assays for an acquisition time of 90 min, and 4 assays for acquisition times between 120 and 180 min (>90 min). <b>B.</b> Pre-acquisition starving has no effect on TuMV transmission. Aphids were starved for 60, 120, or 180 min before being allowed a 60-min acquisition period on TuMV-infected protoplasts and subsequent transfer to test plants for inoculation. The graph shows that there is no measurable effect of pre-acquisition starving on transmission efficiency (<i>P</i> = 0.193; df = 2; Kruskal-Wallis test). 6 assays of 24 test plants were tested for 60 min starvation, 13 assays for the 120 min time point, and 4 assays for 180 min starvation. <b>C.</b> TuMV acquisition from protoplasts absolutely requires living protoplasts. Aphids were allowed to feed for 60 min on infected intact (control) or dead (sheared) protoplasts before transfer to test plants for inoculation. The difference between transmission rates from living and sheared protoplasts is highly significant (<i>P</i><0.001; Mann-Whitney test). The graph shows data from 18 assays (9 for each condition) of 24 test plants each. <b>D.</b> Shearing does not inactivate TuMV. Infected protoplasts were homogenised by repeated passage through a syringe needle and then rub-inoculated to turnip test plants. The graph shows that virions from sheared protoplasts were as infectious as those from intact control protoplasts. Three 24 plant cultivation trays were inoculated per condition. All graphs present mean values ± standard deviation.</p

Experimental set-up of a typical VAPA (virus-acquisition phenotyping assay) transmission experiment.

<p><b>A.</b> To construct the aphid harvesting device, a 1000 µl pipette tip with the utmost 2–3 mm cut off is attached to a silicon tube, with a Miracloth net squeezed in between. The tube is connected to a vacuum source (mechanical pump or human respiratory system) and aphids are sucked up into the pipette tip by negative pressure and retained by the net. <b>B.</b> The virus-acquisition phenotyping apparatus consists of a copper ring sealed with a Parafilm M membrane. Aphids placed in the ring are attracted to the membrane by a light source (not shown); protoplasts are then deposited onto the membrane and spread evenly with a cover glass. After a defined acquisition access period, aphids are transferred with an artist's paint-brush to test plants for inoculation.</p