Xavier Romand

Chloé Bernardy
email: cbernardy@chu-grenoble.fr.33628735836

Minh Vu

A, B , Anaïs Courtier B Chuong Nguyen

Anaïs Courtier

Candice Trocme

Margot Clapasson

A,D , Bertrand Toussaint C,D Marie-Hélène Paclet

Bertrand Toussaint

A,B Philippe Gaudin

Athan Baillet

Systemic calprotectin and chronic inflammatory rheumatic diseases

Keywords: calprotectin, biomarker, chronic inflammatory rheumatic diseases

Introduction

Calprotectin (S100A8/A9 or Myeloid Related Protein 8/14) is predominantly expressed by myelomonocytic cells [START_REF] Edgeworth | Identification of p8,14 as a highly abundant heterodimeric calcium binding protein complex of myeloid cells[END_REF], namely monocytes and neutrophils but also in early differentiation stages of macrophage [START_REF] Odink | Two calcium-binding proteins in infiltrate macrophages of rheumatoid arthritis[END_REF]. This protein constitutes, respectively, 40% and 5% of the polymorphonuclear neutrophil cytosolic and monocytes protein content. Calprotectin is an alarmin with important proinflammatory properties mainly secreted by activated neutrophils in a calcium-dependant manner. It is an heterodimeric complex of S100A8 and S100A9 proteins, which are calcium-binding protein with EF hand calcium domains [START_REF] Korndörfer | The crystal structure of the human (S100A8/S100A9)2 heterotetramer, calprotectin, illustrates how conformational changes of interacting alphahelices can determine specific association of two EF-hand proteins[END_REF], constituting the physiologically active conformation of this protein.

Beside calcium concentration and posttranslational modifications such as phosphorylation or nitrosylation influences calprotectin quaternary structure, and therefore its physiological functions [START_REF] Lim | Oxidative modifications of DAMPs suppress inflammation: the case for S100A8 and S100A9[END_REF]. Extracellular calprotectin induces, in particular via binding to Toll like receptor 4 (TLR-4) and Receptor of Advanced Glycation Endproducts (RAGE), a pro-inflammatory response in monocytes/macrophages, neutrophils, lymphocytes and endothelial cell [START_REF] Rahman | TLR4 endogenous ligand MRP8/14 level in enthesitis-related arthritis and its association with disease activity and TLR4 expression[END_REF]. The C-terminal tail of S100A9 represents the region to which carboxyl groups of fatty acid bind. Hence, calprotectin is a unique arachidonic acid reservoir with a potential impact on prostanoids production by the modulation of cyclooxygenases activity.

Calprotectin plays an important role in inflammation as it triggers the innate immunity receptors [START_REF] Rahman | TLR4 endogenous ligand MRP8/14 level in enthesitis-related arthritis and its association with disease activity and TLR4 expression[END_REF].

Hence, calprotectin was identified as a marker of Rheumatoid Arthritis (RA) in the synovial fluid, with synovial concentration differentiating RA from other rheumatic diseases [START_REF] Baillet | Synovial fluid proteomic fingerprint: S100A8, S100A9 and S100A12 proteins discriminate rheumatoid arthritis from other inflammatory joint diseases[END_REF]. Growing body of evidence suggests a role for calprotectin in several Chronic Inflammatory Rheumatic Disorders such as juvenile chronic arthritis [START_REF] Frosch | Myeloid-related proteins 8 and 14 are specifically secreted during interaction of phagocytes and activated endothelium and are useful markers for monitoring disease activity in pauciarticular-onset juvenile rheumatoid arthritis[END_REF]RA [START_REF] Hammer | Calprotectin (a major leucocyte protein) is strongly and independently correlated with joint inflammation and damage in rheumatoid arthritis[END_REF], Behcet disease [START_REF] Oktayoglu | Elevated serum levels of calprotectin (myeloid-related protein 8/14) in patients with ankylosing spondylitis and its association with disease activity and quality of life[END_REF] and ANCA-associated vasculitis (AAV) [START_REF] Thai | Are anti-proteinase-3 ANCA a useful marker of granulomatosis with polyangiitis (Wegener's) relapses? Results of a retrospective study on 126 patients[END_REF].

Several assays are available to measure calprotectin levels such as ELISA, immunochromatography and immunoturbidimetry [START_REF] Whitehead | Between-assay variability of faecal calprotectin enzyme-linked immunosorbent assay kits[END_REF]. At the moment, no guideline indicates the technique which should be used to estimate calprotectin level.

Calprotectin (s100 a8/a9 protein) and inflammatory conditions 2.1 Calprotectin is a danger signal increased upon neutrophil activation

Calprotectin has a critical role in inflammation as it can activate the innate immunity pathway sensed by RAGE or TLR. Calprotectin is released when phagocytes are activated in a calcium-dependant manner. Neutrophils release calprotectin when interaction between monocyte and activated endothelium under inflammatory conditions(3) which leads to calprotectin deposit onto the endothelium cells. S100A9 subunit interacts with endothelial cell through heparan sulfate proteoglycans. Calprotectin also recognizes carboxylated N-glycans expressed on inflammatory activated endothelial cells. These mechanisms favor leucocyte extravasation [START_REF] Rahman | TLR4 endogenous ligand MRP8/14 level in enthesitis-related arthritis and its association with disease activity and TLR4 expression[END_REF]. Calprotectin exerts paracrine and autocrine effects [START_REF] Berthier | Molecular interface of S100A8 with cytochrome b558 and NADPH oxidase activation[END_REF]. Calprotectin activates TLR-4 in myeloid cells. Ligation of TLR4 by calprotectin induces wide-ranging effects on neutrophils including cytokines, matrix metalloproteinases, chemokine production and generation of reactive oxygen species.

Calprotectin enhances neutrophil oxidative burst

S100A8 and S100A9 were recently introduced as partners for phagocyte NADPH oxidase regulation.

They potentiate NADPH oxidase activation as positive allosteric effectors; but they also induce the change of cytochrome b 558 conformation that initiates its activation [START_REF] Berthier | Molecular interface of S100A8 with cytochrome b558 and NADPH oxidase activation[END_REF].

In vitro studies suggested that S100A8 and S100A9 combine into various dimers; either homodimers or tetramers, depending of calcium concentrations. Stimulation of neutrophils by calprotectin activates NAPDH oxidase activity and increases reactive oxygen species (Figure 1).

Calprotectin modulate adaptive immune system

A cross-talk between neutrophils and T cells has been identified. Neutrophils -T cells co-culture showed that, in non-inflammatory conditions, neutrophils mainly modulate T-Cell activity through direct contact. But, a soluble factor, calprotectin, was also identified as a factor involved in suppression of T cell proliferation [START_REF] Thewissen | Neutrophils and T cells: bidirectional effects and functional interferences[END_REF]. Furthermore, calprotectin skew T cell polarization towards a Th17 polarization. An in vitro study showed that S100A8 has a crucial role in stimulating IL-6 expression by RA synoviocytes, and subsequently promotes Th17 differentiation [START_REF] Lee | MRP8 promotes Th17 differentiation via upregulation of IL-6 production by fibroblast-like synoviocytes in rheumatoid arthritis[END_REF], suggesting that neutralizing calprotectin level in RA synovium may be an effective therapeutic strategy.

Ectopic calprotectin production upon inflammatory condition trigger endothelial dysfunction

Calprotectin is also expressed by other cell types upon inflammatory conditions such as keratinocytes in psoriasis [START_REF] Lee | S100A8 and S100A9 are messengers in the crosstalk between epidermis and dermis modulating a psoriatic milieu in human skin[END_REF] and synovial fibroblast in RA [START_REF] Kane | Increased perivascular synovial membrane expression of myeloid-related proteins in psoriatic arthritis[END_REF], suggesting a central role of calprotectin in transendothelial migration of leukocytes in psoriatic arthritis (PsA) and RA.

Calprotectin directly induces a distinct inflammatory, thrombogenic response in microvascular endothelial cells. The inflammatory response is characterized by the secretion of inflammatory cytokines, chemokines, and adhesion molecules and by increased vascular permeability [START_REF] Baillet | Synovial fluid proteomic fingerprint: S100A8, S100A9 and S100A12 proteins discriminate rheumatoid arthritis from other inflammatory joint diseases[END_REF].

Calprotectin exerts potent cytotoxic activities by inducing apoptotic and necrotic alterations in endothelial cells [START_REF] Frosch | Myeloid-related proteins 8 and 14 are specifically secreted during interaction of phagocytes and activated endothelium and are useful markers for monitoring disease activity in pauciarticular-onset juvenile rheumatoid arthritis[END_REF].

Calprotectin is likely secreted in vessels calcification as neutrophils colonize atheroma plaque.

Calprotectin level raised locally in acute myocardial infarction, with an optimal cut off value of 8 mg/ml to identify acute myocardial infarction within 3hours (9).

Rheumatoid arthritis

Disease activity

In most of the trials in early and established RA, calprotectin correlates significantly with inflammatory markers and disease activity score (Table 1). This correlation is often statistically significant and clinically relevant. In a systematic literature review of 17 studies including a total of 1065 patients enrolled, calprotectin levels were high in active disease and were particularly high in rheumatoid factor-positive patients [START_REF] Abildtrup | Calprotectin as a biomarker for rheumatoid arthritis: a systematic review[END_REF]with a significant drop upon treatment. Surrogate markers of subclinical inflammation are currently sought to improve tight control of the disease. Some authors suggested that calprotectin could represent a proxy for local or subclinical inflammation as a correlation between baseline calprotectin levels and Ultrasonography (US) Power Doppler (PD) was found [START_REF] Hammer | The soluble biomarker calprotectin (an S100 protein) is associated to ultrasonographic synovitis scores and is sensitive to change in patients with rheumatoid arthritis treated with adalimumab[END_REF].

Structural progression and ultrasonography activity

Baseline calprotectin levels is correlated with Van der Heijde modified Sharp score (SHS) progression. This correlation was independent of age, gender, Clinical Disease Activity Index (CDAI), Erythrocyte Sedimentation Rate (ESR), C-reactive protein (CRP) levels and Rheumatoid Factor positivity [START_REF] Hammer | Calprotectin (a major leucocyte protein) is strongly and independently correlated with joint inflammation and damage in rheumatoid arthritis[END_REF][START_REF] Hammer | Calprotectin (a major S100 leucocyte protein) predicts 10-year radiographic progression in patients with rheumatoid arthritis[END_REF].

Jonsson et al. showed that calprotectin is a better predictor of structural progression than ESR or CRP [START_REF] Jonsson | Calprotectin as a marker of inflammation in patients with early rheumatoid arthritis[END_REF]. However, in most of trials evaluating calprotectin as a biomarker for structural damages in RA, some of the independent major predictors of structural evolution, such as anti-citrullinated protein antibodies (ACPA) and baseline erosion, was overlooked. In our assay of calprotectin blood levels in the ESPOIR cohort [START_REF] Chevreau | Calprotectin is not independent from baseline erosion in predicting radiological progression in early rheumatoid arthritis. Comment on « Calprotectin as a marker of inflammation in patients with early rheumatoid arthritis[END_REF]of early RA, suggested CRP, ACPA, Disease Modifying Anti-Rheumatic Drugs (DMARD) treatment and calprotectin were significantly associated with structural evolution in the univariate analysis. When baseline erosion was removed from the multivariate analysis, calprotectin was the only predictor of the structural evolution over 3 years. When the presence of baseline typical erosion was combined in the multivariate Cox Model, calprotectin was not an independent predictor of structural evolution anymore.

In a large Scandinavian RA cohort, calprotectin was significantly correlated with B-mode synovitis (r = 0.379, p<0.001) and PD synovitis scores (r = 0.419, p<0.001). The multivariate regression analysis showed that calprotectin is a better predictor of the CDAI and synovitis than CRP [START_REF] Hurnakova | Relationship between serum calprotectin (S100A8/9) and clinical, laboratory and ultrasound parameters of disease activity in rheumatoid arthritis: A large cohort study[END_REF]. Similarly, calprotectin had the strongest and most consistent associations with clinical variables and US sum scores in correlation analyses at baseline, among IL-6, S100A12, CRP and ESR [START_REF] Nordal | Calprotectin (S100A8/A9) has the strongest association with ultrasound-detected synovitis and predicts response to biologic treatment: results from a longitudinal study of patients with established rheumatoid arthritis[END_REF]. Calprotectin positively correlated with PDUS synovitis in RA and PsA patients in clinical remission or with low disease activity in a cross-sectional study of 92 patients treated with adalimumab and etanercept [START_REF] Inciarte-Mundo | Calprotectin and TNF trough serum levels identify power Doppler ultrasound synovitis in rheumatoid arthritis and psoriatic arthritis patients in remission or with low disease activity[END_REF].

Prediction of clinical response

In a study published in 2016 based on 87 patients, baseline blood level of calprotectin was significantly different between responders and non-responders to methotrexate (23.99µg/ml, 95% CI [15.39-42.75] versus 9.58µg/ml, 95% CI [6.11-24.93], p=0.0025) with an Area Under the Curve (AUC) of 0.705 (95% CI [0.549-0.862]), a sensitivity of 79% and a specificity of 69% at a threshold of 13.70µg/ml [START_REF] Patro | Myeloid-related Protein 8/14 Levels in Rheumatoid Arthritis: Marker of Disease Activity and Response to Methotrexate[END_REF]. In another prospective cohort of patient with RA there was no significant difference between calprotectin baseline level in responders and non-responders to methotrexate (23)(Table 2).

Many studies have investigated the predictive value of calprotectin for clinical response to bDMARD

(biologic Disease Modifying Anti-Rheumatic Drugs) with conflicting results. Several studies showed calprotectin as an independent predictive marker of good response to anti-TNF alpha [START_REF] Obry | Identification of S100A9 as biomarker of responsiveness to the methotrexate/etanercept combination in rheumatoid arthritis using a proteomic approach[END_REF][START_REF] Choi | MRP8/14 serum levels as a strong predictor of response to biological treatments in patients with rheumatoid arthritis[END_REF] [START_REF] Choi | MRP8/14 serum levels as a strong predictor of response to biological treatments in patients with rheumatoid arthritis[END_REF]. Another prospective study showed some predictive value of calprotectin for clinical response of adalimumab or etanercept treatment although the multivariate analysis did not include DAS28-CRP [START_REF] Tweehuysen | Predictive value of serum calprotectin (S100A8/A9) for clinical response after starting or tapering anti-TNF treatment in patients with rheumatoid arthritis[END_REF]. A decrease of calprotectin within the first month of treatment with bDMARD was predictive of both EULAR response and decreased sum PD scores [START_REF] Nordal | Calprotectin (S100A8/A9) has the strongest association with ultrasound-detected synovitis and predicts response to biologic treatment: results from a longitudinal study of patients with established rheumatoid arthritis[END_REF].

However, in a large Caucasian cohort of RA patients, there was no evidence that calprotectin is predictive of EULAR response to etanercept [START_REF] Smith | The predictive value of serum S100A9 and response to etanercept is not confirmed in a large UK rheumatoid arthritis cohort[END_REF]. Obry et al. showed that S100A9 subunit predicts response to etanercept, with 83% sensitivity and 70% specificity at the threshold of 2.59 ng/ml whereas calprotectin did not [START_REF] Obry | Identification of S100A9 as biomarker of responsiveness to the methotrexate/etanercept combination in rheumatoid arthritis using a proteomic approach[END_REF].

Spondyloarthritis

Diagnosis

Magnetic resonance imaging (MRI) is the most sensitive imaging test to detect early SpA and thus helps to limit diagnostic wandering [START_REF] Rudwaleit | The Assessment of SpondyloArthritis International Society classification criteria for peripheral spondyloarthritis and for spondyloarthritis in general[END_REF]. Moreover, Assessment of SpondyloArthritis international Society (ASAS) classification criteria include, in addition to radiographic sacroiliac joints, active sacroiliitis on MRI as a diagnostic criterion for axial SpA. However, sacroiliac joint bone marrow oedema is not only specific of SpA but can be found in other disorders responsible for chronic low back pain such as sacroiliac joints degenerative diseases, as well as in healthy subjects [START_REF] De Winter | Magnetic Resonance Imaging of the Sacroiliac Joints Indicating Sacroiliitis According to the Assessment of SpondyloArthritis international Society Definition in Healthy Individuals, Runners, and Women With Postpartum Back Pain[END_REF]. The prevalence of sacroiliac joint degenerative diseases is probably underestimated [START_REF] Baillet | Bone Marrow Edema in the Sacroiliac Joint-Degenerative Sacroiliac Joint Disease Might Be More Likely Than Spondyloarthritis: Comment on the Article by Turina et al[END_REF]. The definition of a positive MRI for spondyloarthritis remains controversial adding complexity to the SpA diagnosis.

Other biomarkers are probably necessary to facilitate the diagnosis. C-reactive protein (CRP), a systemic inflammation biomarker, is a criterion included in the ASAS criteria. However, about half of SpA patients have normal CRP levels [START_REF] Benhamou | Clinical relevance of C-reactive protein in ankylosing spondylitis and evaluation of the NSAIDs/coxibs' treatment effect on C-reactive protein[END_REF] limiting the interest of this biomarker for the diagnosis.

Serum calprotectin could reflect the local activation of inflammatory innate immune cells involved in enthesitis and arthritis. Over 20 years ago, Hammer et al. described calprotectin as a marker of inflammation and treatment response in reactive arthritis [START_REF] Hammer | A longitudinal study of calprotectin as an inflammatory marker in patients with reactive arthritis[END_REF]. Calprotectin serum levels are significantly increased in axSpA (axial Spondyloarthritis) fulfilling ASAS criteria or AS (Ankylosing Spondylitis) [START_REF] Oktayoglu | Elevated serum levels of calprotectin (myeloid-related protein 8/14) in patients with ankylosing spondylitis and its association with disease activity and quality of life[END_REF][START_REF] Cypers | Elevated calprotectin levels reveal bowel inflammation in spondyloarthritis[END_REF][START_REF] Gupta | Elevated levels of serum MRP8/14 in ankylosing spondylitis: associated with peripheral arthritis and active disease[END_REF][START_REF] Huang | Discriminating Value of Calprotectin in Disease Activity and Progression of Nonradiographic Axial Spondyloarthritis and Ankylosing Spondylitis[END_REF][START_REF] Klingberg | Calprotectin in ankylosing spondylitis--frequently elevated in feces, but normal in serum[END_REF][START_REF] Turina | Calprotectin serum level is an independent marker for radiographic spinal progression in axial spondyloarthritis[END_REF]versus healthy control or with early inflammatory back pain (40) not fulfilling axial SpA ASAS criteria whereas CRP levels could be normal. Nevertheless, calprotectin is not helpful to discriminate early axSpA from nonspecific inflammatory back pain with a sensitivity of 10.0%, a specificity of 90% and a Positive Predictive Value (PPV) of 38.7% for a threshold of 412.40ng/mL [START_REF] Turina | Serum inflammatory biomarkers fail to identify early axial spondyloarthritis: results from the SpondyloArthritis Caught Early (SPACE) cohort[END_REF].

In another cross-sectional study, a serum calprotectin level over 0.9 mg/L display a sensitivity of 95.3%, a specificity of 82.2%, a positive likelihood ratio (LR) of 5.35 and a negative LR of 0.057 for the diagnosis of chronic inflammatory rheumatic diseases including patients with RA, SpA, PsA and healthy controls [START_REF] Grossi | Serum calprotectin levels as a marker of arthritis: an italian experience[END_REF].

Subsequently, calprotectin was found to be elevated in the feces of patients with SpA. In patient with inflammatory bowel disease, especially with ulcerative colitis, fecal calprotectin level is correlated with clinical activity and endoscopic lesion [START_REF] Mosli | C-Reactive Protein, Fecal Calprotectin, and Stool Lactoferrin for Detection of Endoscopic Activity in Symptomatic Inflammatory Bowel Disease Patients: A Systematic Review and Meta-Analysis[END_REF]. Serum calprotectin independently predicts a diagnosis of inflammatory bowel disease with sensitivity of 0.69, a specificity of 0.90 and a positive LR of 7.06 at the threshold of >852 ng/ml. With a combination of biomarker, especially CRP or albumin, the positive LR grow to 24.14. Another predictive model using CRP, serum and fecal calprotectin level detects microscopic bowel inflammation in SpA patients with an AUC of 74.4% (0.639-0.849%, p<0.001) [START_REF] Cypers | Elevated calprotectin levels reveal bowel inflammation in spondyloarthritis[END_REF]. Predictive model including serum calprotectin could be an interesting tool to determine patient with a high risk of inflammatory bowel disease in SpA. The validation of these predictive models must be replicate in other large cohorts. However, serum calprotectin didn't always correlate with fecal calprotectin [START_REF] Fukunaga | Detection of calprotectin in inflammatory bowel disease: Fecal and serum levels and immunohistochemical localization[END_REF]suggesting that serum calprotectin derived predominantly from circulating neutrophils, and not from the neutrophil infiltration in the gut.

Disease activity

Serum levels of calprotectin are also highly significantly increased in axial SpA and AS. Treatment with anti-TNF and anti-IL-17A agents significantly decrease calprotectin levels [START_REF] Turina | Calprotectin (S100A8/9) as serum biomarker for clinical response in proof-of-concept trials in axial and peripheral spondyloarthritis[END_REF][START_REF] Baeten | Anti-interleukin-17A monoclonal antibody secukinumab in treatment of ankylosing spondylitis: a randomised, double-blind, placebo-controlled trial[END_REF]. The correlation between serum calprotectin and disease activity seems to be weaker than in RA (Table 3). However, one might emphasize the wide broad of median levels of serum calprotectin in studies evaluating calprotectin as a proxy for Disease activity with discordant results (Table 4). A possible explanation for such heterogeneity in calprotectin levels in SpA cohorts, besides concerns raised on internal reproducibility of calprotectin assays, is a variable gut production of calprotectin which depends on subclinical gut inflammation and inflammatory bowel disease associated with SpA.

Structural progression

Baseline calprotectin serum levels was found to be significantly higher in AS patients with radiologic spinal progression at 2 years assessed with modified Stoke Ankylosing Spondylitis Spinal Score (mSASSS) (table 4). Serum calprotectin in this study could predict the mSASSS worsening by ≥2units at 2 years with a threshold of >0.5 µg/mL with a sensitivity of 80%, a specificity of 62% and an Odds ratio (OR) of 6.2 (95% CI 1.6-24.2). Calprotectin in PsA was associated with presence of peripheral radiographic damage (46)but did not perform better than traditional biomarkers to assess the disease activity.

Juvenile idiopathic arthritis

In patients with polyarticular juvenile arthritis, calprotectin was much higher in synovial fluid than in serum (42 800 mg/L versus 2 060 mg/L) and significantly decreased after intraarticular triamcinolone therapy, suggesting that serum levels of this biomarkers is determined by joint production of calprotectin by myeloid cells [START_REF] Frosch | Myeloid-related proteins 8 and 14 are specifically secreted during interaction of phagocytes and activated endothelium and are useful markers for monitoring disease activity in pauciarticular-onset juvenile rheumatoid arthritis[END_REF]. Concentrations of calprotectin in serum displayed a strong correlation with disease activity. The exceptionally high serum levels of calprotectin in active systemic-onset juvenile arthritis suggests that calprotectin may be a marker for monitoring disease activity and response to treatment [START_REF] Frosch | Expression of myeloidrelated proteins 8 and 14 in systemic-onset juvenile rheumatoid arthritis[END_REF].

Despite correlation between calprotectin levels and disease activity in juvenile idiopathic arthritis [START_REF] Frosch | Myeloid-related proteins 8 and 14 are specifically secreted during interaction of phagocytes and activated endothelium and are useful markers for monitoring disease activity in pauciarticular-onset juvenile rheumatoid arthritis[END_REF][START_REF] Frosch | Expression of myeloidrelated proteins 8 and 14 in systemic-onset juvenile rheumatoid arthritis[END_REF] , calprotectin could not predict Macrophage Activation Syndrome, which is a lifethreatening complication the systemic form of the disease. A higher rate of calprotectin has been found in patient with juvenile arthritis in terms of response to methotrexate [START_REF] Moncrieffe | A subgroup of juvenile idiopathic arthritis patients who respond well to methotrexate are identified by the serum biomarker MRP8/14 protein[END_REF]. Remarkably serum levels of calprotectin were significantly higher in patients who subsequently developed flares than in patients with stable remission [START_REF] Abildtrup | Calprotectin as a biomarker for rheumatoid arthritis: a systematic review[END_REF].

Anca associated vasculitis (aav)

AAV disease mechanisms and calprotectin

Granulomatosis with polyangiitis (GPA) and microscopic polyangitis (MPA) are severe conditions that share the features of necrositing vasculitis of small vessels in multiple organ systems and characteristic autoantibodies anti-neutrophil cytoplasmic antibodies (ANCAs) and thus can be described together as AAV [START_REF] Thai | Are anti-proteinase-3 ANCA a useful marker of granulomatosis with polyangiitis (Wegener's) relapses? Results of a retrospective study on 126 patients[END_REF].

ANCA activate neutrophils and monocyte in vivo [START_REF] Falk | Anti-neutrophil cytoplasmic autoantibodies induce neutrophils to degranulate and produce oxygen radicals in vitro[END_REF]inducing a calprotectin release. In inflammatory tissue, calprotectin is deposited onto the endothelium cells. Besides apoptosis, neutrophil may display another cell death program leads to the production of extracellular traps (NETs) which are highly immunogenic triggering adaptive immune response leading to ANCA formation [START_REF] Sangaletti | Neutrophil extracellular traps mediate transfer of cytoplasmic neutrophil antigens to myeloid dendritic cells toward ANCA induction and associated autoimmunity[END_REF]. Previous investigations showed the presence of calprotectin in NET and the antimicrobial activity of calprotectin [START_REF] Urban | Neutrophil extracellular traps contain calprotectin, a cytosolic protein complex involved in host defense against Candida albicans[END_REF]. In vivo, calprotectin is partly bound to NETs [START_REF] Bjarnason | Subclinical intestinal inflammation and sacroiliac changes in relatives of patients with ankylosing spondylitis[END_REF]. Although no ANCA were detected against calprotectin [START_REF] Schultz | ANCA against bactericidal/permeability-increasing protein, azurocidin, calprotectin and defensins in rheumatic and infectious diseases: prevalence and clinical associations[END_REF]. In renal biopsy of patient with AAV, calprotectin was found in glomerular lesions with endocapillary proliferation whereas chronic sclerotic lesions are generally negative [START_REF] Pepper | Leukocyte and serum S100A8/S100A9 expression reflects disease activity in ANCA-associated vasculitis and glomerulonephritis[END_REF].

Calprotectin plays a crucial role in the development of glomerulonephritis by exerting a proinflammatory effect on macrophages, intrinsic renal endothelial and mesangial cells [START_REF] Pepper | S100A8/A9 (calprotectin) is critical for development of glomerulonephritis and promotes inflammatory leukocyte-renal cell interactions[END_REF]. Patient with AAV have elevated levels of cell surface calprotectin on neutrophils and monocytes and the level of expression increase with the activity of the vasculitis [START_REF] Pepper | Leukocyte and serum S100A8/S100A9 expression reflects disease activity in ANCA-associated vasculitis and glomerulonephritis[END_REF]. This biomarker may help to manage the treatment in AAV because the course of disease after initial treatment is highly variable with frequently relapse. The monitoring of the disease by a biomarker could avoid an overtreatment with its risk of side effects.

Disease activity, relapse and response to treatment

Serum calprotectin correlate weakly with CRP (r=0.22, p=0.016), total white cell count (r=0.23, p=0.01) and Birmingham Vasculitis Activity Score (BVAS) score (r=0.27, p=0.02) [START_REF] Pepper | Association of Serum Calprotectin (S100A8/A9) Level With Disease Relapse in Proteinase 3-Antineutrophil Cytoplasmic Antibody-Associated Vasculitis[END_REF]. Pepper et al. [START_REF] Pepper | Leukocyte and serum S100A8/S100A9 expression reflects disease activity in ANCA-associated vasculitis and glomerulonephritis[END_REF]have shown that calprotectin levels at 1 or 6 months could predict future relapses, with a sensitivity of 78.6%, a specificity of 92.3% and a likelihood ratio of 10.3 at a threshold of >626 ng/ml at 1 month and a sensitivity of 78.6% and specificity of 92.3% at a threshold of >454 ng/ml at 6 months, suggesting a relationship both statistically significant and clinically relevant. Calprotectin seems significantly better in the prediction of relapses than ANCA titer rising [START_REF] Pepper | Leukocyte and serum S100A8/S100A9 expression reflects disease activity in ANCA-associated vasculitis and glomerulonephritis[END_REF]. In the PR3-ANCA patient of RAVE (Rituximab versus Cyclophosphamide for ANCA-associated Vasculitis) trial, the increase in ANCA at month 2 of the induction treatment compared to baseline was not associated with the risk of a future relapse (hazard ratio (HR) =0.85 (95% CI [0.55-1.13]) whereas the increase in serum calprotectin was significantly associated (HR=2.2, 95% CI [1.17-4.26]) [START_REF] Pepper | Association of Serum Calprotectin (S100A8/A9) Level With Disease Relapse in Proteinase 3-Antineutrophil Cytoplasmic Antibody-Associated Vasculitis[END_REF]. Hence, calprotectin could be a help to identify patients requiring more intensive or prolonged induction treatment [START_REF] Pepper | Association of Serum Calprotectin (S100A8/A9) Level With Disease Relapse in Proteinase 3-Antineutrophil Cytoplasmic Antibody-Associated Vasculitis[END_REF].

Area of uncertainty

These data support a relevant role of calprotectin as a key molecule of inflammation, which could play a role in structural immunological disorders and tissue damage in chronic inflammatory rheumatic diseases. This protein is released locally in inflammatory joints in contrast with CRP, which is produced by hepatocyte. Contrary to CRP which reflects systemic inflammation in response to local production of IL-6(57), calprotectin seems to be a good proxy for neutrophil activation in a biological fluid. Therefore its measurement is of particular interest within a biological fluid "closed to" the inflammation site such as synovial fluid in RA [START_REF] Baillet | Synovial fluid proteomic fingerprint: S100A8, S100A9 and S100A12 proteins discriminate rheumatoid arthritis from other inflammatory joint diseases[END_REF] and serum in AAV [START_REF] Pepper | Association of Serum Calprotectin (S100A8/A9) Level With Disease Relapse in Proteinase 3-Antineutrophil Cytoplasmic Antibody-Associated Vasculitis[END_REF]. Calprotectin showed its ability to predict radiological progression in RA and AS. Serum calprotectin can predict the risk of relapse in AAV and in association with other biomarkers, the risk of inflammatory bowel disease in spondyloarthritis. However, it is necessary to replicate these data in other large cohort to confirm these results. Researchers should be aware of the lack of the assay standardization, especially in the context of SpA studies, as shown in Table 2. Some ELISA kits are design for research use only whereas some are approved for in vitro diagnosis. Calprotectin might be of valuable interest to monitor and predict the evolution of chronic inflammatory rheumatic diseases, especially in a predictive model including multiple biomarkers. Better standardization of the assays is nevertheless necessary in order to be able to compare the studies and to have stronger conclusions.

  . Choi et al. showed a predictive value of calprotectin at baseline with adalimumab (OR 3.30, 95% CI [1.14 to

	9.60], p=0.028), infliximab (OR 9.75, 95% CI [1.93 to 49.33], p=0.006) and rituximab (OR 55, 95% CI
	[4.30 to 703.43], p=0.002)

Table 1 . Blood calprotectin correlates with disease activity in Rheumatoid arthritis.

 1

	Study	CRP	ESR	DAS28 SJC	Blood	Assay ; Mean calprotectin level
						samples
	Hammer HB et al.	0.59	0.67	N/A	N/A EDTA	Home-made ELISA [67] ;
	2010 (19)					plasma	Median 2.2, IQR (1.1-4.2) mg/l
	Hammer HB et al.	0.57	0.50	0.55	0.49 EDTA	Home-made ELISA [67];
	2007 (8)					plasma	Median 1.8, Range (0.3-8.7) mg/l
	Andres Cerezo L	0.44	N/A	0.50	0.44 Serum	BÜHLMANN	LABORATORIES	AG,
	et al. 2011 (58)						SWITZERLAND;
							Mean±SD 5.99±0.88 mg/L and 1.92± 1.16
							mg/L respectively before and after
							treatment
	Jonsson MK et al.	0.66	0.50	N/A	N/A EDTA	CALPROLAB, NORWAY;
	2017 (20)					plasma	Median 1.05, IQR (0.57, 2.24) mg/L
	Hurnakova J et al.	0.56	0.38	0.35	N/A Serum	BÜHLMANN	LABORATORIES	AG,
	2017 (22)						SWITZERLAND;
							Median (range) 2.9 (0.8-3.4) and 1.7 (0.4-
							3.4) µg/mL
	Nordal HH et al.	N/A	N/A	0.49	0.47 EDTA	CALPRO AS, NORWAY
	2017 (23)					plasma	Median 1.15, IQR (0.70-1.95) mg/L
	CRP: C-reactive protein, ESR: Erythrocyte Sedimentation Rate, DAS28: Disease Activity Score of 28
	joints, SJC: Swollen Joint Count, EDTA: Ethylenediaminetetraacetic acid, N/A not assessed, ELISA:
	Enzyme-Linked Immunosorbent Assay, IQR: Interquartile Range 25,75 percentile, SD: Standard
	Deviation.

Table 2 . Calprotectin as a biomarker of DMARD response in rheumatoid arthritis.

 2

	Study	Treatment (n	Blood samples	Calprotectin in	Threshold	Results
		patients)	(Assay)	responders vs. non
				responders
	Nordal et al.	ETN 40%, RTX	Plasma	Higher baseline value	ND	ND
	2017 (23)	22%, TCZ 9%, IFX	(ELISA	of calprotectin in
		9%, CZP 8%, ADA	Calpronorway)	EULAR responders at 3
		4%, ABA 3%		and 12 months
	Patro et al.	MTX	Serum	Responders	13.7 µg/ml	AUC 0.70
	2016 (25)		(ELISA BMA	23.99µg/ml, 95%CI		95%CI [0.54; 0.86]
			Biomed)	(15.39; 42.75)		Se 79%
				Non responders		Spe 69%
				9.58 µg/ml 95%CI		VPP 73%
				(6.11; 24.93), p=0.003
	Obry et al.	MTX + ETN	Serum	Responders	ND	ND
	2014 (26)		(ELISA	510±130ng/ml
			Cusabio)	Non responders
				770±210ng/ml, p=0.32
	Choi et al.	ADA (n= 86)	Serum	Responders	995 ng/ml	Se 57%
	2015(27)		(ELISA)	1100ng/ml, IQR (711-		Spe 71%
				1615)
				Non responders
				730 ng/ml, IQR (575-
				1065), p=0.010
		IFX (n= 60)	Serum	Responders	2027 ng/ml	Se 62%
			(ELISA)	2650 ng/ml, IQR (1483-		Spe 86%
				4120)
				Non responders
				1220 ng/ml, IQR (1053-
				1533), p=0.001
		RTX (n= 24)	Serum	Responders	1665 ng/ml	Spe 91%
			(ELISA)	2811 ng/ml, IQR (1945-		Se 85%
				4525)
				Non responders
				1050 ng/ml, IQR (780-
				1290), p <0.001
	Tweehuysen	ADA (n= 50)	Serum	Responders	ND	AUC 0.61
	et al.	ETN (n= 75)	(ELISA	985 ng/ml, 95%CI (558;		95%CI (0.50; 0.71)
	2018(28)		University of	1417]
			Münster)	Non responders
				645 ng/ml, 95%CI (415;
				973), p =0.04
	Smith et al.	ETN (n= 236)	Serum	Good responders	ND	OR 1.0
	2017(29)		(ELISA	165.5 ng/ml		95%CI (0.99, 1.00)
			Cusabio,	Moderate responders
			China)	183.7 ng/ml
				Non responders
				183.8ng/ml, p=0.957

ETN: etanercept, RTX: rituximab, TCZ: tocilizumab, IFX: infliximab, CZP: certolizumab, ADA: adalimumab, ABA: abatacept, MTX : methotrexate, ELISA: Enzyme-Linked Immunosorbent Assay, EULAR: European League Against Rheumatism, ND: Non Defined, 95%CI : 95% Confidence interval, AUC : Area Under the Curve OR: Odds Ratio, Se : Sensitivity, Spe : Specificity, PPV : Positive Predictive Value, IQR : Interquartile Range

Table 3 . Serum calprotectin correlates weakly with disease activity in axial spondyloarthritis.

 3

	Study	Disease	CRP	BASDAI	ASDAS-CRP SJC	Structural
	J.Huang et al.	AS mNY+	0.431 ***	0.481 ***	0.378 ***	N/A	SPARCC-SI
	2017 (37)	Nr-axSpA ASAS +					0.405 ***
							mSASSS
							-0.033
	Turina et al.	AS mNY + or ax SpA	0.382 ***	N/A	N/A	N/A	N/A
	2014 (39)	with one
		syndesmophyte
	Cypers et al.	axSpA ASAS +	0.386 ***	NS	N/A	NS	N/A
	2016 (35)	or
		SpA peripheral
		ASAS+
	Gupta et al.	axSpA ASAS +	0.279 **	0.15 NS	0.23 **	0.38 **	N/A
	2016 (36)
	Oktayoglu et	AS mNY +	-0.234 NS	-0.278 NS	0.243 NS	N/A	N/A
	al. 2014 (9)
	Klingberg et	AS mNY +	0.337 ***	NS	0.162 NS	N/A	N/A
	al. 2012 (38)
	Turina et al.	SpA ESSG +	0.646 *	0.325 *	N/A	N/A	N/A
	2014 (44)
	CRP: C-reactive protein, BASDAI: Bath Ankylosing Spondylitis Disease Activity Index, ASDAS-CRP:
	Ankylosing Spondylitis Disease Activity Score with CRP, SJC : Swollen Joint Count, AS: Ankylosing
	Spondylitis, mNY+: patient fulfilling modified New York criteria, SpA: spondyloarthrites, axSpA: axial
	Spondyloarthritis, Nr-ax SpA: Non radiological axial Spondyloarthritis, ASAS+: patient fulfilling
	Assessment of Spondyloarthritis international Society, SPARCC-SI: Spondyloarthritis Research
	Consortium of Canada Sacroiliac joint score, mSASSS: modified Stoke Ankylosing Spondylitis Spinal
	Score, ESSG : patient fulfilling European Spondylarthropathy Study Group criteria, N/A: Not Assessed,
	NS: Not Significant. *p<0.05, **p<0.01, ***p<0.001

Table 4 . Serum calprotectin level assessed with different ELISA kit in axial spondyloarthritis and ankylosing spondylitis.

 4

	Study	ELISA Kit	Disease	Patients (n)	Male (%)	Age, years	Disease duration, years	HLA B27 (%) BASDAI	Serum calprotectin (mg/L)
	Turina et
	al.2017 (40)00/00/		axSpA ASAS +		51 32±8 1.1±0.6	86		0.294 (0.214-0.367)
	0000			119					3.9±2.4
	00:00:00
	Turina et al.2014 (39)	HYCULT BIOTECH, the Netherlands §	AS mNY + or axSpA with syndesmophyte	76	66 38±12 4.6±2.8	82	ND	mSASSS worsening 0.68±0.21 without mSASSS worsening 0.48±0.26
	Turina et al.2014 (44)		SpA ESSG +	37	68	48 (29-66)	8.0 (2.0-34.0)	55	5.3 8.6) (2.8-	SpA 1.760±0.13
	Huang et al. 2017 (37)	CUSABIO BIOTECH Co, China	AS mNY+ or axSpA ASAS +	53 AS, 59 Nr-axSpA	66	AS 32±8, Nr-axSpA 34±8	AS 5.2±3.6, Nr-axSpA 5.0±4.1	AS 86.7 Nr-axSpA: 81.3	AS 3.4±1.1 Nr-axSpA 4.2±1.4	AS 0,015±0,007 Nr-axSpA 0.018±0.009
										Without bowel
	Cypers et al.2016 (35)		axSpA ASAS + or peripheral SpA ASAS +	45 AS, 59 Nr-ax SpA, 21peripheral SpA ASAS +	ND	ND	ND	AS 86, Nr-axSpA 70, peripheral SpA 55	ND	inflammation 3.022 (0.741-10.106), With bowel inflammation 3.948 (0.782-
										17.246)
	Levitova et al.2016 (59)	BÜHLMANN LABORATORIES AG, Schoenenbuch, Switzerland § §	axSpA ASAS +	40 All axSpA ASAS+ 18 Nr-axSpA 22 AS	All axSpA ASAS + 67.5, Nr-axSpA 50, AS 82	All axSpA ASAS + 37±1, Nr-axSpA 37±2, AS 37±1	All axSpA ASAS+ 9.9±1.3, Nr-axSpA 7.1±2.2, AS 12.3±1	All axSpA ASAS+ 90.0, Nr-axSpA 89 AS 91	All axSpA 2.63± ASAS+ AS 2.78± 0.31, Nr-axSpA 2.98± 0.28,	All axSpA ASAS+ 2.408±0.183, Nr-axSpA 2.379±0.243 AS 2.430±0.270
									0.35
	Gupta et al. 2016 (36)	BIOLEGEND, USA §	axSpA ASAS +	99	91	30 (16-62)	6.0 (0.3-30.0)	90	ND 34.1 (17.94-264.58)
	Oktayoglu et al.2014 (9)	IMMUNDIAGNOS	AS mNY +	31	74	29.7± 8.1	5.5±4.5	71	4.2±1.7	0.819±0.553
	Klingberg	TIK, Germany
	et al. 2012		AS mNY+	205	57 50±13 24±13	87	3.6±2.1	0.660±0.860
	(38)

ELISA: Enzyme-Linked Immunosorbent Assay, HLA B27: Human Leukocyte Antigen B27, BASDAI: Bath Ankylosing Spondylitis Disease Activity Index, AS: Ankylosing Spondylitis, mNY: modified New York criteria, AxSpA: Axial Spondyloarthritis, Nr-axSpA: Non radiological axial Spondyloarthritis, ASAS: Assessment of Spondyloarthritis international Society criteria, mSASSS: modified Stoke Ankylosing Spondylitis Spinal Score, ESSG: European

Acknowledgement:

The authors thank Sylvie Papacatzis for her help in the study.

Disclosure of interest

The authors declare that they have no competing interest.

Funding: None


Yuxin Ma

Armelle Marais

Marie Lefebvre

Chantal Faure

Thierry Candresse
email: thierry.candresse@inra.fr

Metagenomic analysis of virome cross-talk between cultivated Solanum lycopersicum and wild Solanum nigrum

Keywords: metagenomics, virome, double stranded RNA (dsRNA), tomato, spillover

Wild plants and weeds growing close to crops constitute a potential reservoir for future epidemies or for the emergence of novel viruses but the frequency and directionality of viral flow between cultivated and wild plants remains poorly documented in many cases.

Here, we studied the diversity of viral populations between tomato (Solanum lycopersicum) and neighboring european black nightshade (Solanum nigrum) using high throughput sequencing (HTS) based metagenomics. A large variability in virome richness with only 17.9% shared Operational Taxonomy Units between tomato and nightshade, but this richness could not be linked to a particular host or to local conditions. A detailed population analysis based on assembled contigs for potato virus Y (PVY), broad wilt bean virus 1 and a new ilarvirus tentatively named Solanum nigrum ilarvirus 1 provides information on the circulation of these viruses between these two Solanum species and enriches our knowledge of the tomato virome.

INTRODUCTION

Through the past decade, metagenomics based on high throughput sequencing (HTS) has been widely used in the plant virology field, advancing our knowledge on the diversity of plant viruses. Specifically, metagenomics allowed to discover unknown viruses, explore the intraspecific genetic diversity of known viruses, and study virus ecology and epidemiology [START_REF] Massart | Current impact and future directions of high throughput sequencing in plant virus diagnostics[END_REF][START_REF] Roossinck | Plant Virus Metagenomics: Advances in Virus Discovery[END_REF][START_REF] Villamor | High Throughput Sequencing For Plant Virus Detection and Discovery[END_REF]. Plant viruses cause epidemics on all major cultures of agronomic importance, representing a serious threat to global food security. As a consequence, virologists have for a long time focused their efforts on economically important crops, often neglecting bordering weeds and wild plants [START_REF] Wren | Plant Virus Biodiversity and Ecology[END_REF]. However, agro-ecosystems are complex environments in which crop plants sometimes interact with the in-plot and bordering weeds and wild plants. Viruses may be transferred between wild plants/weeds and crops and vice versa by a variety of mechanisms and vectors. Thus wild plants or weeds may constitute "reservoirs" of viruses that may subsequently spread to cultivated plants while crops may constitute a source from which viral infections may spillover to the wild plants/weeds compartment [START_REF] Power | Pathogen Spillover in Disease Epidemics[END_REF].

Overall, our understanding of the details of fluxes of viruses from crops to weeds and from weeds to crops remains frequently limited. A role of a weed population as a reservoir or, alternatively, virus spillover from a crop are often assumed. Unfortunately, the techniques most frequently used for viral populations characterization, in particular serological ones, frequently do not provide sufficient intra-specific resolution to provide unambiguousl conclusions on such aspects. It is then difficult to ensure that the co-occurrence of a virus in crops and weeds reflects the transfer of isolates rather than the existence separate viral populations adapted to the two host populations.

Tomato (Solanum lycopersicum) is one of the most popular and extensively consumed vegetable crops. There are at least 136 characterized viral species that are capable of infecting tomato and due to global climate changes and increased international trade, the spread of known viruses to new geographic areas and the emergence of new viruses have been frequently detected in particular in recent years [START_REF] Brunt | Plant Viruses Online: Descriptions and Lists from the VIDE Database[END_REF][START_REF] Hanssen | Emerging viral diseases of tomato crops[END_REF].

Torradoviruses (family Secoviridae) are an example of a group of recently emerged plant viruses, many of which affect tomato. These include for example tomato torrado virus (ToTV), which was first described from tomato in Mexico (Verbeek et al., 2008) and reported more recently in France [START_REF] Verbeek | First report of Tomato torrado virus in tomato crops in France[END_REF] and in other host plant species (van der [START_REF] Van Der Vlugt | Torradoviruses[END_REF], as well as tomato marchitez virus (ToMarV; (Verbeek et al., 2008)) and tomato chocolàte virus (ToChV; [START_REF] Verbeek | Tomato chocolate virus: a new plant virus infecting tomato and a proposed member of the genus Torradovirus[END_REF]). Another example of recent emergence of a virus in tomato concerns tomato brown rugose fruit virus (ToBRFV), a tobamo like virus which was discovered from tomato in Israel in 2014 [START_REF] Luria | A new Israeli Tobamovirus isolate infects tomato plants harboring Tm-22 resistance genes[END_REF] and that has spread since then to many countries including Jordan, Mexico, the United States (Southern California), Germany, Italy, Turkey, the Netherlands and Saudi Arabia. The source(s) and cause(s) of the emergence of such novel agents is(are) frequently unknown but weed and wild plants are often considered as a major sources of future emerging viruses than may occasionally be transferred to crops [START_REF] Anderson | Emerging infectious diseases of plants: pathogen pollution, climate change and agrotechnology drivers[END_REF][START_REF] Elena | Evolution and emergence of plant viruses[END_REF][START_REF] Mcleish | Evolution of plant-virus interactions: host range and virus emergence[END_REF].

Recently, during a study characterizing the virome of 170 field-grown tomatoes collected in China by small RNAs sequencing, [START_REF] Xu | Diversity, Distribution, and Evolution of Tomato Viruses in China Uncovered by Small RNA Sequencing[END_REF] showed that the tomato viral community is dominated by a few species, most of them being positive-sense ssRNA viruses. Multiple infections were found to be frequent as well as recombination events in viral genomes [START_REF] Xu | Diversity, Distribution, and Evolution of Tomato Viruses in China Uncovered by Small RNA Sequencing[END_REF].

European black nightshade (Solanum nigrum), a wild species in the same botanical genus as tomato is a widespread weed in many countries. However, in southern India it is widely consumed and cultivated on a commercial scale [START_REF] Jamuna | First Report of Natural Infection of Solanum nigrum with Tomato mosaic virus in India[END_REF] and sometimes also used as for its medicinal properties [START_REF] Javed | In-vitro antiviral activity of Solanum nigrum against Hepatitis C Virus[END_REF]. S. nigrum is known to harbor a wide range of viruses such as begomoviruses, orthotospoviruses, potyviruses, tobraviruses under field conditions, and has often been suspected to act as a reservoir host for viruses or for recombinant isolates infecting solanaceous crops (Holm et al., Garcia-Andrès et al., 2006;[START_REF] Holm | A geographical atlas of world weeds[END_REF][START_REF] Jamuna | First Report of Natural Infection of Solanum nigrum with Tomato mosaic virus in India[END_REF].

In the present study, using a metagenomics approach, we investigated and compared the virome in tomato samples and in the related S. nigrum populations collected either in tomato fields or in various other environments. The comparison of these viromes provides novel insight into the viral fluxes between these two species.

MATERIALS AND METHODS

Study sites and plant samples

Virome richness and composition were analyzed in tomato (Solanum lycopersicum) and in European black nightshade (Solanum nigrum) that were growing either close to the sampled tomato crops or in other sites, unmanaged or involving unrelated crops (sunflower, maize, sorghum and alfalfa, Table S1). In total, tomato crops were sampled in seven sites and nightshade in six of the seven tomato sites, plus in five non-tomato sites (Table S1).

For each sampled plant populations, leaves from a total of 100 individual plants were collected in summer 2017 or 2018 and assembled in two pools corresponding to fifty individual plants (0.1g of leaf/plant) for nucleic acids extraction. No specific efforts were made to select symptomatic plants, but plants with obvious fungal attack, insect colonization or necrotized parts were excluded.

Double-stranded RNAs purification, library preparation and Illumina HiSeq sequencing

Double-stranded RNAs were purified from each plant pool by two rounds of CF11 cellulose chromatography and converted to cDNA according to the protocol described by [START_REF] Marais | Viral Double-Stranded RNAs (dsRNAs) from Plants: Alternative Nucleic Acid Substrates for High-Throughput Sequencing[END_REF]. In parallel, a negative control blank was similarly prepared using only buffer. Whole genome amplifications (WGAs) were performed on each cDNA sample (using the same MID tag for the two pools of each sampling site), the PCR products were purified using the MinElute PCR Purification Kit (Qiagen) and their concentration determined spectrophotometrically [START_REF] Marais | Viral Double-Stranded RNAs (dsRNAs) from Plants: Alternative Nucleic Acid Substrates for High-Throughput Sequencing[END_REF]. Equal quantities of the amplification products from the two pools of each sampling site were then regrouped and independent sequencing libraries prepared for each site and sequenced in multiplexed format (2×150 bp) on an Illumina HiSeq 3000 system at the GenoToul platform (INRA Toulouse, France). Cleaned virome sequence reads have been deposited on the INRA National Data Portal under the identifier https://doi.org/10.15454/S486RR.

Bioinformatics analyses: Reads cleaning, contigs assembly and annotation, Operational Taxonomic Units (OTU) clustering

Following demultiplexing, adapters and MID tags were removed with cutadapt [START_REF] Martin | Cutadapt removes adapter sequences from high-throughput sequencing reads[END_REF], and reads were quality trimmed (minimum quality score 20, minimum length 70 nucleotides). In order to limit inter-sample cross talk associated with index-hopping [START_REF] Illumina | Effects of index misassignment on multiplexing and downstream analysis[END_REF][START_REF] Van Der Valk | Index hopping on the Illumina HiseqX platform and its consequences for ancient DNA studies[END_REF], only reads having identical MID tags on both pair members were retained for further analyses (Table S1). Contigs were de novo assembled for each library using IDBA-UD (https://academic.oup.com/bioinformatics/article/28/11/1420/266973).

All contigs were annotated using BlastN and BlastX against the NCBI Genbank non redundant nucleotide (nt) or protein databases with a conservative e-value cut-off of 10 -4 . In this way, contigs were assigned to one of the following categories: virus, eukaryote, bacteria, algae, and unknown. For viral contigs, a family-level annotation was derived from the NCBI taxonomic information for the first Blast hit.

A clustering approach [START_REF] Lefebvre | The VirAnnot pipeline: a resource for automated viral diversity estimation and operational taxonomy units (OTU) assignation for virome sequencing data[END_REF] was used to define operational taxonomy units, following the strategy highlighted by [START_REF] Simmonds | Methods for virus classification and the challenge of incorporating metagenomic sequence data[END_REF]. Briefly, a search of RNA-dependent RNA polymerase (RdRp) conserved protein motifs was performed in all contigs using Reversed Position Specific Blast (RPS-Blast) [START_REF] Altschul | Gapped BLAST and PSI-BLAST: a new generation of protein database search programs[END_REF] against the pfam database [START_REF] Bateman | The Pfam protein families database in 2019[END_REF]. The contigs encoding a viral RdRp motif (Table S1) were retrieved and aligned with reference sequences and distance matrices computed with the ETE3 toolkit [START_REF] Huerta-Cepas | ETE 3: Reconstruction, Analysis, and Visualization of Phylogenomic Data[END_REF]. These matrices were used to perform a clustering allowing to regroup in a single OTU all contigs differing by less than a set cut-off divergence value [START_REF] Murtagh | Ward's Hierarchical Agglomerative Clustering Method: Which Algorithms Implement Ward's Criterion?[END_REF]. We used a 10% divergence cut-off value, because it has been shown to generate in many viral families OTUs that are a relatively good approximation of taxonomic species [START_REF] Lefebvre | The VirAnnot pipeline: a resource for automated viral diversity estimation and operational taxonomy units (OTU) assignation for virome sequencing data[END_REF]. OTUs were thus defined for each RdRp family, allowing to generate an OTU table indicating for each sampling site the presence/absence and the number of reads integrated in each identified OTU (Table S1).

Further viral genome assemblies, sequence comparisons and phylogenetic analyses

When needed, contigs were extended by repeated rounds of mapping of quality-trimmed reads using CLC Genomics Workbench 11 (CLC-GWB). For some isolates/viruses, genomic scaffolds were assembled by mapping contigs and/or reads on a reference genome using CLC-GWB. Long contigs or scaffolds showing more than 75% completeness for cucumber mosaic virus (CMV), southern tomato virus (STV), broad wilt bean virus 1 (BBWV1, both genomic RNAs), the new ilarvirus (all three genomic RNAs) and potato virus Y (PVY) were used for phylogenetic analyses and have been deposited in Genbank (Accession numbers MN216356 to MN216369 (Table S2).

Multiple sequence alignments of contigs/scaffolds obtained from HTS data and of reference isolates retrieved from Genebank (or alignments of deduced encoded proteins)

were performed using the ClustalW algorithm [START_REF] Thompson | CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice[END_REF] as implemented in MEGA 6.0 [START_REF] Kumar | MEGA: A biologist-centric software for evolutionary analysis of DNA and protein sequences[END_REF]. Phylogenetic trees were reconstructed in MEGA 6.0, using the Maximum Likelyhood (ML) algorithm. Branch support was evaluated by bootstrap analysis (100 replicates).

RESULTS

Comparison of the tomato and nightshade viromes at different sampling sites

A total of 20 viral families were discovered by Blast annotation taking into account the different libraries (18 sampled plant populations) with an average of 4.3±3.3 families per library, but with a very large variability between the sampled plant populations. The tomato sample from the TOM3 site showed the highest number of viral families (13, Figure 1)

followed by another tomato sample (TOM7, 9 viral families) and nightshade samples from the TOM2 and NIG3 sites (8 viral families). The Potyviridae family was represent in a total of 13 samples including both tomato (six samples) and nightshade (seven samples, of which five were from tomato sites; Figure 1). The family Totiviridae was represented in eight samples while at the other extreme the Tombusviridae family was represented in a single tomato sample from the TOM3 site. Given the high between-populations variability it was not possible to establish statistically meaningful differences in family-level richness between the tomato and nightshade populations (Figure 1).

Taking into account all sampling sites, a total of 87 unique RNA-dependent RNA polymerase (RdRp) OTUs were detected (Table S1). Similar to the family-level analysis, a very large variability was observed in the number of OTUS detected per site. The richer viromes were found in the TOM7 site tomato population and in the NIG3 nightshade population, with respectively 38 and 27 OTUs, followed by 26 OTUs for the TOM3 site tomato population. In all other samples less than 8 RdRp OTUs were detected (Table S1).

In total, 62 OTUs were identified from tomato samples and 44 from nightshade ones but this difference is largely the consequence of a single tomato sample (TOM7) which is particularly rich in unique mycovirus-like OTUs (Table S1). Nineteen OTUs (21.8% of total) were shared between the two plant species, most of them from the families Totiviridae, Partitiviridae and Chrysoviridae as well as unclassified mycovirus-like OTUs.

RdRP_1-OTU_8 which corresponds to potato virus Y was the most widely shared OTU (Table S1, see below). It explains the wide prevalence of the Potyviridae family described above. Twenty-five OTUs were found to be nightshade-specific, among which RdRP_2-OTU_13 corresponds to cucumber mosaic virus (CMV) and RdRP_1-OTU_14 to broad bean wilt virus 1 (BBWV1) (Table S1, see below). Fourty-three OTUs were found to be tomato-specific, some of which have extremely high identity levels with known viruses such as Sclerotinia sclerotiorum hypovirus 1, Sclerotinia sclerotiorum umbra-like virus 2 or Botrytis virus F and very likely correspond to these agents (Table S1).

There were overall only very few OTUs shared between tomato and nightshade samples for a given sampling site, with PVY being the most frequent. In five sites out of six, no OTU (sites TOM2 and TOM6) or only one OTU (sites TOM1, TOM4, and TOM5) were shared, whereas in site TOM3, four OTUs were shared (Table S1, Figure S1).

Near complete genome reconstruction for selected viral agents

For several viruses, long, high quality contigs were obtained during the initial trimmed reads assembly. This concerned in particular several single-stranded RNA viruses: cucumber mosaic virus (CMV), broad wilt bean virus 1 (BBWV1, both genomic RNAs), potato virus Y (PVY), and a new ilarvirus (all three genomic RNAs) as well as a double-stranded RNA virus of the Amalgaviridae family, southern tomato virus (STV). In a few cases, the viral genome was unambiguously covered by a few contigs that were either non-overlapping or had only a short overlap and which were therefore manually assembled into a scaffold by mapping contigs on a reference genome. All contigs and scaffolds were validated by visual inspection of read mappings at high stringency to ensure the absence of assembly artifacts. The corresponding sequences have been deposited in Genbank (Accession numbers MN216346 to MN216389, Table S2).

Multiple alignments and phylogenetic analyses (see below) were used to identify representative contigs for the various phylogenetic clusters of each virus. These representative contigs were in turn used a targets for the mapping of the trimmed reads of all libraries at high stringency. This allowed to evaluate the representation of each virus/variant in the virome of each sampled plant population. The low background of viral reads observed in the negative control, probably resulting from low level experimental contamination or from inter sample cross talk due to index-hopping [START_REF] Illumina | Effects of index misassignment on multiplexing and downstream analysis[END_REF], was substracted from the mapped reads numbers of each library. The results of this analysis are presented in Table S3.

CMV was detected, by high read numbers, at a single sampling site (TOM1), in the nightshade population but not in the corresponding tomato population (Table S3). [START_REF] Jacquemond | L'ARN satellite du virus de la mosaïque du concombre I. -Comparaison de l'aptitude à induire la nécrose de la tomate d'ARN satellites isolés de plusieurs souches du virus[END_REF]. Nucleotide identity levels of respectively 99.5%, 99.4% and 99.5% for genomic RNA1, 2 and 3 (respectively 18, 18 and 11 point mutations) are thus observed between the 1981 IF17 isolate and the contigs from HTS data on a 2017 sample, highlighting both the quality of the HTS assemblies and the relative stability of the CMV population over more than 35 years.

In the case of southern tomato virus (STV), unique long contigs representing nearly complete genomes were obtained from several plant populations, representing 92.6%-99.4%

of the full length genome. Coherent with the low diversity identified so far in this virus, these contigs are nearly identical to each other (<0.4% nucleotide divergence) with the exception of one contig, which diverges by 2.6%-2.8% from the others. Identity levels with isolates present in Genbank range from 100% to 95.9%, again highlighting the quality of the contigs assembled from the HTS data. Overall STV was detected in five of the seven tomato pools, an observation in accordance with the presence of this virus in a wide range of tomato varieties [START_REF] Sabanadzovic | Southern tomato virus: The link between the families Totiviridae and Partitiviridae[END_REF]. On the other hand, a surprising result is the detection, with higher reads number than for the tomato pools (Table S3) of STV in a nightshade pool (Nightshade-TOM5), extending the host range of this relatively recently discovered virus. The nightshade STV sequence belongs to the group of closely related isolates and does not present obvious specific molecular properties (data not shown).

Broad bean wilt virus 1 populations diversity

Broad bean wilt virus 1 (BBWV1) was detected in six of the sampled nightshade populations (out of a total of 11 populations, 55%) and was not detected in any of the sampled tomato populations (seven populations, Table S3). The assembly of the BBWV1 reads from the various nightshade populations highlighted a complex viral population structure with a total of five RNA1 clusters and three RNA2 clusters identified (Table S3, see below). On average, the reconstructed genomic sequences represented 94.3% +/-3.9%

of the BBWV1 RNA1 (87.8%-99.3%, depending on the contigs) and 87.8% +/-11.9% of the BBWV1 RNA2 (73.4%-96.6%). For one sample, it was not possible to reconstruct more than 60% of the RNA2 sequence and the corresponding scaffold was therefore not included in further analyses. The average nucleotide divergence between the RNA1 clusters, calculated on representative isolates is 16.6% +/-0.3% (13.4%-17.6%), explaining the effective separate assembly in cases of mixed infection by isolates belonging to different clusters. For the three RNA2 clusters, the corresponding divergence values are 15.8% +/-0.6% (10.5%-18.6%). Mapping of reads at high stringency on contigs representative of the various clusters allowed to describe the BBWV1 population present in the various plant populations. Isolates representative of between one and four RNA1 clusters and of one or two RNA2 clusters could thus be detected at individual sampling sites, with some sites providing evidence of only a single RNA1-RNA2 combination, while at the other extreme, one site showed the presence of four RNA1 clusters and a single RNA2 one. Another site showed the presence of a single RNA1 cluster but of two RNA2 ones (Table S3). Taken together, these elements suggest the frequent occurrence of reassortment between BBWV1 genomic segments in the sampled nightshade populations.

Phylogenetic analyses performed on the RNA1 and RNA2 sequences derived from the HTS data and from all full length isolates present in Genbank (Figure 2A and 2B) demonstrate that the BBWV1 isolates present in the nightshade populations sampled here largely expand the known BBWV1 diversity. Indeed, the HTS-derived sequences cluster separately from reference full-length sequences available to date and are, on average, highly divergent from them with an average intergroup distance of 17.0% +/-0.4% for RNA1 and 19.0% +/-0.6% for RNA2.

Presence of a novel ilarvirus in the sampled nightshade and tomato populations

Long, high quality contigs representative of an ilarvirus were identified in several libraries. analyses performed on all genome encoded proteins (Figure 3A and 3B, Figure S2).

However, these phylogenetic trees demonstrate that the virus is not substantially more related to PMoV than to any other approved species in that small ensemble. The significant divergence of the virus from PMoV is confirmed by sequence comparisons, the deduced proteins being only 81.8% (P1) to 53.9% (CP) identical with those of PMoV while the genomic RNAs show only 73.2% (RNA1) to 58.6% (RNA3) nucleotide identity (Table S4).

Taken together these results suggest that the detected larvirus is a new subgroup 1 member for which the name Solanum nigrum ilarvirus 1 (SnIV1) is proposed.

Mapping of the reads from each plant population on the SnIV1 genomic RNAs showed that this virus was present in eight of them, corresponding to 6/11 nightshade populations (54.5%) and, represented by relatively low read numbers, to 2/7 tomato populations (28%) (Table S3).

Analysis of PVY populations in the sampled nightshade and tomato populations

As for the other viruses, long, high quality contigs were obtained in most cases for PVY. In a few cases, probably resulting from low reads numbers or from the simultaneous presence of closely related isolates in the sampled plant populations, only short PVY contigs were obtained for some isolates. However, from all plants populations with high PVY read numbers, one to three long contigs could be assembled presenting on average 95.4% +/-4.8%

of the full length PVY genome (85.3%-99.9%). A phylogenetic analysis of these contigs, together with representative reference isolates retrieved from Genbank (Figure 4) shows a very contrasted situation, with on the one hand, a large number of sequences forming a very tight cluster corresponding to PVY-NTN and, on the other, a much more diverse second cluster corresponding to PVY-C. No isolates representative of the PVY-O and PVY-N strains were observed nor some of their frequent recombinants such as PVY-Wilga (Figure 4). In total, 10 contigs were obtained for PVY-NTN (five from tomato and five from nightshade) and four for PVY-C (three from tomato and one from nightshade).

The reads from all plant populations were then mapped on selected contigs representative of PVY-NTN and of the three PVY-C variants identified, using stringent parameters so as to limit cross-mapping between isolates. Under these conditions, from one to four PVY variants could be detected in the analyzed plant populations. Some populations showed extremely low read numbers (<90), which is suggestive of an absence or a very low prevalence of PVY in the corresponding plant populations. Remarkably, this situation corresponds to 2/7 (28.6%) tomato populations, to 2/6 (33.3%) nightshade populations growing side by side with tomato but to 4/5 (80%) of the nightshade population growing away from tomato.

As judged from the mapping results, the two most frequent PVY strains were PVY-NTN, which was detected in all tomato and nightshade populations in which PVY was detected, and isolates with mapping affinities with isolate TOM7-C, which clusters together with the French PVY-C1 SON41 pepper isolate (Table S3 and Figure 4). By contrast, isolates corresponding to the two other PVY-C mapping references used were only detected in one to three of the sampled plant populations. The frequency of detection of the various clades does not seem to differ much between tomato and nightshade (Table S3).

DISCUSSION

The viromes characterized in the present work vary greatly between the sampled plant populations and, for some of them, showed only a limited number of OTUs or of viral families despite the size of the composite plant samples analyzed. This might reflect the impact of fungicide treatments in the sampled crops which might have reduced fungal diversity and in turn the ability to detect mycoviral communities associated with the sampled plants. It should however be stressed that the OTU-based analysis provides a lower bound estimate of viral diversity since viruses for which the genome region encoding the conserved RdRp motif is not represented in the assembled contigs will not be identified by a corresponding OTU. On the other hand, competition between the sequence of different viruses for representation in the sequencing reads is unlikely to have adversely impacted the richness of the identified viromes since the three richest viromes were identified in plant populations for which the percentage of mapped viral reads was not obviously higher (or lower) than that observed in samples with a much lower viral diversity (Tables S1 andS2).

In contrast to a recent virome study of 170 tomato samples which indicated that diverse ssRNA viruses represented 77% of the identified viruses [START_REF] Xu | Diversity, Distribution, and Evolution of Tomato Viruses in China Uncovered by Small RNA Sequencing[END_REF], they represented only 12.6% of the viral OTUs identified here (Table S1). The corresponding value for dsRNA viruses is 26.4% while unassigned or unannotated agents accounted for a cumulated 60.9%. Whether this difference is a consequence of differences in the methodology used or actually reflects differences in the analyzed viromes cannot easily be ascertained. However, some frequent viruses of tomato such as PVY, CMV or STV were detected in both studies [START_REF] Xu | Diversity, Distribution, and Evolution of Tomato Viruses in China Uncovered by Small RNA Sequencing[END_REF].

Despite the use of complex plant pools composed of 100 individual plants, we were able to assemble long, high quality contigs for some viruses (PVY, BBWV1, STV, and the new such long contigs could not be assembled, possibly as a consequence of too low coverage and read numbers, or because mixed infection involving closely related variants created problems during contig assembly. Indeed, there is some evidence that at least one additional clade of PVY existed in some tomato samples as judged by the detection of some partial contigs diverging from the fully assembled genomes (data not shown).

For read mapping, stringent parameters were used so that there is no or extremely limited cross talk between isolates of different clades, as seen by reads numbers in the case of BBWV1 and PVY (Table S2). At the same time, it is difficult to know precisely how to interpret the samples with a very low number of reads mapped. Even if the background observed in the negative control was substracted, this cross-talk background likely due to index hoping [START_REF] Illumina | Effects of index misassignment on multiplexing and downstream analysis[END_REF][START_REF] Van Der Valk | Index hopping on the Illumina HiseqX platform and its consequences for ancient DNA studies[END_REF] may not be completely uniform from sample to sample. These low read numbers may therefore either reflect an absence of the virus but a low, slightly uneven cross-talk with other samples or a true, very low prevalence of the virus in the sampled population. It is not possible to decide between these two options here.

A very large and unexpected BBWV1 diversity was identified in the sampled nightshade populations. The analysis of BBWV1 populations suggests the existence of frequent reassortment between RNA1 and RNA2 variants, an observation in line with the results of [START_REF] Ferriol | Genetic variability and evolution of broad bean wilt virus 1: role of recombination, selection and gene flow[END_REF]. BBWV1 is a Fabavirus with a relatively wide host range and which is pathogenic on a range of crops including broad bean, pea, spinach, lettuce, pepper and, occasionally, tomato [START_REF] Blancard | Tomato diseases: identification, biology and control: A Colour Handbook[END_REF][START_REF] Carpino | A comparative study of viral infectivity, accumulation and symptoms induced by broad bean wilt virus 1 isolates[END_REF][START_REF] Taylor | Broad bean wilt virus 1[END_REF]. It is therefore surprising that this aphid-transmitted virus was only detected from nightshade samples in this study. This observation suggest the existence of a biological or epidemiological barrier limiting the spread of BBWV1 from nightshade to tomato. In this respect, it is noteworthy that during a recent comparison of BBWV1 isolates, infection rates in tomato following artificial inoculations ranged only from 40% to 60% for four genetically different BBWV1 isolates [START_REF] Carpino | A comparative study of viral infectivity, accumulation and symptoms induced by broad bean wilt virus 1 isolates[END_REF].

The novel ilarvirus here named Solanum nigrum ilarvirus 1 (SnIV1) was detected in both tomato and nightshade samples. However, both the prevalence and, with one exception, the read numbers of SnIV1 appear to be higher in the nightshade populations than in the tomato ones. On the other hand, the presence of SnIV1 in nightshade samples does not seem to be affected by whether they were growing side by side with tomato or not (respectively 3/6 and 3/5 cases, Table S2). Interestingly, reanalysis of metagenomics data showed that this virus was already present in 2011 at the TOM3 site,in S. villosum (hairy nightshade) a close relative of S. nigrum. Whether this novel ilarvirus is pathogenic to tomato or whether it has the potential to emerge at some point as a tomato pathogen in the same fashion as its close relatives Parietaria mottle virus [START_REF] Roggero | Necrotic disease in tomatoes in Greece and southern Italy caused by the tomato strain of Parietaria mottle virus[END_REF] and tomato necrotic spot virus [START_REF] Batuman | Characterization of Tomato necrotic spot virus (ToNSV), a new ilarvirus species infecting processing tomatoes in the Central Valley of California[END_REF] remains to be evaluated.

The main PVY strains identified in this study were PVY-NTN and PVY-C1. PVY-C1

isolates were mainly detected from tomato, with one isolate shared between tomato and nightshade in the TOM3 site (Figure 4). On the other hand, PVY-NTN isolates were found in both tomato (5/7 samples) and nightshade samples (6/11 samples) from a total of seven of the 12 sampling sites. Interestingly, PVY populations at the TOM3 site had been studied 2011-2012 using specific RT-PCR assays [START_REF] Moury | Host groups of Potato virus Y: Vanishing barriers[END_REF]. At the time, PVY-C1 and recombinant isolates likely to represent PVY-NTN were detected in tomato, while a more diverse population involving PVY-O, PVY-NTN, PVY-N and PVY-C1 was detected in nightshade and in the related S. villosum [START_REF] Moury | Host groups of Potato virus Y: Vanishing barriers[END_REF]. The results reported here therefore suggest a simplification of the PVY nightshade population at that site, with the loss of PVY-O and PVY-N, possibly as a consequence of the competition with PVY-NTN and C1.

A noteworthy observation concerns PVY prevalence in nightshade populations at tomato sites (4/6 sites, 66.6%) and at non-tomato sites (1/5 sites, 20%). This suggests that infection in nightshade is greatly increased by the presence of tomato, reflecting a likely spillover effect from tomato crops to the wild nightshade population [START_REF] Power | Pathogen Spillover in Disease Epidemics[END_REF].

Taken together the results reported here provide evidence for viral exchanges between tomato and nightshade populations growing side by side (such as the extremely closely related tomato and nightshade PVY isolates shared at the TOM3 site or the low detection of the new ilarvirus in tomato only at sites were it is also present in nightshade). At the same time, our results also highlight situations where an expected transfer is not observed, likely as a consequence of unforeseen biological or ecological barriers. This concerns in particular BBWV1 only found in nightshade when there are numerous indications that this virus should be able to infect tomato [START_REF] Carpino | A comparative study of viral infectivity, accumulation and symptoms induced by broad bean wilt virus 1 isolates[END_REF]. These results also highlight the power of metagenomics to analyze viral exchanges in complex plant populations, from the overall virome structure down to the intra-specific variability level, revealing unknown novel agents but also unforeseen biological processes.

LEGENDS TO THE FIGURES

  The contigs corresponding to the three genomic RNAs were further extended and validated for the NIG4 sampling site, allowing to reconstruct near complete molecules. Indeed, a comparison with the genomic RNAs of Parietaria mottle virus (PMoV), the closest characterized ilarvirus (see below) indicated that all five open reading frames (ORFs)[coding respectively for P1 (RNA1), P2 and P2b (RNA2) and the movement (MP) and coat proteins (CP) (RNA3)] were complete, with the exception of ORF2 which misses an estimated 62 nt (21 N-terminal amino acids missing from the P2 protein sequence). The contigs are respectively 3,445, 2,757 and 2,257 nt long for RNA1, RNA2 and RNA3, representing respectively 97.9%, 94.4% and 100.5% of the length of the corresponding genomic RNAs of the reference PMoV isolate (NC_005848, -49 and -54). These genomic sequences have been deposited in Genbank under Accession numbers MN216370 to MN216378. Blast analyses indicated that the virus is most closely related to PMoV and to several other subgroup 1 ilarviruses and this proximity was confirmed by phylogenetic

Figure 1 .

 1 Figure 1. Barplot illustrating the presence/absence data based on Blast annotation for identified viral families in each sampled plant population.

Figure 2 .

 2 Figure 2. Maximum Likelyhood trees reconstructed from the alignment of near complete nucleotide sequences of RNA1 (A) and RNA2 (B) of broad bean wilt virus 1 (BBWV1) isolates and other Fabavirus members. Statistical significance of the branches was evaluated by bootstrap analysis (100 replicates) and only bootstrap values higher than 70% are indicated. The scale bars represent 0.1 substitutions per site. Sequences of BBWV1 determined in this work are indicated by a black diamond. The abbreviations followed by the accession numbers are: BBWV2: broad bean wilt virus 2; GeMV: gentian mosaic virus; LLMV: Lamium mild mosaic virus; PeLaV: peach leaf pitting-associated virus; PrVF: Prunus virus F; ChVF: cherry virus F; GFabV: grapevine fabavirus.

Figure 3 .

 3 Figure 3. Maximum Likelyhood trees reconstructed from the alignment of amino acid sequences of the P1 protein (A) and coat protein (B) of representative members of the genus Ilarvirus. Statistical significance of branches was evaluated by bootstrap analysis (100 replicates) and only bootstrap values higher than 70% are indicated. The scale bars represent 0.2 substitutions per site. Solanum nigrum ilarvirus 1 (SnIV1) characterized in this study is indicated by a black diamond.

Figure 4 .

 4 Figure 4. Maximum Likelyhood tree of the near complete nucleotide genome sequences of potato virus Y (PVY) isolates determined in this study (indicated by diamonds) and reference sequences. PVY isolates from tomato samples are indicated by red diamonds and those from nightshade samples by blue diamonds. The tree was constructed by the Maximum Likelyhood method and the statistical significance of branches was evaluated by bootstrap analysis (100 replicates). Only bootstrap values higher than 70% are indicated. The scale bar represents 0.05 substitutions per site.

Figure S2 .

 S2 Figure S2. Maximum Likelyhood trees reconstructed from the alignment of amino acid sequences of the P2a, P2b and movement (MP) proteins of representative members of the genus Ilarvirus. Statistical significance of branches was evaluated by bootstrap analysis (100 replicates). Only bootstrap values higher than 70% are indicated. The scale bars represent 0.2 substitutions per site. Solanum nigrum ilarvirus 1 (SnIV1) characterized in this study is indicated by a black diamond.

Figure 3 .

 3 Figure 3.

  Figure 4.

  All three genomic RNAs were assembled into unique long contigs of respectively 3,301 nt (RNA1; ca. 98.2% of the full length molecule), 2,996 nt (RNA2, ca. 98.3% of the full length molecule) and 2,155 nt (RNA3, ca. 97.2% of the full length molecule) but no evidence was found for presence of a CMV satellite. Despite the fact that no specific efforts were made to improve/validate the contigs further, all three genomic RNAs are extremely close to CMV sequences present in Genbank and, in particular to the I17F isolate, a subgroup I isolate characterized from tomato in France at the beginning of the 1980's (Jacquemond

Ma et al., Virology, 32

ACKNOWLEDGMENTS

The authors would like to thank Eric Sclaunich (INVENIO, Sainte Livrade sur Lot, France) for assistance in accessing some of the tomato and nightshade sampling sites and the Genotoul Platform (INRA, Toulouse, France) for the Illumina sequencing. We would like to thank Laurence Svanella-Dumas and other colleagues in the INRA virology team for help in sample processing. Yuxin Ma was supported by a China Scholarship Council PhD grant.

been deposited on the INRA National

numbers MN216346 to MN216389. Cleaned virome sequence reads have Data Portal under the identifier https://doi.org/10.15454/S486RR.