Biophysical Features and Local Early Conformational Propensities in Intrinsically Disordered Regions of Rhabdoviral Glycoproteins

Most harmful rhabdoviruses cause disease that is invariably lethal to humans, animals and plants. Throughout viral infection, protein refolding is a complex process critical to both receptor recognition and membrane-interacting fusion domains mediated by transmembrane rhabdoviral glycoprotein (Gp). However, little is known about the early stages of context-sensitive structural transitions of the rhabdoviral Gp. We hypothesized that these involve local interactions between residues situated in intrinsically disordered regions (IDRs) of both the β-sheet rich lateral domain and the fusion domain. This study investigates the host-specific biophysical IDRs-determinants encoded in the primary amino acid sequence of rhabdoviral glycoproteins, which are predicted to modulate early conformational events. This can be related to host-specific biophysical features of the local backbone and secondary structure propensities close to or in IDRs of Gps. While showing striking differences between IDRs of N- and C-termini, our results give direct insights into the biophysical folding signals located in these regions and are in congruence with independent experimental observations. Furthermore, on a quantitative scale, the biophysical features of these residues tend to become those that interact the most in the folded structure and are often residues that display evolutionary covariation, reflecting a general tendency toward conserved host-specificity. In conclusion, the accurate connection of biophysical structural features with both IDRs conformational propensities and context-sensitive folding data suggests their statistically significant role in local transition with lasting effects on subsequent conformational states during virus-host interactions and disease-related pathogenicity outcomes.Prediction and validation of biological properties of viruses using -omic data through machine learning

Occurrence and Partial Genetic Characterisation of Lettuce big‐vein associated virus and Mirafiori lettuce big‐vein virus infecting lettuce in Jordan

Lettuce big‐vein disease (LBVD) is an economically damaging disease that occurs in most lettuce production areas worldwide. Several studies have associated LBVD with a complex of two viruses, Lettuce big‐vein associated virus (LBVaV) and Mirafiori lettuce big‐vein virus (MiLBVV). Both viruses are transmitted through soil by the zoospores of the chytridiomycete fungus Olpidium virulentus. During surveys carried out in two growing seasons from 2015 to 2016 in the main vegetable growing areas in Jordan (Jordan Valley, Jerash, and Madaba), 179 lettuce plants, 46 lettuce seedlings, and 56 weed plants were collected. RNA extraction was conducted from leaves of all samples. Associated viruses were identified using reverse transcription‐polymerase chain reaction (RT‐PCR) with specific primer pairs targeting part of the coat protein (CP) genes of LBVaV and MiLBVV. RT‐PCR results revealed that, out of 225 lettuce samples tested, 32 (14%) and 44 (20%) were singly infected with LBVaV and MiLBVV, respectively, and 73 (32%) samples were dually infected with LBVaV and MiLBVV. Overall, MiLBVV was detected in more lettuce samples (117) compared with LBVaV which was detected in 105 samples. Moreover, one sample of each weed species of Chenopodium murale and Sonchus oleraceus tested positive for MiLBVV and LBVaV, respectively. The partial nucleotide sequence of the CP genes of LBVaV and MiLBVV Jordanian isolates showed similarities to other isolates available in the NCBI database ranging between 93.9–100% and 88.3–100%, respectively. Nucleotide sequence comparison of the Jordanian isolates and inference from phylogenetic trees with strong support revealed well‐defined sub‐groups with little or no correlation with planting area or geographic origin. The occurrence of both viruses in lettuce plants was associated with the presence of resting spores and zoospores of Olpidium spp. in lettuce plants grown under field and growth chamber conditions. Lettuce plants inoculated with Olpidium spp. zoospores isolated from roots of symptomatic lettuce plants, developed LBVD‐symptoms. At 60 days post‐transplantation, both viruses were detected by RT‐PCR in most of the plants analysed. Biological and molecular assays together with the light microscopy observations of resting spores and zoospores of Olpidium spp., revealed the presence of LBVaV, MiLBVV, and O. virulentus in big‐vein diseased lettuce plants

First Report of Candidatus Phytoplasma solani Infecting New Natural Host Plum (Prunus domestica L.) in Jordan.

peer reviewedPlum (Prunus domestica L., Rosaceae) is among the most important stone fruit species grown in Jordan, especially in the northeastern part of the country. In September 2017, 30 trees from a 2-ha orchard of cultivar ‘Early Queen’ showing possible phytoplasma symptoms of leaf yellowing and reddening, stunted growth, and witches’ broom were observed. Additionally, there were around 20 bindweed plants (Convolvulus arvensis L.) among the plum trees showing stunting and leaf reddening in the Mafraq region in northeast Jordan. Leaf samples were collected from 15 symptomatic and five asymptomatic plum trees and three symptomatic bindweed plants. Total DNA was extracted from all samples and analyzed by PCR using the phytoplasma universal primer pair P1/P7 (16S/23S RNA, Deng and Hiruki 1991; Schneider et al. 1995), followed by nested PCR with primer pair R16F2n and R16R2 (16S rDNA, Gundersen and Lee 1996). The same samples were also tested by PCR using the 16SrXII-specific primers SecY1a and SecYR1 (secY, Lee et al. 2010) and by “stolbur” phytoplasma specific quantitative PCR (16S rDNA, Hren et al. 2007). DNA samples of five out of 15 P. domestica and all three bindweed samples tested positive for the presence of a phytoplasma by qPCR (Ct values of 19.5 to 26.2 and 33 to 34 for plum and bindweed, respectively) and nested PCR, yielding the expected PCR amplicons. Interestingly, no phytoplasma was detected in 10 samples from symptomatic plum trees as well as from all the asymptomatic trees. Bidirectional sequencing (Macrogen, Amsterdam, The Netherlands) was done for each PCR amplicon after gel purification. A BLASTn similarity analysis of the assembled consensus sequences derived from the plum and the bindweed host plants revealed that the sequences of phytoplasmas infecting P. domestica (accession nos. MH085227, MH085228, and MH085229) and C. arvensis (MH085225 and MH085226) in Jordan had identity of 99% to the deposited sequence of ‘Candidatus Phytoplasma solani’ from of Vitis vinifera L. in Jordan (KC835139) and the ‘Ca. P. solani’ strain sequence from Capsicum annuum L. from Serbia (AF248959). The same analysis based on the secY gene confirmed the highest identity (99%) of analyzed sequences of ‘Ca. P. solani’ (MK928324 to MK928326) with strains from Russia and Germany (GU004346 and FO393427, respectively). The identity of ‘Ca. P. solani’ was confirmed in plum and bindweed by virtual RFLP (iPhyClassifier, Zhao et al. 2009) and phylogenetic analyses (BioNumerics, Applied Math, Belgium). These results supported the first occurrence of ‘Ca. P. solani’ in plum in Jordan, where it may represent a potential threat to other already reported susceptible stone fruit hosts, such as peach, almond, and cherry. The reported presence of the bindweed in Jordan (Salem et al. 2013), a well-known phytoplasma natural reservoir (Mehle et al. 2011), and potential insect vectors (e.g., Hyalesthes obsoletus) (Quaglino et al. 2013) along with vegetatively propagated plant material (Bertaccini et al. 2014) may have contributed to the plum infection in Jordan

First Report of Little Cherry Virus 1 Infecting Apricot (Prunus armeniaca) in Africa

peer reviewedLittle cherry disease (LChD) is an important viral disease of many stone fruit species (Prunus spp.), sweet cherry (Prunus avium L.) being the most common host. It is associated with two different virus species belonging to the family Closteroviridae, namely, Little cherry virus 1 (LChV-1, Velarivirus) and Little cherry virus 2 (LChV-2, Ampelovirus). The impact of LChD on sweet cherry production consists in the decrease of yield and fruit quality, which is mainly associated with LChV-2, whereas most of LChV-1 reported infections remain associated with an unclear etiology. Other stone fruit species, such as peach and plum, hosting LChV-1 have been reported (Matic et al. 2007; Šafářová et al. 2017). LChV-1 is mainly transmitted through propagation of infected plant material, and no vector transmission is known (Jelkmann and Eastwell 2011). In 2018, during the early vegetative season, a limited survey was carried out for virus detection in apricot and sweet cherry orchards in the main southern Moroccan stone fruit-producing regions of Agadir, Agdez, and Dayat Aoua. Two sweet cherry trees (P. avium ‘Coeur de Pigeon’ and ‘Bigarreau’) and three apricot trees (Prunus armeniaca L.), all asymptomatic, were sampled (five branches with leaves) from three different orchards. RNA was extracted (both leaves and cambial scrapings) using the Spectrum Plant Total RNA kit (Sigma-Aldrich, Belgium), prior to cDNA synthesis using the iScript Reverse Transcription Kit (Bio-Rad, Belgium). LChV-1 detection was done by reverse transcription PCR (RT-PCR) using the specific primers LCUW7090 (5′-GGTTGTCCTCGGTTGATTAC-3′)/LCUWc7389 (5′-GGCTTGGTTCCATACATCTC-3′) (Bajet et al. 2008), amplifying a 300-bp fragment spanning the ORF1b encoding the RdRp gene, and 1LC_12776F (5′-TCAAGAAAAGTTCTGGTGTGC-3′)/1LC_13223R (5′-CGAGCTAGACGTATCAGTATC-3′) (Glasa et al. 2015), targeting a 456-bp fragment of the CP gene. LChV-2 specific primers were used according to Eastwell and Bernardy (2001). RT-PCR results revealed the presence of LChV-1 in two apricot samples from Agdez. No LChV-1 was detected in the sweet cherry samples. The presence of LChV-1 was confirmed by means of the LChV-1 specific reverse transcription loop-mediated isothermal amplification approach as described by Tahzima et al. (2019). No LChV-2 was detected in any of the samples. The RdRp and CP specific amplification products were bidirectionally sequenced (Genewiz, Leipzig, Germany) and assembled. RdRp and CP partial nucleotide sequences of the Moroccan LChV-1 isolates MOT2 and MOA1 were deposited in GenBank (accession nos. MK905349, MK905350; and MK905351, MK905352, respectively). Based on BLAST analysis of RdRp and CP, the Moroccan LChV-1 sequences shared 99% nucleotide identity (99.55% amino acids) with the No2ISTO isolate (HG792418) from Greece and 97.96% (98.64% amino acids) with the Spanish Ponferrada isolate (KX192367), respectively. Although the presence of LChV-1 has previously been reported in many countries in different continents, to our knowledge, this represents the first detection of LChV-1 in Africa

First Report of ‘Candidatus Phytoplasma aurantifolia’-Related Strains Infecting Potato (Solanum tuberosum) in Jordan

Potato (Solanum tuberosum L. ‘Spunta’, Solanaceae) is an important economic crop in Jordan. In November 2013, potato plants showing symptoms of leaf reddening, aerial and abnormally small and deformed tubers, suggestive of possible phytoplasma infection, were observed in three potato fields with disease incidence of 3 to 5% in the Jordan Valley region. Leaf samples were collected from 14 symptomatic and five asymptomatic potato plants. Total genomic DNA was extracted by a cetyltrimethylammonium bromide protocol (Doyle and Doyle 1987). The 16S rRNA gene was partially amplified using the phytoplasma universal primer pairs P1/P7 followed by nested polymerase chain reaction (PCR) with primer pair R16F2n/R16R2 (Deng and Hiruki 1991; Gundersen and Lee 1996). DNA extracts of three symptomatic potato samples showed positive results for phytoplasma infection, yielding a specific PCR amplicon around 1.25 kbp. No phytoplasma was detected in asymptomatic potato plants that were sampled from the same field. PCR products from the three positive samples where cloned into pGEMT-Easy vector, sequenced (Macrogen, Amsterdam, The Netherlands) and analyzed through BLAST search. The sequences (GenBank accession nos. MH085230, MH085231, and MH085232) from Jordan shared 99% identity with sequences of ‘Candidatus Phytoplasma aurantifolia’ from Thailand (JN006076 and JN006079) and China (JQ923433). Furthermore, the identity among all Jordanian phytoplasma strains of this study was 99%. Additionally, a virtual restriction fragment length polymorphism was done for (sub)group classification (iPhyClassifier, http://plantpathology.ba.ars.usda.gov/​cgi-bin/resource/iphyclassifier.cgi, Beltsville, MD, Zhao et al. 2009) and confirmed the identity of the phytoplasma as a member of subgroup 16SrII. Phylogenetic analysis (BioNumerics, Applied Math, Belgium) based on partial 16S rRNA gene sequences of representative phytoplasma strains placed the Jordanian potato phytoplasma strains in a single distinct cluster together with ‘Ca. P. aurantifolia’ subgroup 16SrII. Taken together, these results confirmed the unique occurrence of ‘Ca. P. aurantifolia’-related strains in potato in Jordan Valley. The presence of potatoes infected by a member of the subgroup 16SrII phytoplasma in Jordan may have serious epidemiological implications on this crop. Our results will open an avenue to future studies on the spread and impact of this phytoplasma and its potential insect vectors. To the best of our knowledge, this is the first report of ‘Ca. P. aurantifolia’-related strains infecting potato in Jordan.Peer reviewe

Viruses of cucurbit crops: current status in the Mediterranean Region

peer reviewedCucurbits are among the most cultivated crops, and the most economically important species are melon (Cucumis melo L.), cucumber (Cucumis sativus L.), watermelon (Citrullus lanatus Thumb.), squash (Cucurbita pepo L.), and pumpkin (Cucurbita spp.). These crops have become important income sources providing export and local consumption commodities in many Mediterranean countries. Increased area of cucurbits has led to the emergence of several viral diseases, which can have impacts on crop production and threaten agricultural sustainability. An overview of the most damaging cucurbit viruses in the Mediterranean area is provided to improve understanding of the diseases they cause and to emphasize effective disease management strategies. An updating of the geographical distribution of these viruses, the symptoms they cause and their means of transmission is also provided. Disease management methods and measures by farmers and phytosanitary authorities to address the virus outbreaks are outlined, including diagnostics, use of tolerant cultivars, and chemical and biological vector control. Mediterranean region farmers have learned many lessons from the damaging pandemics caused by cucurbit viruses, through the extensive published research, and this review provides a basis for managing future outbreaks of newly emerging virus infections

Identification of divergent isolates of Banana Mild Mosaic Virus and development of a new diagnostic primer to improve detection

This study aims to describe the identification and genome sequencing of two isolates of Banana Mild Mosaic Virus, and, based on the virus sequences available in GenBank, to design and test a new diagnostic primer for a routine indexing use

Managing the deluge of newly discovered plant viruses and viroids: an optimized scientific and regulatory framework for their characterization and risk analysis

The advances in high-throughput sequencing (HTS) technologies and bioinformatic tools have provided new opportunities for virus and viroid discovery and diagnostics. Hence, new sequences of viral origin are being discovered and published at a previously unseen rate. Therefore, a collective effort was undertaken to write and propose a framework for prioritizing the biological characterization steps needed after discovering a new plant virus to evaluate its impact at different levels. Even though the proposed approach was widely used, a revision of these guidelines was prepared to consider virus discovery and characterization trends and integrate novel approaches and tools recently published or under development. This updated framework is more adapted to the current rate of virus discovery and provides an improved prioritization for filling knowledge and data gaps. It consists of four distinct steps adapted to include a multi-stakeholder feedback loop. Key improvements include better prioritization and organization of the various steps, earlier data sharing among researchers and involved stakeholders, public database screening, and exploitation of genomic information to predict biological properties

Conceptual Slices and Theoretical Sheaves on Plant Virus Exaptations and Structural Evolutionary Virology

The ongoing Little cherry Disease (LChD) global pandemic has piqued public interest in the properties, evolution, and emergence of its associated plant viruses little cherry virus 1 (LChV-1) and little cherry virus 2 (LChV-2). There is a realistic expectation that global efforts in LChD management combined to new HTS-based diagnostic platforms will bring LChV newly triggered outbreaks under reasonable control. Nonetheless, uncertainties remain about latent infection in long-term association that both viruses can establish with Prunus germplasms or wild species and new potential vectors. Although the epidemiological trajectories are difficult to predict, the current conditions and biological variables that influence epidemiology can be proactively anticipated. Persistence of both endemic plant viruses of sweet and sour cherry, perhaps with seasonal epidemicity, may be fueled by reservoirs of new susceptible cultivated Prunus cultivars or rootstocks and post-infection waning resistance and genetic variability through evolutionary drift that hampers host immune protection. Here, I addressed relevant field observations from surveyed LChD outbreaks and explored patterns of LChV phylogenomic evolution as it adapts mainly during its graft-transmission through diverse horticultural eco-systems. Furthermore, the lack of reliable diagnostics or adequate response often enables the surge of new LChV hotspots. In this research, while proposing new diagnostic solutions, key pieces of data have been unveiled in order to draw evidence-based regulatory plant health decisions. From an evolutionary vantage, hypotheses on phylogenomic origin of viruses members of the family Closteroviridae remain unclear because of their diverse and mosaic evolutionary functional proteomic makeup (we coined for the first time here The Modulome). In the second frame of this thesis, I take full advantage of the abundant protein structural and functional data to explore the Modulome of little cherry viruses in the context of the global plant proteome. The discovery of modularity patterns and their wide-spread distribution further shifted our standard paradigms. Using cutting-edge bioinformatics, our comparative proteome-wide data-driven analyses were extended to the study of all detected conformational fold families and their assigned functions confirming their modulation role in the co-evolutionary processes governing vector-based transmission modes fostered by modular innovation. In light of this structural complexity unique insights prompted a critical redefinition and reclassification of plant viruses. Based on primordial evolution of structural domains in the genomes of Closteroviridae, I made robust inference on the ancestry of viral exaptations traced from their co-evolving hosts and draw novel timescaled hypothetical macroevolutionary scenari based on sheaves of novel robust evidence. From more innovative perspectives, the study of intrinsic disordered regions in the proteins (IDR/Ps) of plant viruses remain untapped. To fill this gap, we used the most widely reliable artificial intelligence-based algorithms to predict IDP signatures in viruses of the Closteroviridae taxon, including LChV-1 and LChV-2 benchmarked against the global Modulome.For the first time, I unraveled the IDRs occurrence and abundance, where they operate important modulatory roles and supplement the multifunctionality of diverse viral structural interacting modules, in particular those involved in vector-based transmission. Our Disordome-wide analysis also uniquely established that conserved IDRs signatures adorned with protein molecular recognition features (MoRFs) in plant viruses and vector modules are likely to promote multifunctionality by encoding more non-optimal codon biases. Collectively, these forays offer novel outlooks on the structural evolution of viral IDP as multifaceted evolutionary forces that shape the global plant virome and predominantly govern viral functional adaptability during interactions with phylogenomically divergent hosts. In conclusion, during this research, the deployed efforts shed new light into the ancestral origins, epidemiology and evolution of closteroviruses and elucidated determinants that resist the widespread acceptance of new views and insights. I proposed a new definition of the little cherry disease that is not restricted to the oversimplified presence or absence LChV-1and/or -2 or symptomatic features. I also discuss general views for how plant viruses likely enduringly modulate their interactions according to the fine-tuned evolutionary proteomic signatures, and unearth the conceptual biases that may limit our understanding of virus evolution and classification. With the prism of functional modular evolution, I discuss how these fundamental aspects have surprisingly remained disputed despite being increasingly used within the reach of scientific thinking. Lastly, I introspect few philosophical concepts evolutionary structural virology, symbiopoeisis and viral disease and their impact on the way we might predict future LChD outbreaks.VLAIO-IWT – Little Cherry Projec