Molecular characterization of poleroviruses

Viruses of the genus Polerovirus are serious pathogens of economically important crops causing severe yield losses. Although some species such as BWYV, CtRLV and PLRV are amongst the first plant viruses characterized worldwide, a rapid increase in poleroviral species has been observed in the last decade. New poleroviral species have been correlated with diseases in “traditional” hosts such as pepper and cucurbits as well as in plant species like cassava and strawberry not known to be hosts of members of the genus. In Greece, studies on poleroviruses are very limited, whereas their association with specific crop diseases and the epidemiology of the virus(es) involved remains largely unknown. The purpose of this thesis was to identify viruses of the genus Polerovirus and their disease complexes with umbraviruses and aRNAs in Greece, to molecular characterize them and to study their phylogenetic relationships. The survey conducted during this study showed the significant presence of poleroviruses in Greece in plant species belonging in different families. However, the incidence of the disease complexes was very low and they were mostly found in species of the family Apiaceae. The variability observed in the polerovirus generic detection RT-PCR amplicon suggested that some isolates were in fact new viral species, which was later on confirmed by their molecular characterization. Two poleroviral phylogenetic groups were selected for further study. The first group includes the virus related to pepper yellowing disease (PYD) and the second one the species involved in carrot motley dwarf disease (CMD). These viral isolates were fully sequenced using the NGS technology, and the results indicated that in Greece three new poleroviral species are associated with CMD and PYD. Furthermore, in order to study the interactions between the viruses involved in the CMD complex, three infectious clones capable to infect different plant species were constructed. However, it was not possible to find an appropriate host in order to study the disease and the interactions between the viruses. This study also revealed the high variability of the poleroviruses, as 10 new viral species were identified. Moreover, one of their most profound characteristic of these viruses is the fact that different viral species can cause the same disease in a host. This fact has a direct impact in epidemiological studies since these viruses can form groups including either closely related viral species or species with different phylogenetic origin. These viruses have different biological properties, thus the disease development is directly correlated to the structure of this viral group, in which it is highly likely that other, yet unidentified, species are also involved.Οι ιοί του γένους Polerovirus αποτελούν, αδιαμφισβήτητα, σοβαρά παθογόνα οικονομικώς σημαντικών καλλιεργειών, καθώς η μόλυνση από ιούς του γένους προκαλεί σημαντικές απώλειες στην παραγωγή. Ορισμένοι, όπως οι BWYV, CtRLV και PLRV, αποτελούν από τους πρώτους φυτικούς ιούς που χαρακτηρίσθηκαν παγκοσμίως, ενώ τα τελευταία χρόνια ο αριθμός των ειδών του γένους Polerovirus εμφανίζει μια ραγδαία αύξηση. Νέα είδη του γένους έχουν πλέον συσχετιστεί με ασθένειες τόσο σε «κλασικούς» ξενιστές, όπως η πιπεριά και τα κολοκυνθοειδή, όσο και σε φυτά τα οποία δεν είχαν αναφερθεί προηγουμένως ως ξενιστές ιών του γένους, όπως η κασάβα και η φράουλα. Στην Ελλάδα η παρουσία και η επιδημιολογία των polero-ιών έχει ελάχιστα μελετηθεί. Σκοπός της παρούσας εργασίας ήταν ο προσδιορισμός των ιών του γένους Polerovirus και των συμπλεγμάτων που σχηματίζουν αυτοί με ιούς του γένους Umbravirus και τα aRNA στην Ελλάδα, ο μοριακός χαρακτηρισμός τους και η μελέτη των φυλογενετικών τους σχέσεων. Η επισκόπηση που πραγματοποιήθηκε έδειξε την σημαντική παρουσία των polero-ιών σε φυτικά είδη που ανήκουν σε διαφορετικές οικογένειες. Αντιθέτως, η παρουσία των συμπλεγμάτων ήταν μικρή και αφορούσε κυρίως τα φυτά της οικογένειας Apiaceae. Η παραλλακτικότητα που εμφάνισαν ορισμένες απομονώσεις στο τμήμα που ενισχύει η μέθοδος γενικής ανίχνευσης polero-ιών υπέδειξε την παρουσία νέων ειδών, η οποία επιβεβαιώθηκε με τον περαιτέρω μοριακό χαρακτηρισμό τους. Από τις φυλογενετικές ομάδες που δημιουργήθηκαν επιλέχθηκαν δυο για περαιτέρω μελέτη. Η πρώτη περιλαμβάνει τον ιό που εμπλέκεται με την ασθένεια του ίκτερου της πιπεριάς (PYD), ενώ η δεύτερη με αυτούς που σχετίζονται με το κοκκίνισμα/ποικιλόχρωση των σκιαδανθών (CMD). Απομονώσεις των ιών αυτών αλληλουχήθηκαν πλήρως με NGS και τα αποτελέσματα έδειξαν την ύπαρξη τριών νέων ειδών του γένους Polerovirus που σχετίζονται με τις ασθένειες CMD και PYD στην Ελλάδα. Στη συνέχεια, για να μελετηθούν οι αλληλεπιδράσεις των ιών της CMD, κατασκευάστηκαν τρεις μολυσματικοί κλώνοι, οι οποίοι είναι ικανοί να μολύνουν διάφορα φυτικά είδη, χωρίς όμως να βρεθεί ένας ξενιστής που να είναι κατάλληλος για την μελέτη της ασθένειας και των αλληλεπιδράσεων των ιών. Συνολικά η μελέτη αυτή έδειξε την υψηλή παραλλακτικότητα που υπάρχει στους polero-ιούς καθώς ταυτοποιήθηκαν 10 νέα ιικά είδη. Επιπλέον, αποδείχθηκε ότι διαφορετικοί polero-ιοί σχετίζονται με την ίδια ασθένεια (συμπτωματολογική εικόνα) σε έναν ξενιστή. Το γεγονός αυτό έχει άμεση επίπτωση στη μελέτη της επιδημιολογίας τους, καθώς οι ομάδες αυτές μπορεί να αποτελούνται είτε από συγγενικούς ιούς, είτε από φυλογενετικά απομακρυσμένα είδη. Ως εκ τούτου τα βιολογικά χαρακτηριστικά των ιών αυτών διαφέρουν, γεγονός που επηρεάζει την εξέλιξη της ασθένειας που προκαλούν, ανάλογα με την σύσταση της ιικής ομάδας, στην οποία είναι πολύ πιθανό να συμμετέχουν αρκετά ακόμα άγνωστα είδη polero-ιών

A novel specific duplex real-time RT-PCR method for absolute quantitation of Grapevine Pinot gris virus in plant material and single mites.

Grapevine Pinot gris virus (GPGV) is a widely distributed grapevine pathogen that has been associated to the grapevine leaf mottling and deformation disease. With the aim of better understanding the disease epidemiology and providing efficient control strategies a specific and quantitative duplex TaqMan real-time RT-PCR assay has been developed. This method has allowed reliable quantitation of the GPGV titer ranging from 30 up to 3 x 108 transcript copies, with a detection limit of 70 viral copies in plant material. The assay targets a grapevine internal control that reduces the occurrence of false negative results, thus increasing the diagnostic sensitivity of the technique. Viral isolates both associated and non-associated to symptoms from Greece, Slovakia and Spain have been successfully detected. The method has also been applied to the absolute quantitation of GPGV in its putative transmission vector Colomerus vitis. Moreover, the viral titer present in single mites has been determined. In addition, in the current study a new polymorphism in the GPGV genome responsible for a shorter movement protein has been found. A phylogenetic study based on this genomic region has shown a high variability among Spanish isolates and points to a different evolutionary origin of this new polymorphism. The methodology here developed opens new possibilities for basic and epidemiological studies as well as for the establishment of efficient control strategies

A novel grapevine badnavirus is associated with the Roditis leaf discoloration disease

Roditis leaf discoloration (RLD), a graft-transmissible disease of grapevine, was first reported in Greece in the 1980s. Even though various native grapevine viruses were identified in the affected vines, the etiology of the disease remained unknown. In the present study, we used an NGS platform for sequencing siRNAs from a twenty-year old Roditis vine showing typical RLD symptoms. Analysis of the NGS data revealed the presence of various known grapevine viruses and viroids as well as a hitherto uncharacterized DNA virus. The circular genome of the new virus was fully reassembled. It is 6988 nts long and includes 4 open reading frames (ORFs). ORF1, ORF2 and ORF4 code for proteins with unknown functions while ORF3 encodes a polyprotein with motifs related to the replication, encapsidation and movement of the virus. Phylogenetic analysis classified the novel virus within the genus Badnavirus, with closest relationship to Fig badnavirus 1. Further studies showed that the new badnavirus is closely related with the RLD disease and the provisional name grapevine Roditis leaf discoloration-associated virus (GRLDaV) is proposed. Our findings extend the number of DNA viruses identified in grapevine, further drawing attention to the potential importance of this virus group on grapevine pathology

A subset of highly responsive transcription factors upon tomato infection by pepino mosaic virus

Plants have evolved well-tuned surveillance systems, including complex defence mechanisms, to constrain pathogens. TFs are master regulators of host molecular responses against plant pathogens. While PepMV constitutes a major threat to the global tomato production, there is still a lack of information on the key TFs that regulate host responses to this virus. A combinatorial research approach was applied relying on tomato transcriptome analysis, RT-qPCR validation, phylogenetic classification, comparative analysis of structural features, cis-regulatory element mining and in silico co-expression analysis to identify a set of 11 highly responsive TFs involved in the regulation of host responses to PepMV. An endemic PepMV isolate, generating typical mosaic symptoms, modified expression of ca. 3.3% of tomato genes, resulting in 1,120 DEGs. Functional classification of 502 upregulated DEGs revealed that photosynthesis, carbon fixation and gene silencing were widely affected, whereas 618 downregulated genes had an impact mainly on plant defence and carotenoid biosynthesis. Strikingly, all 11 highly responsive TFs carried abiotic stress response cis-regulatory elements, whereas five of them were better aligned with rice than with Arabidopsis gene homologues, suggesting that plant responses against viruses may predate divergence into monocots and dicots. Interestingly, tomato C2H2 family TFs, ZAT1-like and ZF2, may have distinct roles in plant defence due to opposite response patterns, similar to their Arabidopsis ZAT10 and ZAT12 homologues. These highly responsive TFs provide a basis to study in-depth molecular responses of the tomato–PepMV pathosystem, providing a perspective to better comprehend viral infections

Variability Studies of Two <em>Prunus</em>-Infecting Fabaviruses with the Aid of High-Throughput Sequencing.

During their lifetime, perennial woody plants are expected to face multiple infection events. Furthermore, multiple genotypes of individual virus species may co-infect the same host. This may eventually lead to a situation where plants harbor complex communities of viral species/strains. Using high-throughput sequencing, we describe co-infection of sweet and sour cherry trees with diverse genomic variants of two closely related viruses, namely prunus virus F (PrVF) and cherry virus F (CVF). Both viruses are most homologous to members of the Fabavirus genus (Secoviridae family). The comparison of CVF and PrVF RNA2 genomic sequences suggests that the two viruses may significantly differ in their expression strategy. Indeed, similar to comoviruses, the smaller genomic segment of PrVF, RNA2, may be translated in two collinear proteins while CVF likely expresses only the shorter of these two proteins. Linked with the observation that identity levels between the coat proteins of these two viruses are significantly below the family species demarcation cut-off, these findings support the idea that CVF and PrVF represent two separate Fabavirus species

High-Throughput Sequencing Reveals Further Diversity of Little Cherry Virus 1 with Implications for Diagnostics

Little cherry virus 1 (LChV1, Velarivirus, Closteroviridae) is a widespread pathogen of sweet or sour cherry and other Prunus species, which exhibits high genetic diversity and lacks a putative efficient transmission vector. Thus far, four distinct phylogenetic clusters of LChV1 have been described, including isolates from different Prunus species. The recent application of high throughput sequencing (HTS) technologies in fruit tree virology has facilitated the acquisition of new viral genomes and the study of virus diversity. In the present work, several new LChV1 isolates from different countries were fully sequenced using different HTS approaches. Our results reveal the presence of further genetic diversity within the LChV1 species. Interestingly, mixed infections of the same sweet cherry tree with different LChV1 variants were identified for the first time. Taken together, the high intra-host and intra-species diversities of LChV1 might affect its pathogenicity and have clear implications for its accurate diagnostics

Real-time RT-PCR cycle threshold (Ct) values for GPGV and <i>PEP</i> internal control, absolute quantitation, symptomatology and presence of other grapevine viruses in GPGV positive samples collected from D.O. Utiel-Requena.

<p>Real-time RT-PCR cycle threshold (Ct) values for GPGV and <i>PEP</i> internal control, absolute quantitation, symptomatology and presence of other grapevine viruses in GPGV positive samples collected from D.O. Utiel-Requena.</p

Sequence alignment of the MP/CP region targeted by the real-time RT-PCR assay of 20 GPGV Spanish isolates using GPGV IT isolate (NC_015782.1).

<p>Two polymorphisms producing a shorter MP: P1 (previously reported by Saldarelli et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197237#pone.0197237.ref003" target="_blank">3</a>]) at position 6,684 and P2 (new polymorphism identified in this work) at position 6,687 are indicated.</p

Sensitivity comparison between TaqMan and SYBR Green GPGV real-time RT-PCR detection methods.

<p>Sensitivity comparison between TaqMan and SYBR Green GPGV real-time RT-PCR detection methods.</p

Absolute quantitation of GPGV in the putative transmission vector <i>C</i>. <i>vitis</i>.

<p>Amplification plots obtained for pools of 15 (purple), 10 (black) and 5 (green) and individual mites (blue) are shown. A negative control of mites fed on GPGV free leaves (brown) is included.</p