Varvara I. Maliogka

Control of Viruses Infecting Grapevine

Grapevine is a high value vegetatively propagated fruit crop that suffers from numerous viruses, including some that seriously affect the profitability of vineyards. Nowadays, 64 viruses belonging to different genera and families have been reported in grapevines and new virus species will likely be described in the future. Three viral diseases namely leafroll, rugose wood, and infectious degeneration are of major economic importance worldwide. The viruses associated with these diseases are transmitted by mealybugs, scale and soft scale insects, or dagger nematodes. Here, we review control measures of the major grapevine viral diseases. More specifically, emphasis is laid on (i) approaches for the production of clean stocks and propagative material through effective sanitation, robust diagnosis, as well as local and regional certification efforts, (ii) the management of vectors of viruses using cultural, biological, and chemical methods, and (iii) the production of resistant grapevines mainly through the application of genetic engineering. The benefits and limitations of the different control measures are discussed with regard to accomplishments and future research directions.

Evolutionary relationships of virus species belonging to a distinct lineage within the Ampelovirus genus

A study of the evolutionary relationships of GLRaV-4,-5,-6 and -9, and two new Ampelovirus isolates (GLRaV-Pr and -De) related to grapevine leafroll disease was conducted based on molecular variability, positive selection analysis and maximum likelihood phylogenetic reconstructions. Sequences corresponding to the N-terminal HSP70h and full CP encoding genes were determined for these viruses and datasets including homologous genomic regions from different members of the Closteroviridae were analyzed. GLRaV-Pr and -De were further characterised as distinct from the other closely related species after determination of a large genomic region (4319-4358 nts). ML phylogenetic topologies for both genes established the closer phylogenetic relationships of GLRaV-4,-5,-6,-9,-Pr and -De in regard to the other ampeloviruses, revealing very low inter-species evolutionary distances for this multitudinous lineage. The HSP70h segment phylogeny and bootstrap analysis enabled the identification of species within this lineage and provides a useful taxonomic tool for the rapid demarcation of these viruses. Estimations of d(N)/d(S) using the CP and HSP70h datasets revealed that, within the Closteroviridae, these viruses are subjected to the strongest constraints against amino acid substitutions. These estimations demonstrated a distinct evolutionary trait for this lineage probably related to its particular ecological niche that involves successful adaptation to the host, transmission through vegetative propagation and lack of vectors with high transmission efficiency.

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.

Detection and Quantification of Infectious Avian Influenza A (H5N1) Virus in Environmental Water by Using Real-Time Reverse Transcription-PCR

ABSTRACT Routes of avian influenza virus (AIV) dispersal among aquatic birds involve direct (bird-to-bird) and indirect (waterborne) transmission. The environmental persistence of H5N1 virus in natural water reservoirs can be assessed by isolation of virus in embryonated chicken eggs. Here we describe development and evaluation of a real-time quantitative reverse transcription (RT)-PCR (qRT-PCR) method for detection of H5N1 AIV in environmental water. This method is based on adsorption of virus particles to formalin-fixed erythrocytes, followed by qRT-PCR detection. The numbers of hemagglutinin RNA copies from H5N1 highly pathogenic AIV particles adsorbed to erythrocytes detected correlated highly with the infectious doses of the virus that were determined for three different types of artificially inoculated environmental water over a 17-day incubation period. The advantages of this method include detection and quantification of infectious H5N1 AIVs with a high level of sensitivity, a wide dynamic range, and reproducibility, as well as increased biosecurity. The lowest concentration of H5N1 virus that could be reproducibly detected was 0.91 50% egg infective dose per ml. In addition, a virus with high virion stability (Tobacco mosaic virus) was used as an internal control to accurately monitor the efficiency of RNA purification, cDNA synthesis, and PCR amplification for each individual sample. This detection system could be useful for rapid high-throughput monitoring for the presence of H5N1 AIVs in environmental water and in studies designed to explore the viability and epidemiology of these viruses in different waterfowl ecosystems. The proposed method may also be adapted for detection of other AIVs and for assessment of their prevalence and distribution in environmental reservoirs.

Virus variants with differences in the P1 protein coexist in a Plum pox virus population and display particular host-dependent pathogenicity features.

Subisolates segregated from an M-type Plum pox virus (PPV) isolate, PPV-PS, differ widely in pathogenicity despite their high degree of sequence similarity. A single amino acid substitution, K109E, in the helper component proteinase (HCPro) protein of PPV caused a significant enhancement of symptom severity in herbaceous hosts, and notably modified virus infectivity in peach seedlings. The presence of this substitution in certain subisolates that induced mild symptoms in herbaceous hosts and did not infect peach seedlings suggested the existence of uncharacterized attenuating factors in these subisolates. In this study, we show that two amino acid changes in the P1 protein are specifically associated with the mild pathogenicity exhibited by some PS subisolates. Site-directed mutagenesis studies demonstrated that both substitutions, W29R and V139E, but especially W29R, resulted in lower levels of virus accumulation and symptom severity in a woody host, Prunus persica. Furthermore, when W29R and V139E mutations were expressed concomitantly, PPV infectivity was completely abolished in this host. In contrast, the V139E substitution, but not W29R, was found to be responsible for symptom attenuation in herbaceous hosts. Deep sequencing analysis demonstrated that the W29R and V139E heterogeneities already existed in the original PPV-PS isolate before its segregation in different subisolates by local lesion cloning. These results highlight the potential complexity of potyviral populations and the relevance of the P1 protein of potyviruses in pathogenesis and viral adaptation to the host.

The VP3 Factor from Viruses of Birnaviridae Family Suppresses RNA Silencing by Binding Both Long and Small RNA Duplexes

RNA silencing is directly involved in antiviral defense in a wide variety of eukaryotic organisms, including plants, fungi, invertebrates, and presumably vertebrate animals. The study of RNA silencing-mediated antiviral defences in vertebrates is hampered by the overlap with other antiviral mechanisms; thus, heterologous systems are often used to study the interplay between RNA silencing and vertebrate-infecting viruses. In this report we show that the VP3 protein of the avian birnavirus Infectious bursal disease virus (IBDV) displays, in addition to its capacity to bind long double-stranded RNA, the ability to interact with double-stranded small RNA molecules. We also demonstrate that IBDV VP3 prevents the silencing mediated degradation of a reporter mRNA, and that this silencing suppression activity depends on its RNA binding ability. Furthermore, we find that the anti-silencing activity of IBDV VP3 is shared with the homologous proteins expressed by both insect- and fish-infecting birnaviruses. Finally, we show that IBDV VP3 can functionally replace the well-characterized HCPro silencing suppressor of Plum pox virus, a potyvirus that is unable to infect plants in the absence of an active silencing suppressor. Altogether, our results support the idea that VP3 protects the viral genome from host sentinels, including those of the RNA silencing machinery.

Molecular Identification, Reverse Transcription-Polymerase Chain Reaction Detection, Host Reactions, and Specific Cytopathology of Artichoke yellow ringspot virus Infecting Onion Crops

ABSTRACT An isometric virus ca. 25 nm in diameter with angular contour was isolated from onion plants showing yellow leaf striping and necrotic tips. The virus was mechanically transmitted onto 28 species of indicator plants belonging to five families, viz. Amaranthaceae, Chenopodiaceae, Cucurbitaceae, Leguminosae, and Solanaceae where it causes ring spots, malformations, and/or tip necrosis. Cytopathological studies in infected Nicotiana benthamiana tissues revealed cytoplasmic inclusions resembling those caused by Artichoke yellow ringspot virus (AYRSV), a member of the family Comoviridae. Host range and symptomatology of the onion virus were also similar to AYRSV. A high seed transmission rate (20%) was found in onion. Reverse transcription-polymerase chain reaction using degenerate primers specific for the family Comoviridae allowed amplification of RNA-dependent RNA polymerase sequences, which upon sequence analysis and comparison with AYRSV isolates from Cynara scolymus (AYRSV-AtG) and Vicia faba (AYRSV-F) were highly similar, thus providing evidence that the nepovirus AYRSV is infecting onion in the field.

Demarcation of ilarviruses based on the phylogeny of RNA2-encoded RdRp and a generic ramped annealing RT-PCR

SummaryIn this study, a generic ramped-annealing (RAN) nested RT-PCR was developed, allowing the simultaneous detection and fast characterization of ilarviruses. The method involves a one-step RT-PCR in which a pair of degenerate primers amplifies a 381-bp part of the polymerase gene (RNA2), followed by a nested PCR amplification that increases detection sensitivity. The sensitivity and detection range of the method were further increased by applying a ramped annealing thermocycling step both in the first RT-PCR and in the subsequent nested PCR. The 371-bp nested amplicons can be sequenced directly, without cloning, to obtain initial sequence information on ilarvirus genomes, or can undergo a restriction enzyme analysis for rapid identification of already known virus species. Phylogenetic relationships among different members of the family Bromoviridae were inferred with maximum likelihood and Bayesian analysis, using published homologous partial amino acid sequences corresponding to the nested amplicon and also to a longer residue data set (432–453 aa) comprising all possible positions of homology among the RNA2-encoded polymerases of members of the family Bromoviridae. The implications of these analyses on the taxonomy of ilarviruses are discussed. The specific partial polymerase sequence, corresponding to the polymerase core palm structure (motifs A–D), was verified as phylogenetically informative and can be used to separate ilarviruses from other members of the family Bromoviridae, providing initial information for ilarvirus species characterization. However, the phylogenetic signal of this region is not reliable for inferring relationships among distantly related ilarviruses.

Viruses of the genus Allium in the Mediterranean region.

Allium species are economically important crops in the Mediterranean basin. Viruses are among the most important pathogens affecting their yield and especially those belonging to the genera Potyvirus, Carlavirus, and Allexivirus. Members of the genus Potyvirus are usually the most abundant and cause most of the damage induced. Nevertheless, coinfections with different viruses are not scarce, especially in garlic, and can have synergistic effects that lead to even greater crop losses. Vegetative propagation of alliums and the transmission of most of their viruses by arthropod vectors have significantly contributed to their wide dissemination in the Mediterranean region and elsewhere in the world. Here, we review the general biological and molecular features, the epidemiology, incidence, and methods of diagnosis of the most widespread allium viruses in the basin. Control measures are proposed depending on the mode of propagation of the various alliums, the epidemiology of their viruses and the cultivation procedures adapted by the Mediterranean farmers. The importance of the production and use of virus-free propagative material in order to combat viral diseases of allium crops is especially highlighted. A final discussion focuses on the main shortages identified in the research area of allium viruses, and proposals are made for putative future developments.

Generic and species-specific detection of viruses belonging to an evolutionary distinct lineage within the Ampelovirus genus

A nested RT-PCR was developed, that allows the generic detection of a subgroup of genetically related viruses with a distinct evolutionary history within the genus Ampelovirus. Members of this lineage are Grapevine leafroll associated virus-4, -5, -6, -9 and two isolates (GLRaV-De and GLRaV-Pr) that have been recently characterized and represent new species. The method involves a one step RT-PCR for the generic detection of Closteroviridae species using degenerate primers that target the HSP70h gene followed by a nested PCR, which detects all virus-members of the lineage and differentiates them from the other grapevine closteroviruses. The 490 bp nested PCR amplicons, corresponding to a phylogenetically informative region, can be sequenced directly to obtain initial genetic information for their partial characterization and rapid classification. Additional primers were designed and successfully used for the specific detection of GLRaV-4, -5, -6, -Pr and -De on respective single or multiplex nested PCR assays. The application of a ramped annealing thermal profile in the nested PCR allowed all amplifications to run in parallel. The developed detection scheme is proposed as a tool that can be used for the enrichment of sequence information of known and uncharacterized ampeloviruses, classified within this lineage, enabling their selective amplification in mixed Closteroviridae virus infections.