The Fine Legacy of Giovanni P. Martelli (1935-2020), a Preeminent Plant Virologist and the Founder of Modern Grapevine Virology.

Giovanni Paolo Martelli passed away on 8 January 2020 [...].

Cross-Sectional Comparative Study on Central Sensitization-Psychosocial Associated Comorbidities and Psychological Characteristics in Breast Cancer Survivors with Nociceptive Pain and Pain with Neuropathic Features and without Pain.

The frequency of a high Central Sensitization Inventory (CSI) total score and the prevalence of pain have already been established among breast cancer survivors (BCS). However, the psychological factors' influence based on the clinical features of pain is still unknown, as well as BCS characteristics with no pain. Thus, our main aim was to evaluate the presence of a high CSI total score in BCS with pain and compare it with BCS without pain and to evaluate the influence of psychosocial factors. A cross-sectional comparative study was designed to compare BCS with nociceptive pain (n = 19), pain with neuropathic features (n = 19) or no pain (n = 19), classified by the Leeds Assessment of Neuropathic Symptoms and Signs (LANSS). CSI, pain catastrophizing, fear of movement, anxiety and depression symptoms were analyzed and compared among the three groups. The CSI total score was higher in both BCS pain groups compared to BCS without pain, but there were no statistical differences between the pain groups. The same observation was made when comparing pain catastrophizing. The neuropathic feature group showed greater levels of fear of movement, anxiety and depression compared to the no pain group. Thus, CS-psychosocial associated comorbidities and pain-catastrophizing thoughts were more prevalent among BCS with pain, regardless of the clinical features of pain. BCS with neuropathic pain features showed greater psychological disturbances.

Functional and Cosmetic Outcome after Reconstruction of Isolated, Unilateral Orbital Floor Fractures (Blow-Out Fractures) with and without the Support of 3D-Printed Orbital Anatomical Models.

The present study aimed to analyze if a preformed "hybrid" patient-specific orbital mesh provides a more accurate reconstruction of the orbital floor and a better functional outcome than a standardized, intraoperatively adapted titanium implant. Thirty patients who had undergone surgical reconstruction for isolated, unilateral orbital floor fractures between May 2016 and November 2018 were included in this study. Of these patients, 13 were treated conventionally by intraoperative adjustment of a standardized titanium mesh based on assessing the fracture's shape and extent. For the other 17 patients, an individual three-dimensional (3D) anatomical model of the orbit was fabricated with an in-house 3D-printer. This model was used as a template to create a so-called "hybrid" patient-specific titanium implant by preforming the titanium mesh before surgery. The functional and cosmetic outcome in terms of diplopia, enophthalmos, ocular motility, and sensory disturbance trended better when "hybrid" patient-specific titanium meshes were used but with statistically non-significant differences. The 3D-printed anatomical models mirroring the unaffected orbit did not delay the surgery's timepoint. Nonetheless, it significantly reduced the surgery duration compared to the traditional method (58.9 (SD: 20.1) min versus 94.8 (SD: 33.0) min, p-value = 0.003). This study shows that using 3D-printed anatomical models as a supporting tool allows precise and less time-consuming orbital reconstructions with clinical benefits.

Targeting Glioma Initiating Cells with A combined therapy of cannabinoids and temozolomide.

Glioblastoma multiforme (GBM) is the most frequent and aggressive type of brain tumor due, at least in part, to its poor response to current anticancer treatments. These features could be explained, at least partially, by the presence within the tumor mass of a small population of cells termed Glioma Initiating Cells (GICs) that has been proposed to be responsible for the relapses occurring in this disease. Thus, the development of novel therapeutic approaches (and specifically those targeting the population of GICs) is urgently needed to improve the survival of the patients suffering this devastating disease. Previous observations by our group and others have shown that Δ9-Tetrahydrocannabinol (THC, the main active ingredient of marijuana) and other cannabinoids including cannabidiol (CBD) exert antitumoral actions in several animal models of cancer, including gliomas. We also found that the administration of THC (or of THC + CBD at a 1:1 ratio) in combination with temozolomide (TMZ), the benchmark agent for the treatment of GBM, synergistically reduces the growth of glioma xenografts. In this work we investigated the effect of the combination of TMZ and THC:CBD mixtures containing different ratios of the two cannabinoids in preclinical glioma models, including those derived from GICs. Our findings show that TMZ + THC:CBD combinations containing a higher proportion of CDB (but not TMZ + CBD alone) produce a similar antitumoral effect as the administration of TMZ together with THC and CBD at a 1:1 ratio in xenografts generated with glioma cell lines. In addition, we also found that the administration of TMZ + THC:CBD at a 1:1 ratio reduced the growth of orthotopic xenografts generated with GICs derived from GBM patients and enhanced the survival of the animals bearing these intracranial xenografts. Remarkably, the antitumoral effect observed in GICs-derived xenografts was stronger when TMZ was administered together with cannabinoid combinations containing a higher proportion of CBD. These findings support the notion that the administration of TMZ together with THC:CBD combinations - and specifically those containing a higher proportion of CBD - may be therapeutically explored to target the population of GICs in GBM.

Next-Generation Sequencing and Genome Editing in Plant Virology.

Next-generation sequencing (NGS) has been applied to plant virology since 2009. NGS provides highly efficient, rapid, low cost DNA, or RNA high-throughput sequencing of the genomes of plant viruses and viroids and of the specific small RNAs generated during the infection process. These small RNAs, which cover frequently the whole genome of the infectious agent, are 21-24 nt long and are known as vsRNAs for viruses and vd-sRNAs for viroids. NGS has been used in a number of studies in plant virology including, but not limited to, discovery of novel viruses and viroids as well as detection and identification of those pathogens already known, analysis of genome diversity and evolution, and study of pathogen epidemiology. The genome engineering editing method, clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system has been successfully used recently to engineer resistance to DNA geminiviruses (family, Geminiviridae) by targeting different viral genome sequences in infected Nicotiana benthamiana or Arabidopsis plants. The DNA viruses targeted include tomato yellow leaf curl virus and merremia mosaic virus (begomovirus); beet curly top virus and beet severe curly top virus (curtovirus); and bean yellow dwarf virus (mastrevirus). The technique has also been used against the RNA viruses zucchini yellow mosaic virus, papaya ringspot virus and turnip mosaic virus (potyvirus) and cucumber vein yellowing virus (ipomovirus, family, Potyviridae) by targeting the translation initiation genes eIF4E in cucumber or Arabidopsis plants. From these recent advances of major importance, it is expected that NGS and CRISPR-Cas technologies will play a significant role in the very near future in advancing the field of plant virology and connecting it with other related fields of biology.

Control of pome and stone fruit virus diseases.

Many different systemic pathogens, including viruses, affect pome and stone fruits causing diseases with adverse effects in orchards worldwide. The significance of diseases caused by these pathogens on tree health and fruit shape and quality has resulted in the imposition of control measures both nationally and internationally. Control measures depend on the identification of diseases and their etiological agents. Diagnosis is the most important aspect of controlling fruit plant viruses. Early detection of viruses in fruit trees or in the propagative material is a prerequisite for their control and to guarantee a sustainable agriculture. Many quarantine programs are in place to reduce spread of viruses among countries during international exchange of germplasm. All these phytosanitary measures are overseen by governments based on agreements produced by international organizations. Also certification schemes applied to fruit trees allow the production of planting material of known variety and plant health status for local growers by controlling the propagation of pathogen-tested mother plants. They ensure to obtain propagative material not only free of "quarantine" organisms under the national legislation but also of important "nonquarantine" pathogens. The control of insect vectors plays an important role in the systemic diseases management, but it must be used together with other control measures as eradication of infected plants and use of certified propagation material. Apart from the control of the virus vector and the use of virus-free material, the development of virus-resistant cultivars appears to be the most effective approach to achieve control of plant viruses, especially for perennial crops that are more exposed to infection during their long life span. The use of resistant or tolerant cultivars and/or rootstocks could be potentially the most important aspect of virus disease management, especially in areas in which virus infections are endemic. The conventional breeding for virus-tolerant or resistant fruit tree cultivars using available germplasm is a long-term strategy, and the development and production of these cultivars may take decades, if successful. Genetic engineering allows the introduction of specific DNA sequences offering the opportunity to obtain existing fruit tree cultivars improved for the desired resistance trait. Unfortunately, genetic transformation of pome and stone fruits is still limited to few commercial genotypes. Research carried out and the new emerging biotechnological approaches to obtain fruit tree plants resistant or tolerant to viruses are discussed.

Historical perspective, development and applications of next-generation sequencing in plant virology.

Next-generation high throughput sequencing technologies became available at the onset of the 21st century. They provide a highly efficient, rapid, and low cost DNA sequencing platform beyond the reach of the standard and traditional DNA sequencing technologies developed in the late 1970s. They are continually improved to become faster, more efficient and cheaper. They have been used in many fields of biology since 2004. In 2009, next-generation sequencing (NGS) technologies began to be applied to several areas of plant virology including virus/viroid genome sequencing, discovery and detection, ecology and epidemiology, replication and transcription. Identification and characterization of known and unknown viruses and/or viroids in infected plants are currently among the most successful applications of these technologies. It is expected that NGS will play very significant roles in many research and non-research areas of plant virology.

Evidence of Grapevine leafroll associated virus-1-3, Grapevine fleck virus and Grapevine virus B Occurring in Himachal Pradesh, India.

During a survey conducted in the grapevine orchards of Himachal Pradesh, variety of symptoms ranging from leaf yellowing, vein greening, reduced leaf size, downward rolling/cup shaped leaves to reduced fruit bearing were observed. Symptomatic leaf samples were collected and analyzed by serological (DAS-ELISA) and molecular methods (RT-PCR, PCR) for viruses and phytoplasma known worldwide on grapevine. DAS-ELISA was used for detection of Grapevine leafroll associated virus 1, 2 and 3 (GLRaV-1, 2 & 3), Grapevine virus A (GVA), Grapevine fan leaf virus (GFLV), Grapevine fleck virus (GFkV) and successfully detected GLRaV-1 & 3 and GFkV. All these samples were complemented with RT- PCR along with GVb and phytoplasma (additional to ELISA) using specific primers. Specific amplification in RT-PCR for GLRaV-1 (~232 bp), GLRaV-3 (~300 bp), GFkV (~179 bp) and GVB (~440 bp) confirmed the presence of these pathogens. Overall, ELISA and RT-PCR results confirmed the presence GLRaV-3 (66.7 %), GLRaV-1& GFkV (50 %), and Grapevine virus B (GVB) (12.5 %) in symptomatic plants. None of the samples were found positive for GFLV, GLRaV-2 and phytoplasma. Mixed infection was common and none of the plants were found virus free. To the best of our knowledge this is the first report of detection of GFkV and GVB in India.

Optimization and improvement of oligonucleotide microarray-based detection of tomato viruses and pospiviroids.

Tomato (Solanum lycopersicum L.) is a vegetable crop which is affected by many viruses and several viroids, causing significant economic loss. Their detection and identification is of critical importance for plant protection and quarantine and certification programs. The potential was examined of an array based on the Combimatrix platform for the detection of 37 viruses belonging to 13 families, one of which is unassigned, together with six pospiviroid species, genus Pospiviroid, family Pospiviroidae. More than 470 oligonucleotide probes (40-mer) were selected for the microarray diagnostic technique developed in this investigation. Most of the virus probes were highly specific and were able to identify tomato viruses. Most pospiviroid probes, however, were non-specific in terms of species, but were specific at the genus level as they hybridized to members of the genus Pospiviroid. Only one probe of the Tomato apical stem viroid was species specific. The repeatability and specificity of the Combimatrix method showed that it can be considered for routine diagnostic use in suspected tomato germplasm since it detected 37 viruses and one pospiviroid at the species level and 5-6 pospiviroids at the genus level. The estimated cost for testing of a single tomato virus is similar to or less than the cost of using ELISA.

Oligonucleotide microarray-based detection and identification of 10 major tomato viruses.

A DNA microarray chip was developed for screening 10 major economically important tomato viruses from infected plants using "Combimatrix" platform 40-mer oligonucleotide probes. A total of 279 oligonucleotide virus probes were specific for simultaneous multiple detection, identification, differentiation and/or genotyping of each of the following tomato RNA viruses and/or strains and a virus satellite: Cucumber mosaic virus, Cucumber mosaic virus satellite RNA, Tomatoinfectiouschlorosisvirus, Tomato chlorosisvirus, Tomato spotted wilt virus, Pepino mosaic virus, Potato virus Y, Tobacco mosaic virus and Tomato mosaic virus. This selection included both positive and negative single-stranded RNA viruses. The single-stranded DNA viruses, Tomato yellow leaf curl virus and Tomato yellow leaf curl Sardinia virus were detected but were not differentiated using probes designed from their coat protein genes. A sectored oligonucleotide microarray chip containing four sets of 2000 features (4 x 2 K) was designed. In this way, four samples were tested simultaneously in a hybridization event and 16 samples were analyzed by re-using the chip four times. The hybrids had low background signals. Many of the 40-mer oligonucleotide probes were specific for the detection and identification of each RNA viral species, RNA viral satellite and genotyping strains of Cucumber mosaic virus, Pepino mosaic virus and Potato virus Y. Universal probes were developed for strains of the last three viruses and also for the genus Tobamovirus which includes both Tobacco mosaicvirus and Tomato mosaic virus.

The need for culture collections to support plant pathogen diagnostic networks.

Plant-pathogenic microorganisms, by virtue of their size, similarity in disease symptoms and closely related morphologies, are notoriously difficult to diagnose and detect. Diagnosis gives proof as to the causal agent of disease and is important for developing appropriate control measures. Detection shows the presence of a microorganism and is of importance for safeguarding national and international trade. Live reference collections are required to characterize the taxonomy and function of microorganisms as a prerequisite to development of tools for diagnosis and detection. Two case studies will be presented in this paper to demonstrate the importance of microorganism collections for facilitating knowledge sharing and the development of identification methods. Fusarium wilt of banana caused by Fusarium oxysporum f. sp. cubense and sharka disease of stone fruits caused by plum pox virus (PPV) are considered. Both diseases consist of different races/strains with different host specificities, but Fusarium wilt poses a threat to food security, while PPV poses a threat to trade due to its classification as a quarantine pest, since there is no anti-virus treatment available to control sharka disease in orchards. It is only through comprehensive collections of correctly identified and well-maintained strains representing the genetic diversity of a target organism that robust, specific, reliable and efficient diagnostic and detection tools can be developed.

Viroids and RNA silencing: mechanism, role in viroid pathogenicity and development of viroid-resistant plants.

Viroids are autonomously replicating, small single-stranded circular RNA pathogens that do not code for proteins and may cause diseases in infected, susceptible plants. They have the ability to induce both RNA-mediated transcriptional gene silencing (TGS) and post-transcriptional gene silencing (PTGS), or RNA silencing, in infected plants. Their induced RNA silencing has also been demonstrated in a wheat germ extract system. A possible role of RNA silencing in viroid pathogenicity and evolution has been discussed. It is suggested that RNA silencing can be employed to engineer plants for viroid resistance and attempts to produce these plants have been also discussed.

Oligonucleotide microarray-based detection and genotyping of Plum pox virus.

Plum pox virus (PPV) is the most damaging viral pathogen of stone fruits. The detection and identification of its strains are therefore of critical importance to plant quarantine and certification programs. Existing methods to screen strains of PPV suffer from significant limitations such as the simultaneous detection and genotyping of several strains of PPV in samples infected with different isolates of the virus. A genomic strategy for PPV screening based on the viral nucleotide sequence was developed to enable the detection and genotyping of the virus from infected plant tissue or biological samples. The basis of this approach is a long 70-mer oligonucleotide DNA microarray capable of simultaneously detecting and genotyping PPV strains. Several 70-mer oligonucleotide probes were specific for the detection and genotyping of individual PPV isolates to their strains. Other probes were specific for the detection and identification of two or three PPV strains. One probe (universal), derived from the genome highly conserved 3' non-translated region, detected all individual strains of PPV. This universal PPV probe, combined with probes specific for each known strain, could be used for new PPV strain discovery. Finally, indirect fluorescent labeling of cDNA with cyanine after cDNA synthesis enhanced the sensitivity of the virus detection without the use of the PCR amplification step. The PPV microarray detected and identified efficiently the PPV strains in PPV-infected peach, apricot and Nicotiana benthamiana leaves. This PPV detection method is versatile, and enables the simultaneous detection of plant pathogens.