ICTV Virus Taxonomy Profile: Benyviridae.

The Benyviridae is a family of multipartite plant viruses with rod-shaped virions. Genomes are segmented and comprised of single-stranded, positive-sense RNAs, each with a 5' m7G cap. Unlike rod-shaped viruses classified in the Virgaviridae family, the genome segments have a 3' polyA tract and there is post-translational cleavage of the viral replicase. The better-known members are transmitted by root-infecting vectors in the Plasmodiphorales family, once described as fungi but now classified as Cercozoa. The family has a single genus. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of Benyviridae, which is available at www.ictv.global/report/benyviridae.

ICTV Virus Taxonomy Profile: Virgaviridae.

The family Virgaviridae is a family of plant viruses with rod-shaped virions, a ssRNA genome with a 3'-terminal tRNA-like structure and a replication protein typical of alpha-like viruses. Differences in the number of genome components, genome organization and the mode of transmission provide the basis for genus demarcation. Tobacco mosaic virus (genus Tobamovirus) was the first virus to be discovered (in 1886); it is present in high concentrations in infected plants, is extremely stable and has been extensively studied. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of the Virgaviridae, which is available at www.ictv.global/report/virgaviridae.

Transmission of grapevine Pinot gris virus by Colomerus vitis (Acari: Eriophyidae) to grapevine.

Grapevine Pinot gris virus (GPGV) is a new virus reported in Europe and several other grape-growing countries. In an attempt to identify a vector for GPGV, samples of the eriophyid mite Colomerus vitis collected from buds and erinea in GPGV-infected vines were analysed by RT-PCR, using specific primers. Molecular analysis revealed the presence of GPGV in C. vitis. Transmission trials were conducted using C. vitis collected from GPGV-infected vines. Mites were able to transmit GPGV to healthy grapevines, suggesting that C. vitis is a potential vector of this virus.

Semi-continuous cultivation of EPS-producing marine cyanobacteria: A green biotechnology to remove dissolved metals obtaining metal-organic materials.

Given the necessity for bioprocesses scaling-up, the present study aims to explore the potential of three marine cyanobacteria and a consortium, cultivated in semi-continuous mode, as a green approach for i) continuous exopolysaccharide-rich biomass production and ii) removal of positively charged metals (Cu, Ni, Zn) from mono and multi-metallic solutions. To ensure the effectiveness of both cellular and released exopolysaccharides, weekly harvested whole cultures were confined in dialysis tubings. The results revealed that all the tested cyanobacteria have a stronger affinity towards Cu in mono and three-metal systems. Despite the amount of metals removed per gram of biomass decreased with higher biosorbent dosage, the more soluble carbohydrates were produced, the greater was the metal uptake, underscoring the pivotal role of released exopolysaccharides in metal biosorption. According to this, Dactylococcopsis salina 16Som2 showed the highest carbohydrate productivity (142 mg L-1 d-1) and metal uptake (84 mg Cu g-1 biomass) representing a promising candidate for further studies. The semi-continuous cultivation of marine cyanobacteria here reported assures a schedulable production of exopolysaccharide-rich biosorbents with high metal removal and recovery potential, even from multi-metallic solutions, as a step forward in the industrial application of cyanobacteria.

Rapid and specific detection of wheat spindle streak mosaic virus using RT-LAMP in durum wheat crude leaf extract.

To accurately determine the spread of any pathogen, including plant viruses, a quick, sensitive, cost-effective, point-of-care diagnostic assay is necessary. Wheat spindle streak mosaic virus (WSSMV) is a Bymovirus, transmitted by the plasmodiophorid Polymyxa graminis Led, which causes yellow mosaic and reduces the grain yield in wheat. Currently, detection protocols for WSSMV use ELISA or more sensitive PCR-based approaches requiring specialized laboratory and personnel. A protocol for reverse transcription loop mediated isothermal amplification (RT-LAMP) has been developed and optimized for the rapid detection of viruses using crude extracts from wheat leaves. The protocol was specific for WSSMV detection, while no reaction was observed with SBCMV or SBWMV, the non-target viruses transmitted by the same vector. The RT-LAMP assay was shown to be as sensitive as the one-step WSSMV specific RT-PCR. The RT-LAMP assay can be performed under field conditions using a portable instrument, and can help the actual spread of WSSMV, an aspect of this virus not yet well understood, to be explored.

The benyvirus RNA silencing suppressor is essential for long-distance movement, requires both zinc-finger and NoLS basic residues but not a nucleolar localization for its silencing-suppression activity.

The RNA silencing-suppression properties of Beet necrotic yellow vein virus (BNYVV) and Beet soil-borne mosaic virus (BSBMV) cysteine-rich p14 proteins have been investigated. Suppression of RNA silencing activities were made evident using viral infection of silenced Nicotiana benthamiana 16C, N. benthamiana agroinfiltrated with green fluorescent protein (GFP), and GF-FG hairpin triggers supplemented with viral suppressor of RNA silencing (VSR) constructs or using complementation of a silencing-suppressor-defective BNYVV virus in Chenopodium quinoa. Northern blot analyses of small-interfering RNAs (siRNAs) in agroinfiltration tests revealed reduced amounts of siRNA, especially secondary siRNA, suggesting that benyvirus VSR act downstream of the siRNA production. Using confocal laser-scanning microscopy imaging of infected protoplasts expressing functional p14 protein fused to an enhanced GFP reporter, we showed that benyvirus p14 accumulated in the nucleolus and the cytoplasm independently of other viral factors. Site-directed mutagenesis showed the importance of the nucleolar localization signal embedded in a C4 zinc-finger domain in the VSR function and intrinsic stability of the p14 protein. Conversely, RNA silencing suppression appeared independent of the nucleolar localization of the protein, and a correlation between BNYVV VSR expression and long-distance movement was established.

Traditional Approaches and Emerging Biotechnologies in Grapevine Virology.

Environmental changes and global warming may promote the emergence of unknown viruses, whose spread is favored by the trade in plant products. Viruses represent a major threat to viticulture and the wine industry. Their management is challenging and mostly relies on prophylactic measures that are intended to prevent the introduction of viruses into vineyards. Besides the use of virus-free planting material, the employment of agrochemicals is a major strategy to prevent the spread of insect vectors in vineyards. According to the goal of the European Green Deal, a 50% decrease in the use of agrochemicals is expected before 2030. Thus, the development of alternative strategies that allow the sustainable control of viral diseases in vineyards is strongly needed. Here, we present a set of innovative biotechnological tools that have been developed to induce virus resistance in plants. From transgenesis to the still-debated genome editing technologies and RNAi-based strategies, this review discusses numerous illustrative studies that highlight the effectiveness of these promising tools for the management of viral infections in grapevine. Finally, the development of viral vectors from grapevine viruses is described, revealing their positive and unconventional roles, from targets to tools, in emerging biotechnologies.

Cauliflower mosaic virus: Virus-host interactions and its uses in biotechnology and medicine.

Cauliflower mosaic virus (CaMV) was the first discovered plant virus with genomic DNA that uses reverse transcriptase for replication. The CaMV 35S promoter is a constitutive promoter and thus, an attractive driver of gene expression in plant biotechnology. It is used in most transgenic crops to activate foreign genes which have been artificially inserted into the host plant. In the last century, producing food for the world's population while preserving the environment and human health is the main topic of agriculture. The damage caused by viral diseases has a significant negative economic impact on agriculture, and disease control is based on two strategies: immunization and prevention to contain virus spread, so correct identification of plant viruses is important for disease management. Here, we discuss CaMV from different aspects: taxonomy, structure and genome, host plants and symptoms, transmission and pathogenicity, prevention, control and application in biotechnology as well as in medicine. Also, we calculated the CAI index for three ORFs IV, V, and VI of the CaMV virus in host plants, the results of which can be used in the discussion of gene transfer or antibody production to identify the CaMV.

A Guide to Cannabis Virology: From the Virome Investigation to the Development of Viral Biotechnological Tools.

Cannabis sativa cultivation is experiencing a period of renewed interest due to the new opportunities for its use in different sectors including food, techno-industrial, construction, pharmaceutical and medical, cosmetics, and textiles. Moreover, its properties as a carbon sequestrator and soil improver make it suitable for sustainable agriculture and climate change mitigation strategies. The increase in cannabis cultivation is generating conditions for the spread of new pathogens. While cannabis fungal and bacterial diseases are better known and characterized, viral infections have historically been less investigated. Many viral infection reports on cannabis have recently been released, highlighting the increasing threat and spread of known and unknown viruses. However, the available information on these pathogens is still incomplete and fragmentary, and it is therefore useful to organize it into a single structured document to provide guidance to growers, breeders, and academic researchers. This review aims to present the historical excursus of cannabis virology, from the pioneering descriptions of virus-like symptoms in the 1940s/50s to the most recent high-throughput sequencing reports. Each of these viruses detected in cannabis will be categorized with an increasing degree of threat according to its potential risk to the crop. Lastly, the development of viral vectors for functional genetics studies will be described, revealing how cannabis virology is evolving not only for the characterization of its virome but also for the development of biotechnological tools for the genetic improvement of this crop.

Investigating the effects of essential oils and pure botanical compounds against Eimeria tenella in vitro.

Essential oils (EO) and natural bioactive compounds are well-known antibacterial and anti-inflammatory factors; however, little is known about their anticoccidial activity and mode of action. EO deriving from basil (BEO), garlic (GAR), oregano (OEO), thyme (TEO), and their main bioactive compounds were investigated for their anticoccidial proprieties and compared to salinomycin (SAL) and amprolium (AMP) in vitro. The invasion of Eimeria tenella sporozoites was studied on 2 cell models: Madin-Darby Bovine Kidney (MDBK) cells and primary chicken epithelial cells (cIEC). Invasion efficiency was evaluated at 2 and 24 h postinfection (hpi) with counts of extracellular sporozoites and by detection of intracellular E. tenella DNA by PCR. Results show that at both timepoints, the EO were most effective in preventing the invasion of E. tenella with an average reduction of invasion at 24 hpi by 36% in cIEC and 55% in MDBK. The study also examined cytokine gene expression in cIEC at 24 hpi and found that AMP, BEO, OEO, TEO, carvacrol (CAR), and thymol (THY) significantly reduced interleukin (IL)8 expression, with CAR also reducing expression of IL1ß and IL6 compared to the infected control. In addition, this work investigated the morphology of E. tenella sporozoites treated with anticoccidial drugs and EO using a scanning electron microscope. All the treatments induced morphological anomalies, characterized by a reduction of area, perimeter and length of sporozoites. SAL had a significant impact on altering sporozoite shape only at 24 h, whereas CAR and THY significantly compromised the morphology already at 2 hpi, compared to the untreated control. OEO and GAR showed the most significant alterations among all the treatments. The findings of this study highlight the potential of EO as an alternative to traditional anticoccidial drugs in controlling E. tenella invasion and in modulating primary immune response.

Beet necrotic yellow vein virus subgenomic RNA3 is a cleavage product leading to stable non-coding RNA required for long-distance movement.

Beet necrotic yellow vein virus (BNYVV) is a multipartite RNA virus. BNYVV RNA3 does not accumulate in non-host transgenic Arabidopsis thaliana plants when expressed using a 35S promoter. However, a 3'-derivative species has been detected in transgenic plants and in transient expression assays conducted in Nicotiana benthamiana and Beta macrocarpa. The 3'-derivative species is similar to the previously reported subgenomic RNA3 produced during virus infection. 5' RACE revealed that the truncated forms had identical 5' ends. The 5' termini carried the coremin motif also present on BNYVV RNA5, beet soil-borne mosaic virus RNA3 and 4, and cucumber mosaic virus group 2 RNAs. This RNA3 species lacks a m(7)Gppp at the 5' end of the cleavage products, whether expressed transiently or virally. Mutagenesis revealed the importance of the coremin sequence for both long-distance movement and stabilization of the cleavage product in vivo and in vitro. The isolation of various RNA3 5'-end products suggests the existence of a cleavage between nt 212 and 1234 and subsequent exonucleolytic degradation, leading to the accumulation of a non-coding RNA. When RNA3 was incubated in wheatgerm extracts, truncated forms appeared rapidly and their appearance was protein- and divalent ion-dependent.

A new PCR based molecular method for early and precise quantification of parasitization in the emerging olive pest Dasineura oleae.

BACKGROUND: Dasineura oleae (Angelini 1831) (Diptera: Cecidomyiidae) was considered a minor pest in olive orchards, but in recent years severe outbreaks have been registered in several Mediterranean countries. Damage is caused by the feeding activity of larvae that induce gall formations and alters the physiological activity of the leaves. In Italy, this pest may be controlled by four Hymenoptera parasitoid species belonging to Platygaster and Mesopolobus genera such as Platygaster demades Walker 1835, Platygaster oleae Szelenyi 1940 (Hymenoptera: Platygastridae), Mesopolobus aspilus (Walker 1835) and Mesopolobus mediterraneus (Mayr 1903) (Hymenoptera: Pteromalidae), but parasitization becomes evident only after gall dissection. RESULTS: In this study, we aim to: (i) design a primer for the detection of specimens belonging to Platygaster and Mesopolobus genera; (ii) develop a multiplex quantitative polymerase chain reaction (qPCR) protocol combined to a fast samples DNA extraction method; (iii) apply the developed protocol to field-collected specimens and compare this method with traditional techniques based on visual estimation of parasitism rate on larvae. Primers were designed to anneal with cytochrome oxidase subunit I (COI) sequences of Platygaster and Mesopolobus genera while protocols were developed to be fast and capable to process several samples at the same time. Molecular analyses demonstrated to provide almost double of the parasitism rate assessed by visual inspection. Furthermore, on second instar larvae the PCR-based method was able to detect ten-fold times the parasitization rate estimated by visual inspection. CONCLUSION: The application on a greater scale of this newly developed method could be fundamental in the determination of the biological control potential in olive orchards.

Fast and Sensitive Detection of Soil-Borne Cereal Mosaic Virus in Leaf Crude Extract of Durum Wheat.

Soil-borne cereal mosaic virus (SBCMV) is a furovirus with rigid rod-shaped particles containing an ssRNA genome, transmitted by Polymyxa graminis Led., a plasmodiophorid that can persist in soil for up to 20 years. SBCMV was reported on common and durum wheat and it can cause yield losses of up to 70%. Detection protocols currently available are costly and time-consuming (real-time PCR) or have limited sensitivity (ELISA). To facilitate an efficient investigation of the real dispersal of SBCMV, it is necessary to develop a new detection tool with the following characteristics: no extraction steps, very fast results, and high sensitivity to allow pooling of a large number of samples. In the present work, we have developed a reverse transcription loop-mediated isothermal amplification (RT-LAMP) protocol with such characteristics, and we have compared it with real-time PCR. Our results show that the sensitivity of LAMP and real-time PCR on cDNA and RT-LAMP on crude extracts are comparable, with the obvious advantage that RT-LAMP produces results in minutes rather than hours. This paves the way for extensive field surveys, leading to a better knowledge of the impact of this virus on wheat health and yield.

Essential Oils and Hydrolates: Potential Tools for Defense against Bacterial Plant Pathogens.

The essential oils (EOs) of Origanum compactum and Satureja montana chemotyped (CT) at carvacrol, two Thymus vulgaris CT at thujanol and thymol, and Hydrolates (Hys) of S. montana and Citrus aurantium var. amara were chosen for studying their bactericidal efficacy against few phytobacterial pathogens. The Minimal Inhibitory Concentration (MIC) and Bactericidal Concentration (MBC) were found by microdilution assay. The essential oils of O. compactum (MBC 0.06% v/v), T. vulgaris CT thymol (MBC 0.06% v/v), and Hy of C. aurantium (MBC 6.25% v/v) resulted in being the most effective against Erwinia amylovora; thus, they were used as starting concentrations for ex vivo assays. Despite the great in vitro effectiveness, the disease incidence and the population dynamic ex vivo assays showed no significant results. On the other hand, EO of O. compactum and Hy of C. aurantium (at 0.03% and 4.5% v/v, respectively) showed resistance induction in tomato plants against Xanthomonas vesicatoria infections; both treatments resulted in approximately 50% protection. In conclusion, EOs and Hys could be promising tools for agricultural defense, but further studies will be necessary to stabilize the EOs emulsions, while Hys application could be an effective method to prevent bacterial diseases when used as resistance inducer by pre-transplantation treatment at roots.

Resistance to Soil-borne cereal mosaic virus in durum wheat is controlled by a major QTL on chromosome arm 2BS and minor loci.

Soil-borne cereal mosaic (SBCM) is a viral disease, which seriously affects hexaploid as well as tetraploid wheat crops in Europe. In durum wheat (Triticum durum Desf.), the elite germplasm is characterized by a wide range of responses to SBCMV, from susceptibility to almost complete resistance. In this study, the genetic analysis of SBCMV resistance was carried out using a population of 181 durum wheat recombinant inbred lines (RILs) obtained from Meridiano (resistant) × Claudio (moderately susceptible), which were profiled with SSR and DArT markers. The RILs were characterized for SBCMV response in the field under severe and uniform SBCMV infection during 2007 and 2008. A wide range of disease reactions (as estimated by symptom severity and DAS-ELISA) was observed. A large portion of the variability for SBCMV response was explained by a major QTL (QSbm.ubo-2BS) located in the distal telomeric region of chromosome 2BS near the marker triplet Xbarc35-Xwmc661-Xgwm210, with R(2) values ranging from 51.6 to 91.6%. The favorable allele was contributed by Meridiano. Several QTLs with minor effects on SBCMV response were also detected. Consistently with the observed transgressive segregation, the resistance alleles at minor QTLs were contributed by both parents. The presence and effects of QSbm.ubo-2BS were validated through association mapping in a panel of 111 elite durum wheat accessions.

De novo Sequencing of Novel Mycoviruses From Fusarium sambucinum: An Attempt on Direct RNA Sequencing of Viral dsRNAs.

An increasing number of viruses are continuously being found in a wide range of organisms, including fungi. Recent studies have revealed a wide viral diversity in microbes and a potential importance of these viruses in the natural environment. Although virus exploration has been accelerated by short-read, high-throughput sequencing (HTS), and viral de novo sequencing is still challenging because of several biological/molecular features such as micro-diversity and secondary structure of RNA genomes. This study conducted de novo sequencing of multiple double-stranded (ds) RNA (dsRNA) elements that were obtained from fungal viruses infecting two Fusarium sambucinum strains, FA1837 and FA2242, using conventional HTS and long-read direct RNA sequencing (DRS). De novo assembly of the read data from both technologies generated near-entire genomic sequence of the viruses, and the sequence homology search and phylogenetic analysis suggested that these represented novel species of the Hypoviridae, Totiviridae, and Mitoviridae families. However, the DRS-based consensus sequences contained numerous indel errors that differed from the HTS consensus sequences, and these errors hampered accurate open reading frame (ORF) prediction. Although with its present performance, the use of DRS is premature to determine viral genome sequences, the DRS-mediated sequencing shows great potential as a user-friendly platform for a one-shot, whole-genome sequencing of RNA viruses due to its long-reading ability and relative structure-tolerant nature.

Cauliflower Mosaic Virus TAV, a Plant Virus Protein That Functions like Ribonuclease H1 and is Cytotoxic to Glioma Cells.

Recent comparisons between plant and animal viruses reveal many common principles that underlie how all viruses express their genetic material, amplify their genomes, and link virion assembly with replication. Cauliflower mosaic virus (CaMV) is not infectious for human beings. Here, we show that CaMV transactivator/viroplasmin protein (TAV) shares sequence similarity with and behaves like the human ribonuclease H1 (RNase H1) in reducing DNA/RNA hybrids detected with S9.6 antibody in HEK293T cells. We showed that TAV is clearly expressed in the cytosol and in the nuclei of transiently transfected human cells, similar to its distribution in plants. TAV also showed remarkable cytotoxic effects in U251 human glioma cells in vitro. These characteristics pave the way for future analysis on the use of the plant virus protein TAV, as an alternative to human RNAse H1 during gene therapy in human cells.

A Rapid Protocol of Crude RNA/DNA Extraction for RT-qPCR Detection and Quantification.

Most of the molecular diagnostic protocols used for phytoplasmas detection are based on the purification of total nucleic acids and on the use of genomic DNA of the pathogen as the target of amplification. Here we describe a diagnostic approach that, avoiding the purification of nucleic acids and exploiting the amplification of the abundant phytoplasma ribosomal RNA molecules produced during the infectious process, allows reducing the time and the costs necessary for the analysis, without affecting sensitivity and specificity. This is useful in particular when high numbers of analyses are required, as in certification programs, to monitor phytoplasmas classified as quarantine or quality pathogens. The protocol here described can be used for the detection and quantification of Candidatus Phytoplasma mali, Ca. P. pyri, Ca. P. prunorum, Ca. P. vitis, and Ca. P. solani by qPCR, RT-qPCR, ddPCR, and ddRT-PCR techniques based on TaqMan chemistry.

Long-distance movement of helical multipartite phytoviruses: keep connected or die?

All living organisms have to preserve genome integrity to ensure the survival of progeny generations. Viruses, though often regarded as 'non living', protect their nucleic acids from biotic and abiotic stresses, ranging from nuclease action to radiation-induced adducts. When the viral genome is split into multiple segments, preservation of at least one copy of each segment is required. While segmented and monopartite viruses use an all-in-one strategy, multipartite viruses have to address in the cell at least one of each viral particle in which the split positive stranded RNA genome is individually packaged. Here, we review and discuss the biology of multipartite helical RNA phytoviruses to outline our current hypothesis on a coordinated genomic RNA network RNP complex that preserves an all-in-one strategy and genome integrity.

Beet Necrotic Yellow Vein Virus Noncoding RNA Production Depends on a 5'→3' Xrn Exoribonuclease Activity.

The RNA3 species of the beet necrotic yellow vein virus (BNYVV), a multipartite positive-stranded RNA phytovirus, contains the 'core' nucleotide sequence required for its systemic movement in Beta macrocarpa. Within this 'core' sequence resides a conserved "coremin" motif of 20 nucleotides that is absolutely essential for long-distance movement. RNA3 undergoes processing steps to yield a noncoding RNA3 (ncRNA3) possessing "coremin" at its 5' end, a mandatory element for ncRNA3 accumulation. Expression of wild-type (wt) or mutated RNA3 in Saccharomyces cerevisiae allows for the accumulation of ncRNA3 species. Screening of S.cerevisiae ribonuclease mutants identified the 5'-to-3' exoribonuclease Xrn1 as a key enzyme in RNA3 processing that was recapitulated both in vitro and in insect cell extracts. Xrn1 stalled on ncRNA3-containing RNA substrates in these decay assays in a similar fashion as the flavivirus Xrn1-resistant structure (sfRNA). Substitution of the BNYVV-RNA3 'core' sequence by the sfRNA sequence led to the accumulation of an ncRNA species in yeast in vitro but not in planta and no viral long distance occurred. Interestingly, XRN4 knockdown reduced BNYVV RNA accumulation suggesting a dual role for the ribonuclease in the viral cycle.