Nanobody‐mediated resistance to Grapevine fanleaf virus in plants

Since their discovery, single‐domain antigen‐binding fragments of camelid‐derived heavy‐chain‐only antibodies, also known as nanobodies (Nbs), have proven to be of outstanding interest as therapeutics against human diseases and pathogens including viruses, but their use against phytopathogens remains limited. Many plant viruses including Grapevine fanleaf virus (GFLV), a nematode‐transmitted icosahedral virus and causal agent of fanleaf degenerative disease, have worldwide distribution and huge burden on crop yields representing billions of US dollars of losses annually, yet solutions to combat these viruses are often limited or inefficient. Here, we identified a Nb specific to GFLV that confers strong resistance to GFLV upon stable expression in the model plant Nicotiana benthamiana and also in grapevine rootstock, the natural host of the virus. We showed that resistance was effective against a broad range of GFLV isolates independently of the inoculation method including upon nematode transmission but not against its close relative, Arabis mosaic virus. We also demonstrated that virus neutralization occurs at an early step of the virus life cycle, prior to cell‐to‐cell movement. Our findings will not only be instrumental to confer resistance to GFLV in grapevine, but more generally they pave the way for the generation of novel antiviral strategies in plants based on Nbs.     

A Basic Approach Towards the Development of Bioelectric Bacterial Biosensors for the Detection of Plant Viruses

There is a growing need for virus sensors with improved sensitivity and dynamic range for disease diagnosis, pharmaceutical research, agriculture and homeland security. Membrane‐engineered animal cells bearing antibodies against viral antigens have been previously used for biorecognition biosensors for the ultrarapid (3 min), sensitive (1 ng/ml) detection of plant viruses, such as the cucumber mosaic virus. We here report a new approach for the construction of cell‐based sensors for virus detection, based on membrane (antibody)‐engineered bacteria. The novel method was applied for the detection of tobacco mosaic virus (TMV) and cherry leaf roll virus (CLRV) using sensors containing modified Escherichia coli XL‐1Blue MRF’ bacteria. E. coli membranes have been engineered with electro‐inserted, virus‐homologous antibodies. The detection principle was based on the measurement of changes in the bacterial membrane potential as a result of virus–antibody binding. After optimization of the membrane‐engineering process, the virus detection limit for TMV and CLRV with the bacteria‐based biosensor system was 1 pg/ml, representing a 1000‐fold improvement over currently available methods. Although the novel biosensor is still in its proof‐of‐concept stage of development, its sensitivity and speed (assay time: 60–100 s) could make it a very promising tool for high throughput, field‐based virus screening.     

Structural insights into viral determinants of nematode mediated Grapevine fanleaf virus transmission

Many animal and plant viruses rely on vectors for their transmission from host to host. Grapevine fanleaf virus (GFLV), a picorna-like virus from plants, is transmitted specifically by the ectoparasitic nematode Xiphinema index. The icosahedral capsid of GFLV, which consists of 60 identical coat protein subunits (CP), carries the determinants of this specificity. Here, we provide novel insight into GFLV transmission by nematodes through a comparative structural and functional analysis of two GFLV variants. We isolated a mutant GFLV strain (GFLV-TD) poorly transmissible by nematodes, and showed that the transmission defect is due to a glycine to aspartate mutation at position 297 (Gly297Asp) in the CP. We next determined the crystal structures of the wild-type GFLV strain F13 at 3.0 Å and of GFLV-TD at 2.7 Å resolution. The Gly297Asp mutation mapped to an exposed loop at the outer surface of the capsid and did not affect the conformation of the assembled capsid, nor of individual CP molecules. The loop is part of a positively charged pocket that includes a previously identified determinant of transmission. We propose that this pocket is a ligand-binding site with essential function in GFLV transmission by X. index. Our data suggest that perturbation of the electrostatic landscape of this pocket affects the interaction of the virion with specific receptors of the nematode''s feeding apparatus, and thereby severely diminishes its transmission efficiency. These data provide a first structural insight into the interactions between a plant virus and a nematode vector.     

Multiplex Detection of Plant Pathogens Using a Microsphere Immunoassay Technology

Plant pathogens are a serious problem for seed export, plant disease control and plant quarantine. Rapid and accurate screening tests are urgently required to protect and prevent plant diseases spreading worldwide. A novel multiplex detection method was developed based on microsphere immunoassays to simultaneously detect four important plant pathogens: a fruit blotch bacterium Acidovorax avenae subsp. citrulli (Aac), chilli vein-banding mottle virus (CVbMV, potyvirus), watermelon silver mottle virus (WSMoV, tospovirus serogroup IV) and melon yellow spot virus (MYSV, tospovirus). An antibody for each plant pathogen was linked on a fluorescence-coded magnetic microsphere set which was used to capture corresponding pathogen. The presence of pathogens was detected by R-phycoerythrin (RPE)-labeled antibodies specific to the pathogens. The assay conditions were optimized by identifying appropriate antibody pairs, blocking buffer, concentration of RPE-labeled antibodies and assay time. Once conditions were optimized, the assay was able to detect all four plant pathogens precisely and accurately with substantially higher sensitivity than enzyme-linked immunosorbent assay (ELISA) when spiked in buffer and in healthy watermelon leaf extract. The assay time of the microsphere immunoassay (1 hour) was much shorter than that of ELISA (4 hours). This system was also shown to be capable of detecting the pathogens in naturally infected plant samples and is a major advancement in plant pathogen detection.     

Gene silencing and virus resistance based on defective interfering constructs in transgenic Nicotiana benthamiana is not linked to accumulation of siRNA

RNA interference (RNAi) was established in Nicotiana benthamiana plants by introducing constructs containing a defective interfering (DI) sequence from Tomato bushy stunt virus (TBSV) in combination with a conserved sense-sequence from the target Grapevine fanleaf virus (GFLV). Silencing in plants was confirmed by Agrobacterium-mediated infiltration of a GFP-sensor containing the GFLV-derived target sequence. The transgene-induced RNAi led to silencing of the GFP-sensor and GFP fluorescence was absent post-infiltration. In plants without GFP fluorescence after infiltration with the GFP-sensor, siRNA specific to GFP and the target virus sequence could not be detected. In contrast, infiltrated leaves of wild type and transgenic plants showing GFP fluorescence after infiltration revealed accumulation of siRNA specific to the sequence of the sensor. Silencing could be inhibited by co-infiltration using a p19 silencing suppressor construct together with the GFP-sensor, which always resulted in bright GFP fluorescence. In parallel, virus resistance of transgenic Nicotiana benthamiana was investigated via challenge inoculation with GFLV. Our results indicate that efficient RNAi in transgenic plants does not necessarily lead to a detectable accumulation of siRNA.     

Grapevine Viruses' Detection and Sanitary Selection in Grapevine Germplasm of Calabria (Southern Italy)

A survey of grapevine viruses present in the region of Calabria (southern Italy) was carried out, and the sanitary selection was conducted on various indigenous varieties. Serological (ELISA) and molecular (multiplex RT‐PCR) tests were used to detect the viruses included in the Italian certification programme: Arabis mosaic virus (ArMV), Grapevine fanleaf virus (GFLV), Grapevine leafroll associated virus 1 (GLRaV‐1), Grapevine leafroll associated virus 2 (GLRaV‐2), Grapevine leafroll associated virus 3 (GLRaV‐3), Grapevine virus A (GVA), Grapevine virus B (GVB) and Grapevine fleck virus (GFkV). The frequency with which the above viruses have been detected was 37.4, 32.6, 12.8, 7.7, 7.3, 1.9 and 0.3%, respectively, for GVA, GLRaV‐3, GFLV, GFKV, GLRaV‐1, GLRaV‐2 and GVB. ArMV was never found. The sanitary selection allowed for the detection of 6 putative clones of ‘Arvino’, 2 of ‘Magliocco dolce’ and 2 of the rootstock ‘17–37’ free of the above‐mentioned viruses. The necessary process for the commercialization of these clones as ‘certified’ propagation material was accomplished, and their official approval by the Italian Ministry of Agriculture is currently in progress.     

A Rapid Protocol of Crude RNA/DNA Extraction for RT-qPCR Detection and Quantification of 'Candidatus Phytoplasma prunorum'

Many efforts have been made to develop a rapid and sensitive method for phytoplasma and virus detection. Taking our cue from previous works, different rapid sample preparation methods have been tested and applied to Candidatus Phytoplasma prunorum (‘Ca. P. prunorum’) detection by RT-qPCR. A duplex RT-qPCR has been optimized using the crude sap as a template to simultaneously amplify a fragment of 16S rRNA of the pathogen and 18S rRNA of the host plant. The specific plant 18S rRNA internal control allows comparison and relative quantification of samples. A comparison between DNA and RNA contribution to qPCR detection is provided, showing higher contribution of the latter. The method presented here has been validated on more than a hundred samples of apricot, plum and peach trees. Since 2013, this method has been successfully applied to monitor ‘Ca. P. prunorum’ infections in field and nursery. A triplex RT-qPCR assay has also been optimized to simultaneously detect ‘Ca. P. prunorum’ and Plum pox virus (PPV) in Prunus.     

Grapevine fanleaf virus (GFLV)-specific antibodies confer GFLV and Arabis mosaic virus (ArMV) resistance in Nicotiana benthamiana

Grapevine fanleaf virus (GFLV) is one of the most destructive pathogens of grapevine. In this study, we generated monoclonal antibodies binding specifically to the coat protein of GFLV. Antibody FL₃, which bound most strongly to GFLV and showed cross-reactivity to Arabis mosaic virus (ArMV), was used to construct the single-chain antibody fragment scFvGFLVcp-55. To evaluate the potential of this single-chain variable fragment (scFv) to confer antibody-mediated virus resistance, transgenic Nicotiana benthamiana plants were generated in which the scFv accumulated in the cytosol. Recombinant protein levels of up to 0.1% total soluble protein were achieved. The T₁ and T₂ progenies conferred partial or complete protection against GFLV on challenge with the viral pathogen. The resistance to GFLV in transgenic plants was strictly related to scFvGFLVcp-55 accumulation levels, confirming that the antibody fragment was functional in planta and responsible for the GFLV resistance. In addition, transgenic plants conferring complete protection to GFLV showed substantially enhanced tolerance to ArMV. We demonstrate the first step towards the control of grapevine fanleaf degeneration, as scFvGFLVcp-55 could be an ideal candidate for mediating nepovirus resistance.     

Detection of Grapevine Viruses in Poland

During a 3‐year study, grapevines from 23 vineyards in Poland were surveyed for virus diseases and tested to determine the prevalence of the most economically important viruses by RT‐PCR. The rate of positive samples was 2.2% for grapevine leafroll‐associated virus 1 (GLRaV‐1), 1.9% for grapevine leafroll‐associated virus 2 (GLRaV‐2), 1.5% grapevine leafroll‐associated virus 3 (GLRaV‐3), 1.9% for grapevine virus A (GVA), 0.2% for grapevine virus B (GVB), 0.2% for grapevine virus E (GVE), 0.65% for grapevine fanleaf virus (GFLV), 20.4% for grapevine fleck virus (GFkV) and 71.9% for grapevine rupestris stem pitting‐associated virus (GRSPaV). These viruses were found to occur as single or mixed infections of different combinations in individual grapevines. The overall viral infection rate in the surveyed grapevines was 82.6%. GRSPaV is the most widely distributed virus of all the viruses currently detected in the region. DNA sequencing confirmed the identification of the viruses in selected samples, and analysis indicated that the Polish isolates shared a close molecular identity with the corresponding isolates in GenBank. To our knowledge, this is the first detection of GLRaV‐1, ‐2, ‐3, GVA, GVB, GVE, GFLV, GFkV and GRSPaV in Poland.     

A biolayer interferometry-based assay for rapid and highly sensitive detection of biowarfare agents

Biolayer interferometry (BLI) is an optical technique that uses fiber-optic biosensors for label-free real-time monitoring of protein–protein interactions. In this study, we coupled the advantages of the Octet Red BLI system (automation, fluidics-free, and on-line monitoring) with a signal enhancement step and developed a rapid and sensitive immunological-based method for detection of biowarfare agents. As a proof of concept, we chose to demonstrate the efficacy of this novel assay for the detection of agents representing two classes of biothreats, proteinaceous toxins, and bacterial pathogens: ricin, a lethal plant toxin, and the gram-negative bacterium Francisella tularensis, the causative agent of tularemia. The assay setup consisted of biotinylated antibodies immobilized to the biosensor coupled with alkaline phosphatase-labeled antibodies as the detection moiety to create nonsoluble substrate crystals that precipitate on the sensor surface, thereby inducing a significant wavelength interference. It was found that this BLI-based assay enables sensitive detection of these pathogens (detection limits of 10 pg/ml and 1 × 10⁴ pfu/ml ricin and F. tularensis, respectively) within a very short time frame (17 min). Owing to its simplicity, this assay can be easily adapted to detect other analytes in general, and biowarfare agents in particular, in a rapid and sensitive manner.     

Magnetic biosensor for the detection of Yersinia pestis

A novel type of magnetic-beads based magnetic biosensor is described for the detection of Yersinia pestis. Experiments were performed with the antigen fraction F1 of these bacteria. The magnetic sensor platform offers easy and reliable detection of Y. pestis by the use of magnetic beads for labelling and quantification in a single step due to their paramagnetic features. The system uses antiYPF1 antibodies as capture element on ABICAP columns as core element of the magnetic sensor. Several immobilization methods for antibodies on polyethylene were exploited. The established biosensor has a linear detection range of 25-300 ng/ml Y. pestis antigen F1 and a detection limit of 2.5 ng/ml in buffer and human blood serum. The presented sensor system is small, simple, portable and therefore usable as off-lab detection unit for medical and warfare analytes.     

Simultaneous Detection of Major Pome Fruit Viruses and a Viroid

A rapid and sensitive two-step RT-PCR protocol for simultaneous detection of major apple viruses, namely Apple mosaic virus (ApMV), Apple stem pitting virus (ASPV), Apple stem grooving virus (ASGV), Apple chlorotic leaf spot virus (ACLSV) and Apple scar skin viroid (ASSVd), was developed. Five specific primer pairs were tested and confirmed for these viruses and viroid together in a single tube, giving amplicons of ~198, ~330, ~370, ~547 and ~645 bp corresponding to ASGV, ASSVd, ASPV, ApMV and ACLSV, respectively. Using a guanidinium-based extraction buffer along with a commercial kit resulted in better quality RNA as compared to kit, suited for multiplex RT-PCR. A rapid CTAB method for RNA isolation from apple tissue was developed, which produce good yield and saves time. To the best of our knowledge, this is the first report on the simultaneous detection of five pathogens (four viruses and a viroid) from apple with NADH dehydrogenase subunit 5 (nad5) as an internal control.     

Immunoreagents based on polymer dispersions for immunochemical assays

Immunoreagents based on polymer dispersions consisting of unimodal polyacrolein (PAL) microspheres with diameters in the range 0.3-2.0 microns have been prepared and evaluated by various immunoassay techniques such as immunoradiometric assay of ferritin and microtitre particle agglutination and immunofiltration dot assay of group-specific polysaccharide of S. pyogenes (A-PS) in comparison with conventional carriers and methods. The antibodies were covalently or indirectly bound to the PAL. The coupled antibodies to ferritin retained a high average affinity (Ka = 4.5 x 10(9) M-1). In comparison with microcrystalline cellulose-based immunosorbent, more than an order-of-magnitude lower amount of PAL-IgG was necessary for the analysis of ferritin. Use of PAL-IgG gave a higher sensitivity of assay with a detection limit of 0.7 x 10(-13) M l-1 and a wider concentration range of antigen detection (about four orders of magnitude) without manifestation of the high-dose hook effect. Particle agglutination assay of A-PS in microtitre plate was shown to be a simple, demonstrative and highly sensitive one-step analytical method with a detection limit of 0.05 ng A-PS/ml or 10(4) cells/ml. The sensitivity of immunofiltration assay using both enzyme and latex markers was shown to be approximately the same (50 ng A-PS/ml) and the duration of the assay was 3-5 min. No cross-reaction of latex conjugates with non-A Streptococcus cell lysates were observed.     

Flow-through immunofiltration assay system for rapid detection of E. coli O157:H7

A flow-through amperometric immunofiltration assay system based on disposable porous filter-membranes for rapid detection of Escherichia coli O157:H7 has been developed. The analytical system utilizes flow-through, immunofiltration and enzyme immunoassay techniques in conjunction with an amperometric sensor. The parameters affecting the immunoassay such as selection of appropriate filter membranes, membrane pore size, antibody binding capacity and the concentrations of immunoreagents were investigated and optimized. Non-specific adsorption of the enzyme conjugate was investigated and minimized. A sandwich scheme of immunoassay was employed and the immunofiltration system allows to specifically and directly detect E. coli cells with a lower detection limit of 100 cells/ml. The working range is from 100 to 600 cells/ml with an overall analysis time of 30 min. No pre-enrichment was needed. This immunosensor can be easily adapted for assay of other microorganisms and may be a basis for a new class of highly sensitive bioanalytical devices for rapid quantitative detection of bacteria.     

CRP determination based on a novel magnetic biosensor

The c-reactive protein (CRP) is a very significant human blood marker for inflammatory processes and is routinely determined for many clinical purposes. The widespread and well established detection method for this approximately 115 kDa hepatic protein is the high-sensitivity ELISA assay (hsCRP-ELISA) in blood serum. New approaches in medical CRP diagnosis (e.g. for CVD, inflammatory bowel disease) require rapid quantification in native matrices. A novel CRP determination method based on magnetic detection is described and tested for human blood serum, saliva and urine. The detection principle is based on two different anti-CRP antibodies (monoclonal, IgG) for CRP trapment and labelling. The linear detection range of this immunosensor ranged from 25 ng/ml to 2.5 microg/ml and is therefore much more sensitive than typical hsCRP-ELISA-assays.     

Biotin-Avidin ELISA Detection of Grapevine Fanleaf Virus in the Vector Nematode Xiphinema index

The value of biotin-avidin (B-A) ELISA for the detection of grapevine fanleaf virus (GFLV) in Xiphinema was estimated with field populations and greenhouse subpopulations. Samples consisted of increasing numbers of adults ranging from 1 to 64 in multiples of two. Tests with virus-free X. index populations reared on grapevine and fig plants as negative controls did not reveal a noticeable effect of the host plant. ELISA absorbances of virus-free X. index samples were greater than corresponding absorbances of X. pachtaicum samples. Differences occurred between two X. index field populations from GFLV-infected grapevines in Champagne and Languedoc. In most tests, 1-, 2-, 4-, and 8-nematode samples of virus-free and virus-infected populations, respectively, could not be separated. Consequently, B-A ELISA was not a reliable method for GFLV detection in samples of less than 10 X. index adults, but comparison of the absorbances obtained with increasing numbers may allow differentiation of the viral infectious potential of several populations.     

Incidence of Viruses and Nematode Vectors in Lebanese Vineyards

Surveys for virus diseases and nematode vectors were conducted in 95 commercial vineyards of four different Lebanese districts (Bekaa valley, Mount Lebanon, North and South Lebanon). Out of 915 randomly collected grapevine samples tested by ELISA, 511 (55.8%) were infected by one or more viruses. Grapevine virus A (30.9%) and Grapevine leafroll‐associated virus 3 (23.7%) were the prevailing viruses, followed by Grapevine fleck virus (15.1%), Grapevine leafroll‐associated virus 1 (10.6%) and Grapevine leafroll‐associated virus 2 (8.7%). Arabis mosaic virus was not found whereas Grapevine fanleaf virus (GFLV) and Grapevine virus B were little represented. The most important Lebanese grapevine varieties, i.e. Maghdouchi, Tfeifihi and Beitamouni, had average infection rates between 70% and 87%, whereas varieties of foreign origin had a better sanitary status with the exception of cvs Cinsaut and Thompson (c. 83% infection). Grapevine rupestris stem pitting‐associated virus was detected in 79 of 90 (87.8%) samples tested by RT‐PCR and closteroviruses were recorded in seven of 70 (10%) vines tested. One of these viruses was identified as Grapevine leafroll‐associated virus 5 by ELISA and partial genome sequencing. No nepoviruses other than GFLV were detected in any of 90 samples tested using three different sets of degenerate primers. Xiphinema index was found in 23 of 89 soil samples collected from vineyards, and in three of 15 samples collected primarily under fig trees in fields where no grapevines were grown.