Duplex-RT-PCR assay for the simultaneous detection and discrimination of Brome mosaic virus and Cocksfoot mottle virus in cereal plants.

Brome mosaic virus (BMV) and cocksfoot mottle virus (CfMV) are pathogens of grass species including all economically important cereals. Both viruses have been identified in Poland therefore they create a potential risk to cereal crops. In this study, a duplex-reverse transcription-polymerase chain reaction (duplex-RT-PCR) was developed and optimized for simultaneous detection and differentiation of BMV and CfMV as well as for confirmation of their co-infection. Selected primers CfMVdiag-F/CfMVdiag-R and BMV2-F/BMV2-R amplified 390 bp and 798 bp RT-PCR products within coat protein (CP) region of CfMV and replicase gene of BMV, respectively. Duplex-RT-PCR was successfully applied for the detection of CfMV-P1 and different Polish BMV isolates. Moreover, one sample was found to be co-infected with BMV-ML1 and CfMV-ML1 isolates. The specificity of generated RT-PCR products was verified by sequencing. Duplex-RT-PCR, like conventional RT-PCR, was able to detect two viruses occurring in plant tissues in very low concentration (as low as 4.5 pg/µL of total RNA). In contrast to existing methods, newly developed technique offers a significant time and cost-saving advantage. In conclusion, duplex-RT-PCR is a useful tool which can be implemented by phytosanitary services to rapid detection and differentiation of BMV and CfMV.

Optimizing fluorophore density for single virus counting: a photophysical approach.

In health and environmental research, it is often necessary to quantify the concentrations of single (bio) nanoparticles present at very low concentrations. Suitable quantification approaches that rely on counting and tracking of single fluorescently labelled (bio) nanoparticles are often challenging since fluorophore self-quenching limits the maximum particle brightness. Here we study how the number of labels per nanoparticle influences the total brightness of fluorescently labelled cowpea chlorotic mottle virus (CCMV). We analyze in detail the photophysical interplay between the fluorophores on the virus particles. We deduce that the formation of dark aggregates and energy transfer towards these aggregates limits the total particle brightness that can be achieved. We show that by carefully selecting the number of fluorescent labels per CCMV, and thus minimizing the negative effects on particle brightness, it is possible to quantify fluorescently labelled CCMV concentrations down to fM concentrations in single particle counting experiments.

Complete genome sequence of a novel bromovirus infecting elderberry (Sambucus nigra L.) in the Czech Republic.

The genus Bromovirus currently contains six species whose members have relatively narrow host ranges. In the present work, a new bromovirus infecting elderberry (Sambucus nigra L.) is reported. dsRNA was purified and sequenced by next-generation sequencing, and with minimal additional completion by Sanger sequencing, the full tripartite genome was obtained. RNA1 is 3241 nt long and contains ORF1 (1a protein), RNA2 is 2810 nt long and contains ORF2 (2a protein), and RNA3 is 2244 nt long and contains ORF3a (movement protein) and ORF3b (coat protein, CP), separated by an intercistronic poly(A) stretch. Proteins 1a and 2a showed highest sequence identity (69.9% and 69.4%) to the corresponding proteins of melandrium yellow fleck virus. The coat protein showed highest sequence identity (67.9%) to that of brome mosaic virus. The genome shows a typical bromovirus organisation comprising of all the conserved protein domains within the genus. Phylogenetic analysis supports the assignment of this virus as a new member of the genus Bromovirus, for which the name "sambucus virus S" (SVS) is proposed.

Biophysical analysis of BMV virions purified using a novel method.

Brome mosaic virus (BMV) has been successfully loaded with different types of nanoparticles. However, studies concerning its application as a nanoparticle carrier demand high-purity virions in large amounts. Existing BMV purification protocols rely on multiple differential ultracentrifugation runs of the initially purified viral preparation. Herein, we describe an alternative method for BMV purification based on ion-exchange chromatography and size-exclusion chromatography (SEC) yielding 0.2mg of virus from 1g of plant tissue. Our method is of similar efficiency to previously described protocols and can easily be scaled up. The method results in high-quality BMV preparations as confirmed by biophysical analyses, including cryogenic transmission electron microscopy (cryo-TEM), dynamic light scattering (DLS), static light scattering (SLS), and circular dichroism (CD) measurements and attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) spectroscopy. Our results revealed that purified BMV capsids are stable and monodisperse and can be used for further downstream applications. In this work, we also characterize secondary structure and size fluctuations of the BMV virion at different pH values.

Catching a virus in a molecular net.

A metal-organic molecular net composed of tannic acid (TA) and iron(iii) was constructed around the brome mosaic virus (BMV) particle to determine whether the added net could act as a transport barrier for water, and if the net could stabilize the virus in physically or chemically challenging environments. This new virus engineering strategy is expected to provide benefits both in the study and technological applications of viruses. For instance, a virus wrapped in a thin molecular layer could be extracted from solution either in air or vacuum, and its structure, composition and even internal dynamics could be interrogated by methods not compatible with a liquid environment. Atomic force microscopy (AFM) studies of Fe(iii)-TA coated BMV in liquid and in air supported a marked resistance to dehydration when compared to wtBMV. Native charge detection mass spectrometry (CDMS), was employed to estimate the number of molecules in the molecular net which wrapped the virus. The CDMS data suggested that less than one molecular monolayer wrapped the virus. Additionally, it was found, that this very thin molecular coat was sufficient to render the coated viruses resistant to storage conditions that typically lead to virus disassembly over time. A temporary coat imparting increased resistance to disassembly could be useful in adding time delay control or alleviate required storage conditions of engineered viruses for therapeutic purposes.

A novel set of polyvalent primers that detect members of the genera Bromovirus and Cucumovirus.

Rapid detection and diagnosis of plant virus infection is one of the most important steps in preventing damages caused by viral diseases. Bromoviruses and cucumoviruses belong to the family Bromoviridae, which is one of the most important families of plant viruses, and infect a broad range of host plants including various economically important crops. In this study, an RT-PCR assay was developed for the universal detection of bromoviruses and cucumoviruses using a set of primers designed to target the conserved sequences in viral RNA1. The assay detected three species of Cucumovirus (Cucumber mosaic virus (CMV), Peanut stunt virus (PSV) and Tomato aspermy virus (TAV)) and two species of Bromovirus (Brome mosaic virus (BMV) and Cowpea chlorotic mottle virus (CCMV)) with high specificity and sensitivity. The assay developed in this study is predicted to have the potential to detect all major members of the genera Bromovirus and Cucumovirus and to be used as a routine diagnostic assay.

[Production of immunodiagnosticum preparations based on plant virus strains].

The antigenic properties of virus isolates of tobacco mosaic, cucumber mosaic, bean common and yellow mosaic, soybean mosaic, Y-, X-, and A-potato viruses, Vicia unijuga mosaic, affecting agricultural crops and wild plants in the Far East of Russia were studied. Universal immunodiagnosticums fit for testing the broad range of virus isolates have been obtained on the basis of the most antigen-active strains. They allow to struggle effectively against viruses rendering economic damage to agriculture.

Molecular characterization of the complete genomes of two new field isolates of Cowpea chlorotic mottle virus, and their phylogenetic analysis.

Cowpea chlorotic mottle virus (CCMV, family Bromoviridae) is found worldwide and has been used as a model virus for a long time, but no data is available about the genetic diversity of field isolates. Recently, two new field isolates (Car1 and Car2) of CCMV obtained from cowpea showed distinct phenotypic symptoms when inoculated to cowpea. CCMV-Car1 induced severe mosaic and interveinal chlorosis, while CCMV-Car2 produced mild mottling and leaf rolling. Both isolates produced asymptomatic infection in Nicotiana benthamiana. The complete genome of both isolates was amplified by reverse transcription-polymerase chain reaction using specific primers against the CCMV sequences available in the GenBank database, cloned and sequenced. Both nucleotide and amino acid sequences were compared between the newly sequenced CCMV isolates and the three previously characterized CCMV strains (T, M1, and R). Phylogenetic analysis of the RNA 1 sequence showed that CCMV-Car1 was in a separate branch from the rest of the CCMV isolates while CCMV-Car2 grouped together with CCMV-R. On the basis of RNA 2 and RNA 3 sequences, two major groupings were obtained. One group included CCMV-Car1 and CCMV-Car2 isolates while the other contained CCMV-T, CCMV-M1, and CCMV-R strains. Recombination programs detected a potential recombination event in the RNA 1 sequence of CCMV-Car2 isolate but not in RNA 2 and RNA 3 sequences. The results showed that both mutations and recombination have played an important role in the genetic diversity of these two new isolates of CCMV.

All-atom multiscale simulation of cowpea chlorotic mottle virus capsid swelling.

An all-atom multiscale computational modeling approach, molecular dynamics/order parameter extrapolation (MD/OPX), has recently been developed for simulating large bionanosystems. It accelerates MD simulations and addresses rapid atomistic fluctuations and slowly varying nanoscale dynamics of bionanosystems simultaneously. With modules added to account for water molecules and ions, MD/OPX is applied to simulate the swelling of cowpea chlorotic mottle virus (CCMV) capsid solvated in a host medium in this study. Simulation results show that the N-terminal arms of capsid proteins undergo large deviations from the initial configurations with their length extended quickly during the early stage of capsid swelling. The capsid swelling is a symmetry-breaking process involving local initiation and front propagation. The capsid swelling rate is approximately 0.25 nm/ns (npn) during the early stage of the simulation, and propagation of the structural transition across the capsid is roughly 0.6 npn. The system conditions that affect swelling of the capsid are analyzed. Prospects for creating a phase diagram for CCMV capsid swelling and using predictions to guide experiments are discussed.

A simple technique for separation of Cowpea chlorotic mottle virus from Cucumber mosaic virus in natural mixed infections.

A simple technique was developed to separate Cowpea chlorotic mottle virus (CCMV) from Cucumber mosaic virus (CMV) in natural mixed infections. Sap from cowpea leaves infected naturally with a mixture of CCMV and CMV was inoculated mechanically on the first tri-foliolate leaf of cowpea seedlings. Both inoculated and non-inoculated upper leaves were sampled 3 or 8 days post-inoculation and tested by reverse transcription polymerase chain reaction (RT-PCR) using primers specific to CCMV and CMV. RT-PCR analysis showed the presence of only CCMV in the inoculated leaf and both viruses in the non-inoculated systemically infected upper leaves. Total RNA from the inoculated leaves positive to CCMV only was further confirmed upon re-inoculation to cowpea seedlings. Typical CCMV symptoms were produced within 1 week and RT-PCR analysis showed only the presence of CCMV in both inoculated and non-inoculated systemically infected upper leaves. Systemically infected upper leaves of the same plants were used for CCMV purification. RT-PCR analysis of the purified virion and RNA extracted from the virion further confirmed the absence of CMV contamination. To our knowledge, this is the first report of a method separating CCMV directly from mixed infections with CMV in cowpea.

Rapid and efficient purification of Cowpea chlorotic mottle virus by sucrose cushion ultracentrifugation.

A simple, economical and efficient method for isolation of Cowpea chlorotic mottle virus (CCMV) particles was developed. Abundant viral particles could be obtained with three steps of 10 min duration each of conventional differential centrifugations and one sucrose cushion ultracentrifugation step of 2 h. Characterization of purified virus assessed by transmission electron microscopy, polyacrylamide gel electrophoresis (PAGE) and Western blot revealed the typical isometric particles of CCMV without any visible contamination or degradation. RNA extracted from purified CCMV particles showed all four RNA components by agarose gel electrophoresis. Both purified virus particles and RNA were biologically active and initiated successful infection upon inoculation to cowpea.

Expression and self-assembly of cowpea chlorotic mottle virus-like particles in Pseudomonas fluorescens.

Coat protein of the cowpea chlorotic mottle virus (CCMV), a plant bromovirus, has been expressed in a soluble form in a prokaryote, Pseudomonas fluorescens, and assembled into virus-like particles (VLPs) in vivo that were structurally similar to the native CCMV particles derived from plants. The CCMV VLPs were purified by PEG precipitation followed by separation on a sucrose density gradient and analyzed by size exclusion chromatography, UV spectrometry, and transmission electron microscopy. DNA microarray experiments revealed that the VLPs encapsulated very large numbers of different host RNAs in a non-specific manner. The development of a P. fluorescens expression system now enables production of CCMV VLPs by bacterial fermentation for use in pharmaceutical or nanotechnology applications.

Characterization of a Brome mosaic virus strain and its use as a vector for gene silencing in monocotyledonous hosts.

Virus-induced gene silencing (VIGS) is used to analyze gene function in dicotyledonous plants but less so in monocotyledonous plants (particularly rice and corn), partially due to the limited number of virus expression vectors available. Here, we report the cloning and modification for VIGS of a virus from Festuca arundinacea Schreb. (tall fescue) that caused systemic mosaic symptoms on barley, rice, and a specific cultivar of maize (Va35) under greenhouse conditions. Through sequencing, the virus was determined to be a strain of Brome mosaic virus (BMV). The virus was named F-BMV (F for Festuca), and genetic determinants that controlled the systemic infection of rice were mapped to RNAs 1 and 2 of the tripartite genome. cDNA from RNA 3 of the Russian strain of BMV (R-BMV) was modified to accept inserts from foreign genes. Coinoculation of RNAs 1 and 2 from F-BMV and RNA 3 from R-BMV expressing a portion of a plant gene to leaves of barley, rice, and maize plants resulted in visual silencing-like phenotypes. The visual phenotypes were correlated with decreased target host transcript levels in the corresponding leaves. The VIGS visual phenotype varied from maintained during silencing of actin 1 transcript expression to transient with incomplete penetration through affected tissue during silencing of phytoene desaturase expression. F-BMV RNA 3 was modified to allow greater accumulation of virus while minimizing virus pathogenicity. The modified vector C-BMV(A/G) (C for chimeric) was shown to be useful for VIGS. These BMV vectors will be useful for analysis of gene function in rice and maize for which no VIGS system is reported.

Enhanced local symmetry interactions globally stabilize a mutant virus capsid that maintains infectivity and capsid dynamics.

Structural transitions in viral capsids play a critical role in the virus life cycle, including assembly, disassembly, and release of the packaged nucleic acid. Cowpea chlorotic mottle virus (CCMV) undergoes a well-studied reversible structural expansion in vitro in which the capsid expands by 10%. The swollen form of the particle can be completely disassembled by increasing the salt concentration to 1 M. Remarkably, a single-residue mutant of the CCMV N-terminal arm, K42R, is not susceptible to dissociation in high salt (salt-stable CCMV [SS-CCMV]) and retains 70% of wild-type infectivity. We present the combined structural and biophysical basis for the chemical stability and viability of the SS-CCMV particles. A 2.7-A resolution crystal structure of the SS-CCMV capsid shows an addition of 660 new intersubunit interactions per particle at the center of the 20 hexameric capsomeres, which are a direct result of the K42R mutation. Protease-based mapping experiments of intact particles demonstrate that both the swollen and closed forms of the wild-type and SS-CCMV particles have highly dynamic N-terminal regions, yet the SS-CCMV particles are more resistant to degradation. Thus, the increase in SS-CCMV particle stability is a result of concentrated tethering of subunits at a local symmetry interface (i.e., quasi-sixfold axes) that does not interfere with the function of other key symmetry interfaces (i.e., fivefold, twofold, quasi-threefold axes). The result is a particle that is still dynamic but insensitive to high salt due to a new series of bonds that are resistant to high ionic strength and preserve the overall particle structure.

Natural isolates of Brome mosaic virus with the ability to move from cell to cell independently of coat protein.

Brome mosaic virus (BMV) requires encapsidation-competent coat protein (CP) for cell-to-cell movement and the 3a movement protein (MP) is involved in determining the CP requirement for BMV movement. However, these conclusions have been drawn by using BMV strain M1 (BMV-M1) and a related strain. Here, the ability of the MPs of five other natural BMV strains to mediate the movement of BMV-M1 in the absence of CP was tested. The MP of BMV M2 strain (BMV-M2) efficiently mediated the movement of CP-deficient BMV-M1 and the MPs of two other strains functioned similarly to some extent. Furthermore, BMV-M2 itself moved between cells independently of CP, demonstrating that BMV-M1 and -M2 use different movement modes. Reassortment between CP-deficient BMV-M1 and -M2 showed the involvement of RNA3 in determining the CP requirement for cell-to-cell movement and the involvement of RNAs 1 and 2 in movement efficiency and symptom induction in the absence of CP. Spontaneous BMV MP mutants generated in planta that exhibited CP-independent movement were also isolated and analysed. Comparison of the nucleotide differences of the MP genes of BMV-M1, the natural strains and mutants capable of CP-independent movement, together with further mutational analysis of BMV-M1 MP, revealed that single amino acid differences at the C terminus of MP are sufficient to alter the requirement for CP in the movement of BMV-M1. Based on these findings, a possible virus strategy in which a movement mode is selected in plant viruses to optimize viral infectivity in plants is discussed.

Efficient purification of bromoviruses by ultrafiltration.

Ultrafiltration using polyethersulfone-membranes was evaluated as an efficient and preferred method for purifying Cowpea Chlorotic Mottle Virus (CCMV). Cesium chloride (CsCl) ultracentrifugation and ultrafiltration protocols are described, and comparative UV-spectroscopic and electron micrograph results are presented. CCMV purified by ultrafiltration are shown to be equivalent to CCMV purified by ultracentrifugation, while reducing purification time by two days and avoiding the need for expensive capital overheads such as ultracentrifuges, rotors and toxic CsCl chemical waste.

Comparison of the native CCMV virion with in vitro assembled CCMV virions by cryoelectron microscopy and image reconstruction.

Cryoelectron microscopy and three-dimensional image reconstruction analysis has been used to determine the structure of native and in vitro assembled cowpea chlorotic mottle virus (CCMV) virions and capsids to 25-A resolution. Purified CCMV coat protein was used in conjunction with in vitro transcribed viral RNAs to assemble RNA 1 only, RNA 2 only, RNA 3/4 only, and empty (RNA lacking) virions. The image reconstructions demonstrate that the in vitro assembled CCMV virions are morphologically indistinguishable from native virions purified from infected plants. The viral RNA (vRNA) is packaged similarly within the different types of virions. The centers of all assembled particles are generally devoid of density and the vRNA packs against the interior surface of the virion shell. The vRNA appears to adopt an ordered conformation at each of the quasi-threefold axes.

Structure-based rationale for the rescue of systemic movement of brome mosaic virus by spontaneous second-site mutations in the coat protein gene.

We describe spontaneous second-site reversions within the coat protein open reading frame that rescue the systemic-spread phenotype and increase virion stability of a mutant of brome mosaic virus. Based on the crystal structure of the related cowpea chlorotic mottle virus, we show that the modified residues are spatially clustered to affect the formation of hexamers and pentamers and therefore virion stability.