Dispersed gold nanoparticles potentially ruin gold barley yellow dwarf virus and eliminate virus infectivity hazards

Gold nanoparticles (AuNPs) application melted barley yellow dwarf virus-PAV (BYDV-PAV) spherical nanoparticle capsids. Synergistic therapeutic effects for plant virus resistance were induced by interaction with binding units of prepared AuNPs in a water solution which was characterized and evaluated by zeta sizer, zeta potential and transmission electron microscopy (TEM). The yield of purified nanoparticles of BYDV-PAV was obtained from Hordeum vulgare (Barley) cultivars, local and Giza 121/Justo. It was 0.62 mg/ml from 27.30 g of infected leaves at an A260/A280 ratio. Virus nanoparticle has a spherical shape 30 nm in size by TEM. BYDV-PAV combined with AuNPs to challenge virus function in vivo and in vitro. Dual AuNPs existence in vivo and in vitro affected compacted configuration of viral capsid protein in the interior surface of capsomers, the outer surface, or between the interface of coat protein subunits for 24 and 48 h incubation period in vitro at room temperature. The sizes of AuNPs that had a potentially dramatic deteriorated effect are 3.151 and 31.67 nm with a different intensity of 75.3% for the former and 24.7% for the latter, which enhances optical sensing applications to eliminate virus infectivity. Damages of capsid protein due to AuNPs on the surface of virus subunits caused variable performance in four different types of TEM named puffed, deteriorated and decorated, ruined and vanished. Viral yield showed remarkably high-intensity degree of particle symmetry and uniformity in the local cultivar greater than in Giza 121/Justo cultivar. A high yield of ruined VLPs in the local cultivar than Justo cultivar was noticed. AuNPs indicated complete lysed VLPs and some deteriorated VLPs at 48 h.


Introduction
Nanotechnology is a multilateral term for a broad range of relatively novel technologies; the main unifying theme is that it is concerned with the matter on the nanometer scale (Greek ñanos means dwarf). It is a highly multidisciplinary area that describes a field of applied science and technology focused on the synthesis, characterization and application of materials on this scale (Steinmetz and Evans 2007). It has become a prominent industrial and scientific field, with increasing production and wider applications that are expected to become routinely present in natural ecosystems. Application of nanomaterial-based agrochemicals could lead to widespread intentional environmental dispersion (Hassellöv et al. 2008). The successful application of nano-platforms in medicine under in vitro conditions has generated some interest in agri-nanotechnology which held the promise of agrochemicals and sitetargeted delivery of different macromolecules needed for plant disease resistance (Nair et al. 2010). Growing nanoscale disease-resistant varieties as natural substitution agents in our agriculture environments is the most economical and efficient method of controlling diseases. Resistance to BYDV is of great significance. To protect the plant against the virus, the FAO/WHO meeting in 2011 summarized major issues of nanomaterial applications in crop protection as follows: accurate characterization of nanomaterials in biological matrices for in-depth understanding, nanomaterial interaction with biology, dose-response considerations and exposure assessment and characterization studies. Giljohann et al. 2010 proved the advantage of some properties of modified AuNPs with different surface chemistries such as nucleic acids and peptides, and each kind of chemistry endows the particles with different properties. Virus-based nanotechnology has generated interest in some usage due to the specificity of virus interaction design with inorganic and organic nanoparticles, in addition to multifunctional proteinaceous shell (capsid) structure surrounding the genomic material (Capek 2015). Plant viruses are a valuable addition to the bionanotechnology toolbox for the construction of new materials for use in medical applications. There is currently a knowledgeable interest in the self-assembly of composite materials from metal nanoparticle, proteins and nucleic acids (Niemeyer 2001). DNA molecules, in particular, play a major role in this approach due to their large molecular recognition capabilities (Niemeyer et al. 2003). Nanoconjugates with noble metal nanoparticles have ideal template construction due to the nanosized components of the virus particles, monodispersity, and a variety of chemical groups available for modification. Multifunctional viruses integrated with nanoparticles and other functional additives to generate bioconjugates with different properties-possible antiviral and antibacterial activities-viral nanoparticles, noble metals, and decorated viruses and their nanoconjugates. Successful development of ultrasensitive constructs for bioimaging is a challenging function such as wellcharacterized fine three-dimensional structures, multivalency, orthogonal reactivities, and responsiveness to genetic modifications. Imaging and binding units can find out the alteration tolerances on the surface of protein subunits, which leads to deteriorated VLPs by gold nanoparticles. These findings highlight some recent progress in the applications of viruses in bioimaging in TEM (Li et al. 2010).

Materials and methods
Preparing AuNPs in water solution and TEM examination AuNPs were prepared by using citrate ion as a reducing agent. This process was conducted to produce modestly monodispersed spherical AuNPs which were suspended in water, according to Alkubaisi et al. (2015) and Turkevich et al. (1951). A drop of this particle placed on a carboncoated Formvar grid for 2 min and then examined by TEM with a JOEL 1220.
Characterization of AuNPs according to zeta sizer and zeta potential and BYDV-PAV particles AuNPs were tested for size distribution that was reported by intensity using zeta sizer for Malvern (ZEN3600). Then, the magnitude of the electrostatic or charge repulsion/attraction between the particles was measured by zeta potential which reflects the properties of their effective charge. AuNPs were examined in TEM which obtained from purified crude sap samples of treated barley with AuNPs only.

AuNPs treatment with characterized BYDV-PAV
The purified virus particles were obtained from the infected barley according to Alkubaisi et al. (2015). The partially purified sap was stained using uranyl acetate and examined in TEM. Then, the barley was double treated with both characterized AuNPs and BYDV-PAV in two kinds of Hordeum vulgare cultivars, local and Giza 121/Justo. Both cultivars were obtained from the College of Agriculture, Plant Protection Department, King Saud University. AuNPs were applied first as a protection pre-treatment in vivo on barley plant, followed by purified BYDV-PAV particles (AuNPs/BYDV-PAV), while purified BYDV-PAV particles were applied first on both cultivars followed by AuNPs as a post-treatment in vivo (BYDV-PAV/ AuNPs) for comparative efficacy for AuNPs.

Dual AuNPs effects for 24 and 48 h in vitro by TEM examination
The purified virus particles were obtained from previously treated barley according to Alkubaisi et al. (2015). Then, these were instantly incubated with AuNPs for 24 and 48 h in vitro at room temperature, and then five grids from each similar treatment of AuNPs for both cultivars and time incubation as illustrated in the following abbreviations for each one were negatively stained with uranyl acetate and examined in TEM. Partially Purified sap from the local cultivar was treated with AuNPs/infected by BYDV-PAV and then treated again in vitro by AuNPs for 24 and 48 h and coded as: 1. Local-AuNPs/BYDV-PAV (NPs in vivo)-AuNPs-24 vs 48 h (NPs in vitro).
Partially purified sap from the Justo cultivar that was obtained by treatment with AuNPs/infected by BYDV-PAV and then treated again in vitro by AuNPs for 24 and 48 h was coded as:
Partially Purified sap from the local cultivar was treated with AuNPs/infected by BYDV-PAV and then treated again in vitro by AuNPs for24 and 48 h and coded as:

Results and discussion
Characterization of AuNPs and BYDV-PAV particles by TEM TEM characterized and revealed the actual size of the prepared AuNPs ranging from sizes 3.151 and 31.67 nm with a final concentration of 0.034 mg in 100 ml d. water to have monodispersed spherical AuNPs ranging from 3.151 and 31.67 nm (Figs. 1a, 2a, 3). Negatively stained BYDV-PAV spherical particles showed measurement about 30 nm in size in EM (Fig. 1c). In our study, we interfered the bio Nanoparticles (virus) with metal Nanoparticles (Au) in the plant system to find out ability to capture the chemical electron transfer dynamics of the two molecules, which could provide an entirely new perspective on biology (Hong et al. 2015). We selected the most severe BYDV-PAV isolates having spherical morphological capsid that permit high amplification of optical scattering energy in the presence of AuNPs for nano application.

Particle size distribution and zeta potentials
Zeta sizer chart revealed the concentration with two different dispersed peaks (Fig. 2a) ranging from 3.151 to 31.67 nm in the width of two peaks (1-100 nm) having different intensities of 75.3% for the former and 24.7% for the latter. The zeta potential chart ( Fig. 2b) had three negative heteros different, 10.8, 31.0 and 54.0 mV, respectively, in the solutions. According to spectrophotometry of BYDV-PAV from purified virus preparations, the average of virus yield was 0.62 mg/ml from 27.30 g of infected leaves at A260/A280 ratio ( Fig. 2c) according to Alkubaisi et al. (2015). Electron micrographs ( Fig. 3) illustrated purified AuNPs only that showed well-dispersed particles of size 3.151 and 31.67 nm.
The correlation between the zeta potential value and the zeta sizer is inverse proportion. AuNPs solutions are more active and dispersed. The minuscule size of nanoparticles means they exhibit enhanced or different properties when compared with the bulk material. The large surface area of the nanoparticles produces a large volume. Due to the application on leaf surfaces, they enter through the stomata openings or the base of trichomes and translocate to various tissues (Uzu et al. 2010). According to strong electric fields at the surface, the absorption and scattering of electromagnetic radiation by noble metal nanoparticles are strongly boosted. These unique properties supply the strength of styling photothermal cancer therapy. It is desirable to use agents that are dynamic in the beside-infrared (NIR) zone of the radiation spectrum to reduce the light extinction by intrinsic chromophores in native tissue (Huang et al. 2006).

Gold nanoparticles potentially ruin gold barley yellow dwarf virus in cellular plant tissues
Gold BYDV-PAV particles can exploit the molecular sensing capability of AuNPs optical antennas and the biochemical properties of viral capsids of millions of different capsid topologies. That can terminate the biological hazards and take advantage of using either inherent or endowed biochemical characteristic of the viral capsids to aim subcellular regions (Plummer and Manchester 2013) and in the vivo region. Gold BYDV-PAV particles can potentially function as optical nanosatellites in a biological galaxy, such as cellular tissues and intracellular spaces which exhibit various biomolecular imaging capabilities with detection sensitivity. We propose that the unique and detailed structures of viral capsids can be used as strong plasmonic templates by conjugating novel metals onto their surfaces. The plasmonic metal layer formed in this manner will function as an optical antenna to amplify and convert incident light into confined nanoscale volumes (Hong et al. 2015). Gold viruses can utilize the molecular sensing capability of optical antennas and the biochemical properties of viral capsids (Wu et al.

Local-AuNPs/BYDV-PAV (in vivo)-AuNPs-24 vs 48 h (in vitro) as protective treatment
After 24 h incubation with AuNPs, numerous deteriorated and ruined VLPs sink in dark AuNPs which had a large area of partially vanished VLPs (Fig. 4a). In the 48 h incubation treatment, the major background of lysed VLPs, as well as numerous ruined corrosive VLPs distributed in most micrographs with dark spots of AuNPs (Fig. 4b).
Justo-AuNPs/BYDV-PAV (in vivo)-AuNPs-24 vs 48 h (in vitro) as protective treatment The protective treatment of AuNPs as a first process affecting the virus in vivo followed by more incubation stress of AuNPs for 24 h indicated numerous individual/ aggregation of more than one puffed VLPs decorated with dark AuNPs and largely circulated area of partially vanished VLPs (Fig. 5a). While AuNPs incubation for 48 h indicated large white circulated distant with completely lysed anddeteriorated VLPs (Fig. 5b).

Justo-BYDV-PAV/AuNPs (in vivo)-AuNPs-24 h (in vitro)
AuNPs were integrated with some vanished smashed VLPs. Some individual spherical deformed particles disintegrated (Fig. 8a). Figure 8b illustrates clearing and The first level (1st) is ruined VLPs and the second level (2nd) is lysed VLPs. Scale bar 0.2 lm. b The purified virus was incubated with AuNPs and showed less aggregation of VLPs (white arrows) within AuNPs (black arrows). The yellow numbers indicate the variability of the four major performances of damaged VLPs: 1 puffed particle, 2 deteriorated particle and decorated with AuNPs, 3 ruined particle, 4 vanished particle. Scale bar 0.2 lm Appl Nanosci dissolving of VLPs as well as puffed particles. The AuNPs, concentration treatment had the most efficacy on VLPs in both cultivars, while the degree of dispersed AuNPs and its distribution was ideal in Justo. There was a high yield of ruined VLPs in the local cultivar compare to the Justo cultivar for the previous one.

Justo-BYDV-PAV/AuNPs (in vivo)-AuNPs-48 h (in vitro)
After 48 h incubation with AuNPs, the most puffed VLPs decorated with dark circular AuNPs and deteriorated VLPs which had evacuated parties showed white density b Purified virus that was incubated with AuNPs. Variable performance of VLPs was individually visible in four different types of damage; ruined VLPs: 1 puffed particle, 2 deteriorated particle and decorated with AuNPs, 3 ruined particle, 4 vanished particles. Scale bar 0.2 lm b Purified virus that was incubated with AuNPs treatment showed two different areas that were distinguished and distributed in a wavy dispersed dark look of AuNPs has centered core for grapping three large spherical deteriorated No. 2 of VLPs, while the light area showed clearing and dissolving VLPs. The left upper part of b micrograph revealed puffed particle No. 1 (yellow), while the middle part indicated the first level (1st) of ruined aggregated VLPs in higher magnification. Scale bars (a) and (b) 50 nm (Fig. 9a). Four puffed spherical VLPs were surrounded by AuNPs and located in the same area of dissolving VLPs (Fig. 9b). Previous studies using electron microscopy have demonstrated that virus particles have highly ordered natural nano-architectures, with a protein coat called a capsid (Adrian et al. 1984). The outer surface of a viral capsid usually has protrusions and valleys on the subnanometer scale, the accurate the morphology need for optical antennas. Furthermore, viral reproduction yields a remarkably high degree of symmetry and uniformity that is beyond the capability of current nanotechnology. It was greater in the local cultivar. compared to the Justo cultivar. These features make viral nanoparticles attractive templates for optical antennas of AuNPs in our study. Additionally, infection selectivity was remarkable in the local cultivar. Moreover, elimination of harmful viral effects has enabled virus resistance. The amount of nanoparticle accumulation in plants varies with the reduction potential of ions and the reducing capacity of plants that depends on the presence of various polyphenols and other heterocyclic compounds present in plants. No single barley variety is resistant or susceptible to all the great diseases. Thus, varieties should be selected according to their local adaptability, high yield potential, severe losses in the production and quality of wheat and barley crops and resistance to the most shared and serious diseases (Douglas et al. 2002a, b). The capsids of most plant viruses are modest and of firm construction, consisting of numerous copies of one or a few sort of protein subunits coordinated with either icosahedral or helical symmetry. In many conditions, capsids are produced in huge amounts either by the infection of plants or by the subunit(s) expression of a variety of heterologous systems, given their relative simplicity, stability and ease of production (Lomonossoff and Evans 2011). Thus, the advantage of the morphological characteristics of plant viruses can be taken by integrating AuNPs as a novel metal onto the external surfaces of their capsids for highly sensitive and selective molecular spectroscopic detection such as localized electronic absorption and vibration spectroscopy (Hong et al. 2015). Variable performance of gold capsids was individually visible in four different types of damage or ruined VLPs: (1) puffed particle, (2) deteriorated particle and decorated with AuNPs, (3) ruined particle, and (4) vanished particles (Fig. 7b). Oancea et al. (2009) explain the formation of such chemicals encapsulated in nanocomposites made of layered double hydroxides. In our data, we could relay in this result as a benefit of reverses such phytotoxicity of AuNPs by blending it with virus particles as protein and viral nucleic acid components to accelerate their applicability and effectiveness for virus control. The application of AuNPs particles on the Justo and local cultivars had a great damage on virus particles in TEM (Figs. 4,5,6,7,8,9). It could explain the interaction performance in our results which had different degrees between VLPs and AuNPs illustrated as aggregation, surrounding, integration, and accumulation particles. This kind of interval molecular interaction could lead to disappearance or/and swelling, smashing, deformation, corrosiveness and puffiness of infected virus particles. The self-assembly of substratebound monolayers comprised DNA-modified gold nanoparticles. For the first time, bifunctional particles containing two independently addressable oligomer sequences were used as building blocks for layer assembly. Saini et al. 2008 proved that Chilo iridescent virus provided a biotemplate which contains a bioscaffold for 2-5 nm of AuNPs on the viral capsid. These a bio scaffold allowed AuNPs to act as nucleation sites for Au ions in the surrounding solution by allowing electrolysis deposition to occur at the sites. The size of AuNPs has a potential dramatic effect in our study from 3.151 to 66.74 nm. Giljohann et al. 2010 studied the application of AuNPs and found that the size of AuNPs size (5 nm) could be used as a template for biomimetic high-density lipoprotein structure which makes this size control necessary. Cheung et al. (2003) Proved in this respect that most viruses are in the scale of nanometers, and supermolecules and assembly viruses are of tens of nanometers in size. The AuNPs size ranged between 10 and 40 nm as shown in Fig. 1. Moreover, nanometric building blocks can aid in the survey of the molecularly directed assembly of biomolecular arrays. Hilger et al. 2002 add an alternating magnetic field that could also be used to heat the magnetic nanoparticles, thus making hyperthermia treatment of tumors feasible and a potential simultaneous treatment. This strategy of AuNPs application in our study induced by combining virus particles with AuNPs possibly leads to synergistic therapeutic effects for inducing virus resistance by the interaction performances, as illustrated in TEM. Giljohann et al. (2010) studied the modified AuNPs for inducing gene regulation upon entry into the cytoplasm of the cell; the DNA-AuNPs can bind to the strand preventing translation of the mRNAs corresponding protein. Different sizes of particles are easily distinguishable in electron micrographs, allowing simultaneous multiple labeling experiments In vitro analysis, AuNPs, are used to locate and probe viruses. In vivo applications of gold nanoparticles illustrated procedures for probing as well as diagnosis and hyperthermic treatment that allow the targeted destruction of cells. There is an important key point in the model he explained related to Tm of nucleic acid. The oligonucleotides on the AuNP surface are close enough such that the counterions associated with one oligonucleotide also act to screen the negative charge on adjacent oligonucleotides. This additional cost screening results in greater oligonucleotide duplex stabilization about free DNA strands and explains why the DNA-AuNP aggregates melt at higher temperatures than the same unconjugated oligonucleotide duplexes under the same conditions. Patel et al. (2007) cleared that nuclear localization signal NLS peptide is a virus-derived protein fragment that interacts with intracellular proteins for transport across the nuclear envelope. Goldfarb et al. (1985) interpreted that addition of NLS peptide to the DNA-AuNP did not change the binding properties of the oligonucleotides, but it did alter the intercellular localization of the AuNPs. The way of AuNPs application on the plant was performed in vivo, in-vitro and with both of them The most efficient ones on virus particles are those treated by dual stress effect of AuNPs (Figs. 4b,5b,7,9). Nair et al. (2010) illustrated the mechanism of AuNPs inside the plant. They reported that the formation of nanoparticles, whether they are formed outside in the media and then translocated to plants or whether they conformed with the reduction of metal salts within the plants itself, still needs more clarification. The in vitro assembly of empty virions has been extensively characterized, and these protein cages have great potential to be utilized as constrained reaction vessels for material synthesis and/or entrapment in the field of nanotechnology (Douglas et al. 2002a, b). Recently, plant virus capsids as a protein shell that forms the surface of a typical plant virus particle are useful templates. All virus capsids were assembled from virus-coded protein subunits called capsomeres. Many plant viruses assemble capsids with precise 3D structures providing nanoscale architectures that are highly homogeneous and can produce a great mass. Capsids are adjustable to both genetic and chemical modifications, allowing new functions to be integrated into their structure by the layout. The three capsid appearances, at the interior surface, the outer surface, or the interface between coat protein subunits, can be separately functionalized to produce multifunctional templates (Young et al. 2008). Plant virus-like particles (VLPs) have attracted attention as potent reagents for bionanotechnology. Plant virus-like particles (VLPs) have attracted attention as potent reagents for bio nanotechnology science having genetic materials and chemical alteration of super protein molecular structures (Lomonossoff and Evans 2011). In our study, we obtained four biomolecule alterations of VLPs due to dual AuNPs probing illustratedin Fig. 6b. Homogeneity of virus morphologies in nature and high-throughput reproducibility accelerate the potential for the use of viral particles in optical sensing applications. Moreover, using asymmetric gold coupling leaves parts of the native capsid uncovered that can interact with host cells and biochemically alter functions. Such gold virus nanoparticles can metaphorically work as 'nanosatellites' exploring 'cellular galaxies' (Hong et al. 2015). Therefore, biological structures and biomolecules are promising tools for bionanotechnological applications. The study subject of the area here is to probe BYDV-PAV with AuNPs to diminish its infectivity hazards in plant system and coupling these purified asymmetric gold virus particles once more with AuNPs in vitro to follow up morphological alteration of these gold BYDV-PAV nanostructures as a result of dual AuNPs stress in vivo and in vitro as nanosatellites.

Conclusion
AuNPs and biocompatible nature characters of BYDV nanoparticles have been postulated in the present study. In vivo conditions, sustained effect of AuNPs inside the plant system will have strong anti-virus influence on viral protein. Hence, this nanoparticle may perhaps be recommended as therapeutics while treating plants infected with the virus.