Sharka: The Past, The Present and The Future

Members the Potyviridae family belong to a group of plant viruses that are causing devastating plant diseases with a significant impact on agronomy and economics. Plum pox virus (PPV), as a causative agent of sharka disease, is widely discussed. The understanding of the molecular biology of potyviruses including PPV and the function of individual proteins as products of genome expression are quite necessary for the proposal the new antiviral strategies. This review brings to view the members of Potyviridae family with respect to plum pox virus. The genome of potyviruses is discussed with respect to protein products of its expression and their function. Plum pox virus distribution, genome organization, transmission and biochemical changes in infected plants are introduced. In addition, techniques used in PPV detection are accentuated and discussed, especially with respect to new modern techniques of nucleic acids isolation, based on the nanotechnological approach. Finally, perspectives on the future of possibilities for nanotechnology application in PPV determination/identification are outlined.

represented by Bymovirus, where species are restricted to Poaceae (Graminae) and are transmitted in soil by zoospores of Polymyxa graminis Ledingham. In this case, it is very difficult to study the epidemiology of diseases caused by Bymovirus, because they are not transmitted mechanically, purified preparations demonstrate only low infectivity, and the vector is an obligate parasite of roots that inhabits the individual cells of rhizodermis and cortex [49]. In addition, they frequently occur in a mixture with furoviruses. A new soilborne virus -soybean leaf rugose mosaic virus (SLRMV) -has been isolated and characterized by Kuroda et al. [50]. This virus is closely related to bymoviruses, however, further characterization is necessary. However, Polymyxa graminis is obligate parasite of monocot plants (Glycine max (L.) Merr. is dicot) and soybeans became diseased when grown in virusinfested soil [50]. With exception of Bymovirus with bipartite genome, where open reading frame is divided between two genomic RNAs, all potyviruses have monopartite genome with a genome-linked protein attached to the 5´ end (VPg) and a polyadenosine tail at the 3´ end of the genome. Their genome was characterised also as monocistronic for many years because of the presence of only one functional ORF. However, bioinformatic evidence and experimental verification of the evidence for overlapping coding sequences within the P3 cistron of potyviruses have been presented in the work of  helper component-proteinase (HCpro) has been recognized to be involved in the processes necessary for life, respectively infection cycle, especially in interactions with host proteins as well as other viral proteins. Thus, the presence of HC-pro is essential for all potyviruses [64]. These processes include not only interactions with host proteins, but also polyprotein processing and suppression of antiviral RNA silencing [65,66]. In addition, possible interactions between HCpro and host proteins must be carefully considered. This fact has been demonstrated in work of Shen et al., who observed interactions between HCpro and full-length papaya calcireticulin, the multifunctional protein that regulates intracellular calcium(II) ions levels and protein folding in the endoplasmic reticulum [67]. In conclusion, HCpro has multiple functions. Despite the HCpro multiple function, the understanding of its function is still poor. The work of Guo et al. revealed the crystal function of cysteine protease domain of HCpro from turnip mosaic virus [68]. HCpro cleavages only dipeptide Gly-Gly at its C terminus. A cleaved C domain remains tightly bond at the active site cleft to prevent trans activity. Generally, the structure adopts a compact alpha/beta-fold. The catalytic cysteine and histidine residues constitute an active site and HCpro recognizes a consensus sequence YXGVV around the cleavage site between two glycine residues [68]. There are at least six cleavage sites in cis-/trans-arrangement in the viral polyprotein recognized by NIa proteinase (NIa-Pro). NIa-Pro represents C-terminal domain of NIa and is shown to be analogous to picornavirus 3C protease [69]. The N-terminal domain of NIa is designated as VPg and will be further discussed. The active -catalytic -sites of NIa-Pro are defined by a consensus heptapeptide sequence surrounding each cleavage site containing triad of His-Asp-Cys, which are conserved among the potyviruses [70]. The mutation of the catalytic residues His46, Asp81, and Cys151 resulted in complete loss of activity [71]. In addition, NIa proteinase possesses structural motifs shared with cellular serine proteases with the substitution of serine by a cysteine as the active site nucleophile. The specificity of enzyme and control its activity is discussed in some recently published papers [72][73][74], which are focused on the investigation of recombinant viruses and their host interactions. Mutation in the Lys(27) of NIa-Pro has established the role of this enzyme in the determination of host specificity. On the other hand, Lys(27) mutation did not affect the protease activity of NIa-Pro [75]. The NIa-Pro is a sequence-specific proteinase required for processing of viral polyprotein in the cytoplasm. This fact has been demonstrated in many potyviruses, such as pepper vein banding virus (PVBV), and is generally relevant for all known potyviruses [76]. Its accumulation in nuclei of infected cells manifested as a formation of "inclusion bodies" and has been demonstrated in some papers, for example see work of Anindya et al. [71] or Restrepo et al. [77]. This fact has been established by Knuhtsen, who observed tobacco etch virus-induced intranuclear inclusions [78]. However, this author is not the first to demonstrate the presence of nuclear inclusions in the nuclei of infected cells. In 1968, Shepard used electron microscopy for the characterization of nuclear (and cytoplasmic as well) inclusions in tobacco etch virus-infected Nicotiana tabacum L. cv. Havana 425 [79], and before him in 1941, Sheffied observed formation of nuclear inclusions in infected cells [80]. The NIa-Pro nuclear localization is still investigated. Accumulation of Nia-Pro in nuclei is observable mainly at the end of viral infection cycle. It seems that NIa-Pro executes DNA cleavage activity, respectively nonspecific double-stranded DNA degradation. This fact indicates the NIa-Pro role in viral infection cycle with subsequent degradation of the host DNA [71]. Potential use of NIa-Pro in biotechnologies is indicated in the paper by Fellers et al., where three genes, each consisting of two NIa encoding regions (tobacco vein mottling virus, tobacco etch virus and potato virus Y) were introduced into Nicotiana tabacum L. cv. Burley 21. The authors indicate that "results of abovementioned paper showed that different potyvirus NIa-Pro genes can be used for a protection against potyvirus infection and may protect plants against more than one potyvirus" [81]. Generally, transformation of plants with translatable sequences corresponding to the structural proteins, such as CP, or non-structural proteins such as the NIb or the P1 of potyviruses can make the plants highly resistant to infection with the corresponding virus [82][83][84]. Pathogen-derived resistance to viruses in transgenic plants is typically based on RNA silencing. This fundamental cytoplasmic antiviral defense system can be efficiently directed against viruses by producing homologous RNA from a transgene in plant cells. Plant potyviruses encode two membrane proteins 6K and P3. The role of P3 protein is only poorly understood. The role of P3 in viral infection cycle and as a symptom determinant is shown in the paper by Jenner et al. [85]. Development of symptoms of the disease may be closely connected with interactions between the P3 protein and proteins of host plant. The paper by Lin  . This protein was found to be phosphorylated as a part of the virus particle by the cellular kinase activity, which means that VPg is accessible to protein-protein interactions [110]. Interactions with HCPro were shown in the papers of Yambalo et al. and Roudet-Tavert et al. [111,112].
The potyvirus-interacting protein (PVIP), a plant-specific protein with some homologues has been identified in many plants. PVIP function is still discussed, however, as it probably serves as a control in chromatin remodelling and as an ancillary factor to support potyvirus movement in plants [113]. Co-immunoprecipitation and bimolecular fluorescence complementation (BiFC) assays revealed that P3N-PIPO interacts with host protein PCaP1, a cation-binding protein that attaches to the plasma membrane via myristoylation. PIPO domain of P3N-PIPO binds PCaP1; in this process myristoylation of PCaP1 is unnecessary for interaction with P3N-PIPO. It seems that PCaP1 links a complex of viral proteins and genomic RNA to the plasma membrane by binding P3N-PIPO, enabling localization to the plasmodesmata and cell-to-cell movement [55]. Phosphorylation itself probably plays important role in the VPg-mediated functions during the infection cycle of potyviruses [110]. Interactions between viral RNA polymerase and VPg support the VPg role in a viral RNA synthesis [114]. VPg displays still investigated roles in nuclei of host plants [115,116]. However, the nuclear VPg localization is not the only localization in infected cells. Using green fluorescent protein technologies, VPg was localized in endoplasmic reticulum with the probable role in viral RNA translation [117]. Mutants in the VPg domain demonstrate inhibition of nuclear transport debilitated viral genome amplification. Interactions between VPg and eIF 4E that indicate its role in the initiation of translation of the viral RNA has been demonstrated and represent a critical element for virus production [118][119][120][121][122]. VPg is probably an efficient modulator of eIF 4E biochemical function [123,124] and directs eIF 4E to promote viral RNA expression [125]. Interactions of VPg with next molecules, such as the phosphatidylserine of biomembranes [126]. However, the significance of these interactions must be further investigated. The NIb acts as a RNA-dependent RNA polymerase and is generally localized in the nuclei of infected plants [127]. Interactions between NIb and VPg have been demonstrated in some papers [128][129][130][131]. NIb role in uridylylation of VPg protein was investigated in potato virus A model and it seems that has important function in the regulation of RNA synthesis [131]. NIb introduction into plants (transgenic plants) may pose one of the directions in progress in the field of potyvirusresistant plants [132,133].
The coat protein (CP) represents the multifunctional protein [134]. It is important for transmission of Potyviridae and, in addition, the degree of the identity between coat protein sequences can be used for the determination of relationship within the Potyviridae  Table 2.

Plum pox virus
The plum pox virus was firstly observed on plums in 1915 in Bulgaria and the disease was described in 1932 by Atanasoff [184]. Therefore, Eastern Europe was the localization of the first PPV epidemic. Later, PPV was reported on apricots (1933, by the same author who described the first PPV observation), on peaches (1961) and on sour cherries in the 1980´s [185]. Between 1932 and 1960, the disease caused by PPV moved from Bulgaria into Yugoslavia, Hungary, Romania, Albania, Czechoslovakia, Germany and Russia. Progression of disease in Western Europe was recorded after Word War II from Germany and Austria to The Netherlands, Switzerland, Greece, England and Turkey (1960s), then France, Italy, and Belgium (early 1970s), Spain and Portugal (early 1980s), Egypt, Syria and Cyprus (late 1980s). After 1990s, the disease was recorded in Chile, USA (Adams County, Pennsylvania, in 1999), Jordan, India and Canada. However, new reports about PPV occurrence (including individual PPV strains) still appear [186][187][188][189][190][191]. In Kazakhstan, PPV was firstly detected on plum and apricot trees in 2004 [192]. In 2005, plum pox virus has been found on apricot trees in China (Hunan Province of China, plants with typical yellow rings and diffused chlorotic spots) [193], in 2006, sharka disease in Prunus species was demonstrated in Argentina [194] and Pakistan [195]. One of the latest reports indicates detection of PPV in commercial Japanese apricot trees in Tokyo [196]. For the disease distribution and status, see Table 3.
PPV as a member of Potyviridae, which is a RNA virus with flexuous filamentous particles approximately 750 × 15 nm. It is widely distributed in Europe, North Africa, Asia and both Americas. As a natural host, PPV is restricted only to members of genus Prunus L. with above-mentioned exception. However, many experimental host plants have been identified. PPV is transmitted by several aphid species (see Table 1) [240]; however, it is also graft-transmissible to susceptible Prunus species and sap-transmissible to a wide range of herbaceous species. Symptoms of "sharka" highly depend on sensitivity of host plant and environmental conditions, especially on the weather conditions and age of the trees [241].   [192,195,197,208,[211][212][213][214][215][216][217][218][219][220][221][222][223][224][225][226][227][228][229] Introduced,  [197,208,218,[230][231][232][233][234][235] Introduced, eradicated Belgium, Denmark [197,208] Not present Estonia, Finland, Ireland, Israel, Lebanon, Malta, Morocco, Palestine, Sweden [197,208,236,237] Unknown status China*, Libya [208,238,239] PPV usually affects both the leaves (leaf symptoms) and the fruit (fruit symptoms) of the plants. The intensity of fruit symptoms is usually significantly increased by the age of infected plants. The symptoms include chlorotic spots or rings, oak-leave patterns and vein clearing on leaves (plum), chlorotic pale-green rings and lines on leaves (apricot), small chlorotic blotches and distortion of the leaves (peach) or pale green patters and rings (cherry) [242,243]. Shallow rings and arabesque depressions, sometimes with brownish or reddish necrotic flesh (plums), light colored depressed rings (apricot), pale rings with diffuse bands on the epidermis (peach) or chlorotic and necrotic rings (cherry) are the main symptoms in the fruits [242]. In addition, fruits may drop prematurely [241]. Infection is usually symptomless in almond. On the other hand, in vitro explants lack the presence of symptoms typical for PPV infection; only typical interveinal chlorosis produced by PPV is visible [244]. This fact must be discussed in the light of the composition of cultivation medium, which represents complex matrix composed of macro-and microelements, as well as source of carbon (sugar(s)) and plant growth regulators -phytohormones. Just phytohormones play crucial role in the regulation of physiological processes as well as processes directing to the programmed cell death [245]. Most of the observable symptoms are probably dependent on a complex of virus-host interactions, where several or all viral genes could be involved in some way. On the other hand, the work of Clemente-Moreno et al. brings interesting findings and shows the possible protective role of ferrulic acid [244]. The work of Nagyova et al. revealed the role the 3´ proximal part of the plum pox virus P1 gene in the virus-host interactions resulting in various pathotypes and demonstrated a different relative importance of particular PPV genes for symptom manifestation in different herbaceous host plant species [246]. There was an effort of several working groups to classify PPV in accordance with various characteristics, especially on the symptoms caused in experimental conditions of different plant hosts [27,247]. Based on different experimental plant hosts and symptoms, different strains have been described. Classification based on Chenopodium foetidum Lam. as an experimental plant host ranks PPV isolates to yellow, intermediate and necrotic strains [248]. Classification based on molecular-biological data initially brings the recognition of six strains of plum pox virus, see Table  4. Nevertheless, new isolates still occur and there is an effort for the establishment new PPV strains (for example "PPV-T, PPV-Penn, etc."; however, these are still only isolates, for example PPV-Penn isolate(s) belongs to the PPV-D strain) [249,250]. On the other hand, PPV-EA isolate demonstrates 79-80 %, 80 %, 77 %, and 77 % homology with isolates of strains D/M, Rec, C, and W, respectively) and is classified as a strain [251,252]. The same situation is in the case of PPV-W that represents a new strain that occurs in Canada and Latvia [253].

Plum pox virus genome organization
PPV virions are long, flexuous and rod-shaped, approximately 750 nm (660-750 nm) in length and 15 nm (12.5 -20 nm) in width [294]. PPV as a member of Potyviridae has the structure typical for all potyviruses with exception of Bymovirus. The molecule of ssRNA is of positive polarity. The RNA of PPV has a VPg protein linked to its 5´end and a long poly(A) tail, which is heterogenous in its size at its 3´end [295]. In the case of an aphid non-transmissible PPV, excluding a 3´terminal poly(A) sequence the ssRNA is 9741 nucleotides in length. The 3´noncoding region is 220 nucleotides in length without the poly(A) tail [296]. PPV genome contains a long open reading frame starting at the first AUG codon, nucleotide 36, that is translated upon infection, starting at the second AUG codon as nucleotide 147, into a large polyprotein of 355.5 kDa [70,295,297]. There are still unanswered questions about initiation of potyviruses translation via recognition of the specific viral sequences [125,298]. Because potyviruses including PPV have relatively short 5´non-coding regions with a low content of cytosine and guanine, they avoid a stable secondary structures and lack nonfunctional intraleader opening reading frames [299,300]. Cap-independent internal initiation of translation has been proposed for four members of the genus Potyvirus [301,302]. Work of Simón-Buela et al. presents different in vitro and in vivo evidence of cap-independent leaky scanning as the mechanism of translation initiation of PPV genomic RNA [303]. Originated polyprotein precursor is co-and posttranslationally cleaved by three virus-encoded proteinases into 11 mature proteins -P1, HCpro, P3, P3N-PIPO, 6K1, CI, 6K2, NIa (respectively VPg and NIa-Pro), NIb and CP. CP protein of about 36 kDa forms helically arranged "coat". Similarly to other potyviruses, the PPV P1 proteinase represents together with P3/6K1, 6K2, NIa/VPg and N-terminal domain of CP highly variable protein [285, 304,305]. This variability enables the determination of PPV isolates and their further characterization [285, [304][305][306]. The untranslated region of PPV consists of 147 nucleotides, starting with four adenine residues. It seems that intact 5´end is not essential for PPV replication. PPV 5´ untranslated region that is essential for virus replication is confined to the first 35 residues. Deletion of a long sequence between nucleotide 39 and 145 did not affect the rate of infection and viral accumulation, but affected process of pathogenesis [307,308].

Plum pox virus transmission and cytological, histological and biochemical changes in infected plants
The PPV transmission is widely discussed in the two directions as aphid and non-aphid transmission. PPV is transmitted over short-distances in a non-persistent manner by Aphis fabae Scopoli (Aphididae), Aphis gossypii Glover (Aphidae), Aphis spiraecola Patch (Aphididae), Brachycaudus persicae Passerini (Aphididae), Myzus persicae Sulzer (Aphididae) and next members of Aphididae [309][310][311][312][313]. Long-distance transmission is based on non-aphid transmission, this means on the spreading of infected plants and infected plant parts. The grafting may represent important risk in PPV spreading [24,314]. Milusheva et al. indicates the possibility of PPV transmission by infected seeds in a cultivar-dependent manner [315], however, some previously published papers disproves the possibility of PPV transmission by seeds [316,317]. The next fate of PPV and symptoms expression in infected plants is discussed. In stems of infected plants, PPV is localized in xylem and pith, which may indicate its possible spreading via xylem part of vascular tissue of infected plants [318]. This xylem transport does not suppose the transport for long distances, but for short distances (cell-to-cell) in the horizontal direction, which may be responsible for the localization of PPV in xylem and pith. This type of transport is provided by parenchyma cells. In addition, these cells are interconnected with other parenchyma cells; strict compartmentation is not possible. Later, this localization has been made more accurate. Hoffmann et al. proved the PPV presence in ray and axial parenchyma of vascular tissue of infected plants [319]. In petioles, PPV was demonstrated in epidermis and parenchyma cells of ground tissue, not in xylem [318]. These findings enable to express hypotheses about PPV spreading in plants [320,321]. On the other hand, there are significant differences between resistant and susceptible cultivars. Whereas the long-distance PPV spreading is highly limited in resistant cultivars, PPV susceptible cultivars allow long-distance PPV transport via xylem and the development of symptoms of sharka disease [320,322]. The differences between cell-to-cell (short-distance) and long-distance PPV transport between individual plant cultivars have been recorded, however, there are still unanswered questions [323]. Plum pox virus infection leads to the changes on different levels, i.e. cell and tissue as well as biochemical [1,324]. Whereas the changes in subcellular and cellular levels are connected with subcellular compartmentation of individual PPV proteins [320], changes in biochemistry of infected plants and resistant/susceptible plants may have practical impact in utilization of enzymes/proteins as markers of sharka disease. The paper by Hernandez et al. described the response of differently sensitive apricot (Prunus armeniaca L.) cultivars to plum pox virus infection [325]. This paper is interesting due to choice of cultivars, both resistant (Goldrich) and sensitive (Real Fino), and comparison of responses at antioxidant enzymes levels [325]. The most significant changes were observed in the case of superoxide dismutase(s) (SODs) and ascorbate peroxidase (APX) enzymes. Compared to sensitive cultivar, the significant decrease of APX activity in resistant cultivar was determined. Modifications in peroxidase activity were demonstrated also in Nicotiana clevelandii Gray PPV-infected plants, where the role of gaseous phytohormon ethylene in PPV-induced senescence has been partially revealed [326], and in Chenopodium foetidum Lam. [327]. On the other hand, levels of SODs, glutathione reductase (GR), dihydroascorbate reductase (DHAR) and monodehydroascorbate reductase (MDAR) of resistant cultivar were significantly increased compared to sensitive cultivar. Levels of catalase (CAT) remained unaltered. These results indicate the role of hydrogen peroxide, which is generated by the SOD activity, in response to PPV on the biochemical level. The experimental work continues in the second article of Hernandez et al., in this case focused on the effect of PPV on photosynthesis and again on antioxidant enzymes [328]. These results were confirmed in pea plants (Pisum sativum L.), where PPV infection led to the accumulation of reactive oxygen species in chloroplasts under affecting of photosynthetic processes [329]. This fact is in agreement with papers focused on interactions between potyviral proteins and proteins of photosynthetic apparatus [98].The above-mentioned facts may be a reaction on its damage. In the third study, the same authors investigated response to long-term plum pox virus infection in peach plants (Prunus persica (L.) Batch cv. GF305 with the great susceptibility to PPV) with the focus on oxidative stress [328]. In this study, an oxidative stress and antioxidant mechanisms imbalance in accordance with the progress of PPV infection was determined. This fact has been confirmed by the following study of the authors [330]. Diaz-Vivancos et al. described an oxidative stress as a result of PPV infection in the apoplastic space of only susceptible apricot plants [331,332]. Whereas all above-mentioned papers were focused on oxidative stress caused by PPV infection, Escalettes et al. determined differential gene expression ins PPV-infected apricot cultivar (cv. Goldrich) [333]. The CH4 and CH22 fragments coding for a putative myosin and kinesin, were over-expressed in cv. Goldrich. Both myosin and kinesin are closely associated with cytokinesis, where they serve as motor proteins in the organization of phragmoplast microtubules. In the PPV-infected cv. Goldrich, the transketolase analogue CH15 was over-expressed, while it presented a very similarly modified expression in the susceptible genotype. Expression of the ankyrin-like CH27 was obviously enhanced in PPV-inoculated partially resistant apricot and in the susceptible Prunus genotype. The clear repression of CH29 transcript encoding a putative class III chitinase in the partially resistant genotype suggests a virus-mediated repression of this gene in the Goldrich cultivar [333]. Arabidopsis thaliana (L.) Heynh. was used as a model plant for investigation of gene expression alteration due PPV infection in the following study [334]. Genes associated with soluble sugar, starch and amino acid, intracellular membrane/membrane-bound organelles, chloroplast, and protein fate were up-regulated, while genes related to development/storage proteins, protein synthesis and translation, and cell wallassociated components were down-regulated. These gene expression changes were closely associated with PPV infection and symptom development [334]. The paper by Wang et al. revealed that genes involved in defense, cellular transport, development, protein synthesis, proteins with binding function in the PPV-infected peach leaf tissue are more active than those in PPV-free leaves [335]. The changes of expression of genes (increase of the following gene transcripts: including beta-1,3-glucanase, cytochrome-P450-like protein, cytochrome P450 monooxygenase, heat-shock protein 70, thioredoxin, alcohol dehydrogenase, catalase, cysteine protease inhibitor, translation factor EF-1 alpha, and pathogenesis-related protein (PRI)) in Pisum sativum L. PPV-infected plants support the ability of PPV to induce common stress responses in susceptible plants [335]. The recent studies focused on the changes in growth characteristics and yield of infected plants [336,337]. Similar results were demonstrated by Garcia-Ibarra et al., who investigated changes in protective mechanisms including antioxidant enzymes in peach infected by apple chlorotic leaf spot virus (ACLSV, Flexiviridae). The results show increases in the APX, dehydroascorbate reductase (DHAR), superoxide dismutase (SOD) and glutathione S-transferase (GST) activities, whereas POX suffered a decrease of about 34 % [338]. Induction of oxidative stress by plant viruses is demonstrated in some works, such as Amari et al. [339], Clarke et al. [340], Farkas et al. [341], Fodor et al. [342], and Kiraly et al. [343]. In the lights of these findings, changes in markers of oxidative stress may be useful in the detection of PPV infection. However, further characterization of infected plants is necessary, because changes in antioxidant mechanisms under plant virus infection are indistinctive.

Detection of PPV
The detection of PPV undergoes rapid development, from the usage of very simple methods based on host plants to molecular-biological methods and techniques. However, significant limitations for methods used in routine testing of plants for PPV must be carefully considered.  Table 4.  [208,357] The first, but highly important step in progress of PPV diagnostics was based on the serological assay, respectively on the enzyme-linked immunosorbent assay (ELISA), which takes advantages from antibodies and their subsequent detection. One of the first record about the use of antibodies was published in 1980 [358]. ELISA method underwent progress with some modifications and now, it is one of the most popular and used techniques for PPV detection [359][360][361][362]. Whereas the usage of polyclonal antibodies, which recognize multiple epitopes on any one antigen, is controversial due to problems with specificity and consequently with sensitivity (serum contains a mixture of antibodies of different affinity), monoclonal antibodies, which detect only one epitope on the antigen, helped to overcome above-mentioned problems and are still widely used in PPV diagnostics [363][364][365][366][367]. Monoclonal antibodies are usually obtained after immunization of BALB/c mice with purified PPV isolates [367,368]. Despite the fact that commercial kits had been available and used before the introduction of 5B-IVIA monoclonal antibody, universal and specific detection of any PPV isolate was achieved by the use of 5B-IVIA monoclonal antibody [23,369]. Generally, this antibody enabled the production and use of commercially available kits for PPV detection, which enable specific identification of respective PPV strain. Antibodies are produced especially against non-structural PPV proteins as P3 [169], 6K2 [169], CP and CI [181,370,371], but also HCPro [181,372], NIb [169,181,373], and P1 [169]. However, non-structural proteins antibodies have not found the application in the practice and are used only for scientific purposes. As a secondary antibody, peroxidase-labelled, biotin-streptavidin system or fluorescence probe-labelled secondary antibodies are used [358]. Double Antibody Sandwich Indirect ELISA (DASI-ELISA) and Triple Antibody Sandwich ELISA (TAS-ELISA) have been recently recommended in PPV detection [13,23,266,269,360,374]. They are used especially for the determination of individual PPV strains [23, 365,375,376]. Antibodies may be useful for the investigation of subcellular compartmentation of PPV mature proteins, or, in addition, may be used for subcellular localization of some members of Potyviridae family [181]. In this case, gold-labelled antibodies are used [169,377]. During the years, improved ELISA-based techniques have been proposed and developed. One of the most recent technique is impedimetric immunosensor, which is based on gold electrodes modified with 1,6hexanedithiol, gold nanoparticles, anti-PPV IgG polyclonal antibody and bovine serum albumin. The proposed technique displays very good detection limit (10 pg/ml) and is able to differentiate the samples from healthy plants and the samples containing 0.01 % of infected plant extract [378]. The PCR-ELISA, respectively RT-PCR-ELISA, which is based on immunoenzymatic detection of PCR products, represents next possibility of ELISA/PCR modification with significant increase of sensitivity compared to both ELISA and PCR methods [374,379,380].
Hybridization techniques are based on an establishing a non-covalent, sequence-specific interaction between two or more complementary strands of nucleic acids into a single hybrid. The revolutionary were two works of Wetzel et al. published in 1991. Authors described molecular cloning and partially described the nucleotide sequence of the genomic RNA of PPV-EA. In addition, they compared this sequence to the corresponding sequence of previously sequenced PPV strains [381]. Finally, a sensitive, polyvalent assay based on the polymerase chain reaction (PCR) was developed for plum pox potyvirus (PPV) detection. This technique was adapted for a single tube, the chemical denaturation and reverse transcription of the viral RNA followed by the PCR reaction yielding a 243-base-pair product [382]. These two works started the large-scale application of PCR in the practice. Molecular hybridization techniques as well as different PCR-based assays have been developed to detect the PPV RNA presence in the sample [383][384][385][386]. PCR is widely used to amplify specific nucleic acid regions; thus, there is a necessity to have primers of known nucleotide sequence. In the past, different primers have been proposed and used, mainly on the HCPro [387], C-terminal part of NIb [265,388], Cterminal part of CP [249, 265,389], N-terminal part of CP [390] or 3´-noncoding region [272]. The request to known nucleotide sequence is absolutely necessary also in hybridization techniques, which are popular due to relative simplicity. The paper by Pasquini et al. describes the strategy, development and use of 70-mer oligonucleotide probes specific for determination and genotyping (identification) the individual PPV isolates [391]. Despite the above-mentioned facts, the RNA isolation is the crucial step in these techniques. There are some difficulties connected with PPV RNA isolation from plant tissues, especially higher rate of polyphenolics and polysaccharides, which affect RNA isolation due to formation of complexes with both RNA and proteins. In addition, these compounds are able affect also activity of enzymes (including reverse transcriptase necessary for RNA transcription into cDNA form) used in PCR. One-Step RT-PCR as well as Two-Step RT-PCR is widely used in PPV RNA detection [265,[392][393][394][395][396]. Products of PCR may be visualized by the electrophoretic separation of fragments in agarose gel with subsequent labelling by ethidium bromide or SYBR Green [397][398][399], on the nitrocellulose membrane [400], eventually by immunoenzymatic procedure [379]. There are some techniques, which are based on PCR technique and pose the improvement, especially significant enhancement, of sensitivity. Co-operational PCR (Co-PCR) has been described for sensitive detection of plant viruses and bacteria [401][402][403]. This technique, carried out in a single reaction, minimizes contamination risks and has a level of sensitivity similar to nested PCR and real-time PCR. In addition, it can be coupled with dot blot hybridization, making it possible to characterize the nucleotide sequence [401]. A highly sensitive assay, based on the polymerase chain reaction amplification of cDNA synthesized from the viral RNA of antibody-captured viral particles was developed by Wetzel et al. in 1992 [404]. The immunocapture step, by allowing the use of large sample volumes and by the viral particle pre-purification it achieves, dramatically increased the sensitivity of the assay. Recently, modifications of this technique are widely used. An immunocapture reverse transcription-polymerase chain reaction (IC-RT-PCR) based assay for the detection and identification of plant viruses represents technique using clarified plant extracts with degenerate primers, without necessity of isolation of total nucleic acids. This technique was used for detection and determination of papaya ringspot virus [405,406], zantedeschia mild mosaic virus [407], and sugarcane streak mosaic virus [408]. In addition, this technique was used in identification of PPV isolates [14,199,[265][266][267]269]. A nucleic acid sequence-based amplification method coupled with flow-through hybridization (NASBA-FH) was developed for plum pox virus (PPV) detection with the detection limit 1000 times higher compared to conventional RT-PCR [409,410]. Despite the fact that ELISA followed by PCR techniques is the most frequently used technique in the PPV detection, loop-mediated isothermal amplification (LAMP) will probably become the most frequently applied approach in PPV detection. The great advantage of LAMP is its enormous rate of amplification paired with a very high specificity and low artefact susceptibility, which means great specificity [411]. Spot real-time RT-PCR is a method for detection of plum pox virus using conventional ELISA plant crude extracts immobilized on paper. This method has been developed to overcome the need of RNA isolation [412].
The field of nanotechnology is focused on the study and control of phenomena and materials and length scale below 100 nm, it means 1-100 nm [413,414]. This definition is not the only one, which tries to define nanotechnology. One of the most important criterions consists in accentuation the special properties of nanomaterials due to their nanoscaled proportions, which open their unique properties and features [415]. Nanotechnology found its application especially in industry and medicine, especially in electronics, imaging and drug delivery system for targeted therapy [416][417][418][419][420][421][422][423][424][425][426]. On the other hand, their application in the field of plant biology and phytopathology is limited, especially for cell imaging and manipulation [427]. There are works describing nanotechnology application in agriculture in the crop production in improving the yield and product quality [428][429][430][431][432]. On the other hand, plant viruses are considered as used in the nanotechnology, especially due to highly organized protein capsids, which are useful as scaffolds for building nanomaterials [433][434][435]. Tobacco mosaic virus has found applications as a building block for nanoelectronics as a template for metal deposition, mineralization and the deposition of the silica, such as nanowires and conductive films, as well as in light-harvesting systems [436][437][438][439][440]. Cowpea mosaic virus with its icosahedral protein coat shape can be utilized in non-invasive imaging, biosensors, and in vaccines [434,439,441]. Plant virus nanoparticles may be used in medicine in in vivo targeting and tumor targeting [442,443]. Based on the nanotechnology applications in the field of analytical chemistry [444][445][446][447][448], they represent the principal improvement of serological and immunofluorescence techniques in plant virus isolation and determination. In the field of PPV isolation and identification, there is an important requirement for PPV RNA of the highest quality. Therefore, methods of nanotechnology take advantage of nanoparticles on the basis of surface modifications possibility, which can significantly improve the quality of isolated RNA. Magnetic nanoparticles have been used for isolation and purification of nucleic acids; the use of magnetic nanoparticles provides very rapid and simple isolation of nucleic acids [449][450][451][452][453][454][455][456]. In addition, magnetic nanoparticled surface may be variously modified (these modifications include for example the introduction of silanol, epoxide, diol and carboxyl groups respectively). In comparison with traditional methods, the solid-phase process based on the interaction of nucleic acids with chemically-modified surface of magnetic nanoparticles is characterized by convenience, speed, timesaving, and being amenable to automation [457,458]. Using automation together with simple and rapid PPV RNA isolation under high specificity may represent great progress, especially due to the possibility of analyzing many samples in a short time period [459][460][461]. Generally, in the field of PPV detection, there is only limited number of publications focused on the use of nanoparticles in different isolation/detection steps. A paper by Byzova et al. introduces the possibility of using monoclonal antibodies as gold nanoparticle conjugates (26 nm in diameter) [462]. Using these conjugates with optimal ratio, an express immunochromatographic assay of PPV with a detection limit of 3 ng/ml and duration of 10 min. was developed [462]. Colloidal gold nanoparticles (5 -60 nm) as a carrier system conjugated with corresponding antibody are used in the paper by Safenkova et al. [463]. This paper demonstrates correlation between gold nanoparticles, respectively conjugate size and affinity. An increase of conjugate size leads to the increase in its affinity [463]. In conclusion, not only improvement of those methods used (ELISA, PCR) and the establishment of new detection techniques, but also the development of new, effective methods usable in PPV RNA isolation with subsequent routine detection, will bring new opportunities to routine PPV detection, and thereby provide the possibility of eradicating plum pox disease as an one of the most devastating viral diseases of stone fruits.