Bioinformatics Predicted Linear Epitopes of the Major Coat Protein of the Beet Yellows Virus for Detection of the Virus in the Cell Extract of the Infected Plant.

Beet yellows virus, which belongs to the genus Closterovirus, family Closteroviridae and has a significant negative economic impact, has proven to be challenging to detect and diagnose. To obtain antibodies against BYV, we propose an easier bioinformatics approach than the isolation and purification of the wild virus as an antigen. We used the SWISS-MODEL Workspace (Biozentrum Basel) protein 3D prediction program to discover epitopes of major coat protein p22 lying on the surface of the BYV capsid. Sequences coding these epitopes were cloned into plasmid pQE-40 (Qiagen) in frame with mouse dihydrofolate reductase gene. Fused epitopes were expressed in Escherichia coli and isolated by the Ni-NTA affinity chromatography. Murine antibodies were raised against each epitope and in a combination of both and characterized by dot-ELISA and indirect ELISA. We successively used these antibodies for diagnosis of virus disease in systemically infected Tetragonia tetragonioides. We believe the approach described above can be used for diagnostics of difficult-to-obtain and hazardous-to-health viral infections.

Polyclonal antibodies against potato spindle tuber viroid RNA.

Ribonucleic acid (RNA) can act as a hapten in the direct immunization of animals. For antigen synthesis, 65 mg of viroid RNA were obtained by in vitro transcription of the recombinant DNA. We received a reasonable immune response in mice and rabbits with synthesized conjugate viroid RNA-lysozyme. Analyses of polyclonal mouse and rabbit antisera as well as estimates of antibody specificity were performed by dot-Enzyme Linked Immunosorbent Assay (ELISA), sandwich ELISA, and northern immunoblotting. Antiserum obtained showed strong cross-reactions with cellular RNA. The viroid polyclonal antibody cross-reactions with cellular RNAs were depleted via titration antibodies by the plant cellular or commercial yeast RNA. We successfully used antibodies against the viroid RNA-lysozyme antigen to detect the wild-type potato viroid and diagnose potato viroid infection. We presume that intrinsic cross-reactions of RNA antibodies are potentially dangerous after nucleic acid vaccination. Research into the specificity of antibodies against viral RNAs is underway.

Environmentally friendly method of RNA isolation.

The diversity of organisms, tissues and cells is so great that, to date, no universal method for RNA extraction from these biological materials exist. The RNA isolation technique with a mix of guanidine thiocyanate, phenol, and chloroform is most widely used. Extraction and purification of RNA methods using selling guanidinium-phenol (TRIzol)-based and silica-based column kits have limitations on toxicity, or RNA isolation, particularly for plants, and scaling. The agents' toxicity is particularly relevant when employing for mass analysis in practice while gaining RNA preparations during the pandemics, epizootics, and epiphytotic. In modern diagnostics of infections at the molecular level, powerful RT-PCR technology is used, which amplifies the detection of RNA pathogens by hundreds of millions of times. We proposed obtaining RNA samples from viruses, bacteria, and plants for the reverse transcription reactions with a subsequent amplification of cDNAs by the polymerase chain reaction using potent and nontoxic chaotropic agent ammonium trichloroacetate. The method works in the analytical and preparative range and can be useful in the case of extraordinary circumstances during mass infections. Potentially this method can be adapted for obtaining RNA samples ready for the RT-isothermal PCR in the field.

Chimeric Virus Made from crTMV RNA and the Coat Protein of Potato Leafroll Virus is Targeted to the Nucleolus and Can Infect Nicotiana benthamiana Mechanically.

A genetically engineered chimeric virus crTMV-CP-PLRV composed of the crucifer-infecting tobacco mosaic virus (crTMV) RNA and the potato leafroll virus (PLRV) coat protein (CP) was obtained by agroinfiltration of Nicotiana benthamiana with the binary vector pCambia-crTMV-CPPLRV. The significant levels of the chimeric virus enabled direct visualization of crTMV-CP-PLRV in the cell and to investigate the mechanism of the pathogenesis. Localization of the crTMV-CP-PLRV in plant cells was examined by immunoblot techniques, as well as light, and transmission electron microscopy. The chimera can transfer between vascular and nonvascular tissues. The chimeric virus inoculum is capable to infect N. benthamiana mechanically. The distinguishing feature of the chimeric virus, the RNA virus with the positive genome, was found to localize in the nucleolus. We also investigated the role of the N-terminal sequence of the PLRV P3 coat protein in the cellular localization of the virus. We believe that the gene of the PLRV CP can be substituted with genes from other challenging-to-study plant pathogens to produce other useful recombinant viruses.

Chimeric Virus as a Source of the Potato Leafroll Virus Antigen.

Large quantities of potato leafroll virus (PLRV) antigen are difficult to obtain because this virus accumulates in plants at a low titer. To overcome this problem, we constructed a binary vector containing chimeric cDNA, in which the coat protein (CP) gene of the crucifer infecting tobacco mosaic virus (crTMV) was substituted for the coat protein gene of PLRV. The PLRV movement protein (MP) gene, which overlaps completely with the CP gene, was doubly mutated to eliminate priming of the PLRV MP translation from ATG codons with no changes to the amino acid sequence of the CP. The untranslated long intergenic region located upstream of the CP gene was removed from the construct. Transcribed powerful tobamovirus polymerase of the produced vector synthesized PLRV CP gene that was, in turn, translated into the protein. CP PLRV packed RNAs from the helical crTMV in spherical virions. Morphology, size and antigenic specificities of the wild-type and chimeric virus were similar. The yield of isolated chimera was about three orders higher than the yield of native PLRV. The genetic manipulations facilitated the generation of antibodies against the chimeric virus, which recognize the wild-type PLRV.

Production of platinum atom nanoclusters at one end of helical plant viruses.

Platinum atom clusters (Pt nanoparticles, Pt-NPs) were produced selectively at one end of helical plant viruses, tobacco mosaic virus (TMV) and potato virus X (PVX), when platinum coordinate compounds were reduced chemically by borohydrides. Size of the platinum NPs depends on conditions of the electroless deposition of platinum atoms on the virus. Results suggest that the Pt-NPs are bound concurrently to the terminal protein subunits and the 5' end of encapsidated TMV RNA. Thus, a special structure of tobacco mosaic virus and potato X virus particles with nanoparticles of platinum, which looks like a push-pin with platinum head and virus needle, was obtained. Similar results were obtained with ultrasonically fragmented TMV particles. By contrast, the Pt-NPs fully filled the central axial hole of in vitro assembled RNA-free TMV-like particles. We believe that the results presented here will be valuable in the fundamental understanding of interaction of viral platforms with ionic metals and in a mechanism of nanoparticles formation.

Highly sensitive field test lateral flow immunodiagnostics of PVX infection.

A test system is described and expanded upon for mass field immunochromatography assay on porous membrane carriers for rapid diagnostics of potato virus X (PVX) in potato leaf tissue and sprout extracts using colloidal gold nanoparticles as a marker. Sensitivity of the assay developed for PVX identification is found to be comparable to the sensitivity of solid-phase sandwich-ELISA. Complete assay time does not exceed 15 min, and the lower limit of the PVX detection in non-clarified leaf extract is 2 ng/ml. A single measurement requires 0.1-0.2 ml (3-5 drops) of tested solution only (extracted from 10-20 mg of potato leaf tissue or sprouts). The simplicity and reliability of the method makes it especially efficient in direct rapid monitoring of many infected potato specimens in the field, as verified by field trials of 360 clones of 28 domestic and foreign cultivars of potato. A diagnostic kit for routine analyses of potato viral infections both in the laboratory and in the field is described and expanded upon.

Immunolocalization of Picornavirus RNA in infected cells with antibodies to Tyr-pUp, the covalent linkage unit between VPg and RNA.

The genomic RNA of picornaviruses is attached to a small protein (VPg) via a covalent bond between a tyrosine and a 5'-terminal uridine phosphate. The same structure is present in potyvirus and calicivirus families. VPgs play a key role in initiation of viral replication by acting as primers for RNA synthesis. The model compound [N(Ac),CO(NHMe)]Tyr-(5'P→O)Up-O-(CH(2))(6)NH(2) (mCLU), mimicking this 'covalent linkage unit' (CLU) and containing Tyr-pUp was synthesized in solution following the phosphoramidite scheme and used to raise antibodies for studying picornavirus infection. The antibodies recognized CLU-containing mengovirus RNA and showed minimal cross-reactivity with RNAs lacking CLU. Immunofluorescence staining of cells infected with a human rhinovirus demonstrated co-localization of the signals from anti-mCLU and from anti-VPg antibodies. Efficient synthesis of mCLU and anti-mCLU antibodies might be of great utility for investigating viral replication and identifying yet unknown viral and cellular CLU-containing RNA-protein complexes.

Atomic force microscopy as a tool of inspection of viral infection.

Here we present a short review of application of atomic force microscopy (AFM) for investigation of viruses, accompanied by examples of high-resolution AFM images of different viral particles. The possibility of using AFM to identify viruses is discussed.