Extraction and molecular detection of viral dsRNA from different infected plants

Extraction of viral double stranded RNA (dsRNA) from infected plants is helpful in identification of the viruses involved in infection. To date, there have been several methods developed to isolate dsRNA; however, type of the plant and virus is determinative in extraction efficiency. In this study we extracted dsRNA from different woody and herbaceous plants through a modified method which reduces the costs and time of extraction procedure. This method is based on different affinity of nucleic acids for the cellulose CF-11 in1X STE (Sodium chloride Tris EDTA) buffer containing 16 % ethanol. There is no phenol treatment or mini columns used in the isolation procedure. Extracted dsRNAs were identified by ribonuclease treatment and RT-PCR (Reverse Transcriptase-Polymerase Chain Reaction). We have applied the procedure on five different hosts representing Amaranthaceae, Vitaceae, Fabaceae and Rosaceae infected with four different viruses representing Secoviridae and Bromoviridae.&nbsp

Molecular detection of Grapevine fanleaf Virus by the isolation of ssRNA and dsRNA from Xiphinema index

Xiphinema index is an important grapevine pathogen nematode which also vectors Grapevine fanleaf virus. The viral genes involved in transmission by the vector nematode are mapped to the C-terminal residues of RNA2-encoded polyprotein. To recognize viruliferous nematodes, there are some serological and molecular methods. In this study, we extract RNA and dsRNA of the virus, then Reverse transcription-polymerase Chain Reaction was done with virus specific primers to detect virus in its vector. The virus was detected by visualizing the desired 350 and 750 bp gene fragments in electrophoresis. This study reduces the virus detection time to only couple of hours with least imposed charges, and could be employed in transmission experiments as well.&nbsp

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Absract A pair of degenerate primers, GMPF1 and GMPR1, was designed on the basis of alignment of previously reported Grapevine fanleaf virus (GFLV) movement protein (MP) nucleotide sequences from Iran and other parts of the world. cDNA was synthesized by the use of Oligo d(T)18 from total RNA extraction from each diseased grapevine leaf sample and subjected to polymerase chain reaction (PCR) with the degenerate primers under a range of annealing temperatures from 48 to 62°C. It was revealed that 55°C gave the best result in terms of producing exactly the expected fragment (1044 bp) from as many samples as possible although accompanied by few fade non specific fragments. However, by application of "hot-start" PCR and annealing at 60°C the specific fragment was amplified from 41 out of 86 samples. This was the first amplification of the precise MP cDNA from GFLVs in Iran which is very important as to preparation of recombinant anti-GFLV MP antibody to use in studying the GFLVgrapevine interaction, and also for generating pathogen-derived resistant vines

Serological Methods to Confirm Expression of Coat Protein Gene From an Iranian Isolate of Cucumber Mosaic Virus in Escherichia coli

Background: Cucumber mosaic virus (CMV) has isometric particles with a diameter of about 28 - 29 nm. Detection and prevention are the critical steps in the control of plant viruses. Detection in a large number of samples is still done by serological methods due to their robustness and perhaps low cost. Objectives: To this end, our aim was to express the CMV CP gene in E. coli to be used as the antigen for antibody production in the future. Materials and Methods: Coat Protein (CP) gene cDNA from an isolate (B13) of Cucumber Mosaic Virus (CMV) was subcloned from pTZ57RCMVCP to pET21a expression vector and transformed to E. coli strain Rosetta. Expression of CMV CP was successful and confirmed by Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE), wherein a ~30- kDa protein band was revealed. Induction by Isopropyl-Thiogalactoside (IPTG) at final concentrations of 0.5 to 2 mM appeared to produce similar results as to the amount of the expressed protein, which was judged by intensity of the band on SDS-PAGE. Results: The identity of the expressed protein was confirmed by immunoassays such as western blot, Dot-Immunobinding Assay (DIBA) and Enzyme-Linked Immunosorbent Assay (ELISA) by the use of anti-CMV antibody. Conclusions: This is the first report of expression of CMV CP gene in Iran, which is important for the preparation of anti-CMV antibody and paving the way for the use of the virus coat protein as a nanomaterial. Keywords: Methods; Cloning; Expression; Gene; Escherichia col

Genomic characterization of Ambrosia asymptomatic virus 1 and evidence of other Tymovirales members in the Oklahoma tallgrass prairie revealed by sequence analysis

The Plant Virus Biodiversity and Ecology project was undertaken to better understand the nature of plant-viral interactions and the occurrence of non-pathogenic viruses. Plants from the Tallgrass Prairie Preserve (TPP), Osage County, Oklahoma, were surveyed from 2005 to 2008 for the presence of viruses, resulting in the detection, using a virus-like particle enrichment method, of the genome a novel virus, Ambrosia asymptomatic virus 1 (AAV1), from Ambrosia psilostachya DC (western ragweed). Here, we present the genomic organization and genetic variability of AAV1. The virus has a single-stranded RNA genome of about 7408 nt, which has six open reading frames (ORFs). Phylogenetic analysis of the replicase and coat protein ORFs of the virus indicates strongly that the virus should be placed in the genus Mandarivirus. No evidence of recombination was detected. We also report the detection in the TPP of two known viruses and seven other putative viruses, members of the order Tymovirales

Production of Polyclonal Antibody against Grapevine fanleaf virus Movement Protein Expressed in Escherichia coli

The genomic region of Grapevine fanleaf virus (GFLV) encoding the movement protein (MP) was cloned into pET21a and transformed into Escherichia coli strain BL21 (DE3) to express the protein. Induction was made with a wide range of isopropyl-β-D-thiogalactopyranoside (IPTG) concentrations (1, 1.5, and 2 mM) each for duration of 4, 6, or 16 h. However, the highest expression level was achieved with 1 mM IPTG for 4 h. Identity of the expressed protein was confirmed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western blotting. The expressed 41 kDa protein was purified under denaturing condition by affinity chromatography, reconfirmed by Western blotting and plate-trapped antigen enzyme-linked immunosorbent assay (PTA-ELISA) before being used as a recombinant antigen to raise polyclonal antibodies in rabbits. Purified anti-GFLV MP immunoglobulines (IgGs) and conjugated IgGs detected the expressed MP and GFLV virions in infected grapevines when used in PTA-ELISA, double antibody sandwich-ELISA, and Western blotting. This is the first report on the production of anti-GFLV MP polyclonal antibodies and application for the virus detection

Detection of Russian olive witches’-broom disease and its insect vector in Northwestern Iran

Recently, Russian olive trees showing witches’-broom and little leaf symptoms have been widely observed in northwestern and central Iran. Polymerase chain reaction (PCR) and nested PCR assays using phytoplasma universal primer pairs confirmed phytoplasma symptomatic infection of trees. Sequence analyses showed that ‘Candidatus Phytoplasma asteris’ was the causal agent of the disease in these regions. However, RFLP results using restriction enzymes HpaII, EcoRI, HinfI and AluI indicated that the collected isolates in these regions are genetically different. In addition, leafhopper Macropsis infuscata was recognized as a possible insect vector of the disease for the first time