Prunus necrotic ringspot virus (PNRSV), Apple mosaic virus (ApMV) and Arabis mosaic virus (ArMV) are important rose viruses in the world. The aims of this study were to detect the viruses and evaluate their distribution in damask rose floricultures of the Isfahan, Kerman and Markazi Provinces. To do this, total 749 leaf samples were collected randomly from floricultures of the mentioned provinces in 2012-13. Presence of the viruses in the samples was checked by DAS-ELISA using specific antibodies against each of the viruses. The result of ELISA test on collected samples in 2012 showed that 4.41%, 2.20% and 6.3% of the samples were infected with ArMV, ApMV and PNRSV, respectively. These percentages for samples collected in 2013, were 2.31%, 5.32% and 4.16%. Because of wider distribution of PNRSV in comparison to ArMV and ApMV, COAT PROTEIN gene in four isolates of PNRSV was cloned and sequenced. Phylogenetic tree was drawn and recombination analyses were performed. Based on the phylogenetic tree, Iranian isolates were placed in PV96 phylogroup. This is the first report of genetic diversity of PNRSV in Iran and also the first incidence report of ArMV, ApMV and PNRSV in Isfahan and Markazi provinces.

The Prunus necrotic ring spot virus (PNRSV) poses a significant threat to the global stone fruit industry. In this study, samples suspected of PNRSV infection were gathered from Iran’s primary stone fruit-growing areas. The COAT PROTEIN (CP) gene of the isolates was amplified, and the nucleic acid and amino acid sequences of the PNRSVs were determined. Phylogenetic analysis revealed that these isolates belonged to the PV-96-II, a prominent group of PNRSVs found worldwide. An isolate from this clade was chosen to express the CP gene in Escherichia coli. The CP gene of this isolate was cloned into the pET28 vector for expression, and the resulting COAT PROTEIN was purified as a native PROTEIN using Ni-NTA agarose. The purified PROTEIN served as a recombinant antigen to generate anti-PNRSV-CP antiserum in rabbits. Purified anti-PNRSV-CP IgG and conjugated IgG showed specificity and sensitivity, successfully detecting expressed CP and PNRSV isolates in infected stone fruit trees through various serological and sero-molecular techniques such as plate-trapped antigen enzyme-linked immunosorbent assay (PTA-ELISA), Double Antibody Sandwich-ELISA (DAS-ELISA), and western blotting. These antibodies can be valuable in virus and plant screening studies, as well as serological and sero-molecular tests. This study represents the first documentation of a polyclonal antibody preparation against the CP of an Iranian PNRSV isolate, offering potential assistance in controlling and preventing this economically significant virus in the future.

Background and Aims: Serological assay is considered as one of the best choices for conducting large number of infection tests. Recombinant DNA technology has been used for expression of virus COAT PROTEIN (CP) gene in prokaryotic bacterial cells such as Escherichia coli and the recombinant CP (rCP) is used as immunogen in antibody production. Heterologous CP PROTEIN expression and purification of the full length Prunus necrotic ringspot virus-PNRSV, Ilarvirus genus, from an Iranian isolate as an antigen was the aim of the study. Materials and Methods: A predominant Iranian PNRSV isolate (PK5) was selected and its CP gene was amplified using specific primers and the nucleotide sequence has been determined. The amplicon was cloned into pET28a(+) expression vector. The amplified CP gene and linearized pET-28a(+) were purified from gel, ligated and transformed into BL21 strain of E. coli. Expression of rCP in transformed BL21 competent cells was tested using SDS-PAGE and Western Blot assays. Results: RT-PCR on total RNA extracted from the infected leaves resulted in a DNA fragment of approximately 688 bp corresponding to full PNRSV/CP. BLAST analysis of the obtained nucleotide sequence for PNRSV/CP revealed 97% identity to JW isolate (accession no. DQ983491). The size of pET-PNRSV/CP was about 6000 bp. The E. coli BL21 cells harboring recombinant pET-PNRSV/CP successfully expressed the recombinant CP after IPTG induction. Conclusions: In this study, the recombinant CP gene of a predominant Iranian PNRSV isolates expressed in E. coli. The recombinant CP can be used for producing high quality antibodies against PNRSV.

The use of plant viruses as carriers of anti-cancer drugs in medicine is increasing day after day. One of the most important potato viruses is Potato X Potexvirus (PVX) which is globally expanded and is one of the first plant viruses used as a carrier of anticancer drugs. In this research, in order to produce PVX particles, its COAT PROTEIN (CP) gene was expressed. First, the RT-PCR reaction was performed for CP amplification using specific primers containing the endonuclease digestion sites. Electrophoresis of the PCR product on agarose gel confirmed the amplified fragment with a length of 714 bp. Then the CP gene and pGBKT7 plasmid were digested by BamHI and EcoRI enzymes and after purification, ligation was done between them. Recombinant plasmid pGBKT7-CPPVX was transferred to E. coli bacteria by electroporation. After selecting the bacteria containing the recombinant plasmid by PCR and extracting the plasmids from them, sequencing confirmed the correctness of the cloned gene sequence. Then, the CP gene was cloned into pET-21a expression plasmid by digestion and ligation method. The recombinant plasmid was transferred to BL21 strain of E. coli and cultured. By adding IPTG in the bacterial culture medium, CP PROTEIN was expressed from the plasmid and bacterial PROTEINs were extracted. PROTEIN electrophoresis showed the expression of CP. After purification, this PROTEIN can be used as a carrier of anti-cancer drugs.

During the spring of 2011, several symptomatic hosta plants Hosta sieboldiana with mosaic, mottling and leaf deformation were observed in commercial hosta propagation greenhouses and gardens in Karaj and Tonekabon regions in Iran. A total of 17 symptomatic samples were collected and tested for the presence of Arabis mosaic virus-ArMV, Cucumber mosaic virus-CMV, Tomato spotted wilt virus-TSWV, Impatiens necrotic spot virus-INSV and Tomato ring spot virus-ToRSV by DAS-ELISA method. None of the tested samples had positive reaction with any of the antibodies. Some of symptomatic samples induced necrotic local lesions on Gomphrena globosa indicator plant in mechanical inoculations without inducing any symptom on other inoculated plants. Samples were tested for Hosta virus X (HVX) infection by reverse transcriptase polymerase chain reaction (RT-PCR) using specific primers designed for COAT PROTEIN (CP) gene in this study and a DNA fragment with the expected size about 700 bp was amplified. CP sequences of Iranian HVX isolates showed 98.5 – 99.5 % nucleotide identity with 55 other non-Iranian HVX isolates. This is the first report of HVX occurrence in Iran and CP nucleotide sequence of five Iranian HVX isolates was determined.

Citrus tristeza virus (CTV) is among the most destructive pathogens of citrus and causes substantial economic losses to citrus industry worldwide. Considering recent distribution of this pathogen and its capability of transmission by existing aphid vectors in Iran, detection of this virus should be mandatory for controlling the damage caused by this pathogen in Iran, as one of the major citrus producing countries. Toward this aim, developing a reliable and sensitive detection method such as enzyme- linked immunosorbant assay (ELISA) would be the first step to detect CTV in large scale screenings of field samples. As the serological method requires great amounts of specific antibody, consequently preparation of a large scale antigen source for immunization process would be necessary. In this study the COAT PROTEIN gene of CTV (CP25) was amplified by polymerase chain reaction from a cloned CP25 gene in pTZ57R/T and subcloned in pET26b expression vector and named pET-CP25. Two Escherichia coli strains of BL21 and Rosetta Gami (DE3) were transformed by pET-CP25. Expression of recombinant PROTEIN was induced by IPTG. The authenticity of recombinant PROTEIN was confirmed by western immunoblot analysis using a polyclonal antiserum against CTV particles. The results indicated that CTV COAT PROTEIN gene was expressed in E. coli. This recombinant PROTEIN could be used as a source of antigen for immunization process.

Iranian wheat stripe virus (IWSV) is a tentative member of the genus Tenuivirus. The COAT and nonstructural PROTEINs (CP and NS4, respectively) are the most common PROTEINs of tenuiviruses encoded by the vcRNA3 and vRNA4 segments, respectively. The specific antibodies against those PROTEINs can be used for detection of the viruses and investigation of functions of the related genes. To express CP and NS4 of IWSV, the related genes were amplified using specific primers and cloned into the expression vector pET 28a. Recombinant plasmids were used to transform Escherichia coli strain BL21 (DE3). The transformed bacteria were cultured in IPTG-induced media. SDS-polyacrylamide gel electrophoresis showed two bands with molecular weights 40.9 and 23 kDa which corresponded to the molecular weight of the putative products of the related genes in tenuiviruses. The expression of target PROTEINs was confirmed using specific polyclonal antibodies by western blot analysis. The large-scale production of highly purified PROTEINs by a microbial system represents pure antigen free from the contaminating plant materials that can be used to produce the related antibodies.

Background: The advent of recombinant DNA technology has facilitated heterologous expression of PROTEINs from varioussources in different host systems including Escherichia coli. If a plant virus COAT PROTEIN is expressed in the bacteriumit can be used as the antigen for antibody preparation. Such a recombinant antigen preparation can be particularly usefulwhere equipment such as ultracentrifuge is unavailable to purify virus particles to use as the antigen for conventional antibodypreparation.Objective: Heterologous PROTEIN expression and purification of the full length Potato virus Y (PVY) COAT PROTEIN (CP)from isolate pot187 (an affiliate of strain N) to be used as an antigen was the aim of the study.Materials and Methods: Reverse transcription Polymerase Chain Reaction (RT-PCR) was carried out to amplify an 801bp fragment of the CP gene from PVY-infected potato leaves. The amplicon was cloned into pGEM-T Easy. The clonedfragment was restricted by BamHI+SacI and the purified fragment was cloned into the expression vector pET21a (+)which was restricted with the same enzymes. The generated plasmid was introduced into E. coli strain RosettaTM. Theexpression was induced with isopropyl-b-D-thiogalactopyranoside (IPTG) and its PROTEIN content was subjected to SDSPAGEand western blotting.Results: SDS-PAGE analysis of PROTEIN from the induced bacteria showed a ~35 KDa PROTEIN corresponding to PVY CP.Expression of the recombinant PROTEIN was confirmed by anti-His anitibody.Conclusions: The full-length cDNA of PVY-CP was amplified from the infected potato leaves. The cDNA was heterologouslyexpressed in E. coli. The produced recombinant CP can be used as an antigen to generate polyclonal antibody.

There are many studies related to the production of a ELISA kit for diagnosing virus infections. However, production of most kits depends on purification of whole virus particles, which involves the use of costly equipment and reagents. The purpose of this study was to check out if the anti-CP42 antibodies could be used as a diagnostic assay for detection of Grapevine fanleaf Virus (GFLV). In this study, recombinant GFLV COAT PROTEIN gene related to selected antigenic determinants was inserted into pET-28a bacterial expression vector and the construct (pET-28a CP42) was cloned into E. coli strain (DE3). Expressed PROTEIN was verified with western blotting assay by the use of commercially available anti-GFLV antibody. The recombinant PROTEIN was purified using nickel– nitrilotriacetic acid (Ni– NTA) resin. Balb/c mice were immunized with purified PROTEIN and splenocytes of hyperimmunized mice were fused with murine myeloma Sp2/0 cells. Positive hybridomas were selected by ELISA using CP42 as COATing antigen. The results showed that monoclonal antibody (MAb) specific to CP42 has been successfully generated. Efficiency of produced antibody was analyzed by ELISA and western blotting assay using some confirmed grapevine samples. The infection was confirmed previously based on morphological features and ELISA assay, performed using commercial anti-GFLV antibody. The monoclonal antibody reacted with antigen in ELISA and immunoblot method. Our results demonstrated that anti recombinant CP42 monoclonal antibodies are able to diagnose whole virus in infected grapevine sample using ELISA test.