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Cucumber mosaic cucumovirus (CMV) was isolated from lettuce plants (Lactuca sativa)showing virus like symptoms. Isolation was performed depending on specific polyclonal antibodies and Chenopodium quinoa as a local lesion host. Virus was purified from 200 gm of virus-infected Nicotiana tabacum cv. White Burley leaves giving A 260/280 ratio of 1.21 and a yield of 1.7 mg. Purified virus preparation was used for rabbit immunization to produce specific polyclonal antibodies. IgGs were purified and evaluated by indirect enzyme linked immunosorbant assay (I-ELISA) to determine the dilution end point which found to be 1:512. Electron micrographs showed spherical virus particles of about 30 nm in diameter. Virus coat protein (CP) molecular weight was determined using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), giving a single band of 25 kDa within resolving gel. Immunocapture-reverse transcriptase-polymerase chain reaction (IC-RT-PCR) was used for the amplification of CMV coat protein gene (cp), the appearance of 657 bp bands confirmed the expected size of such gene. Comparing virus cp gene sequence with the sequences of seven overseas isolates confirmed that the under study isolate was related to the CMV subgroup I. ª 2013 Production and hosting by Elsevier B.V. on behalf of Faculty of Agriculture, Ain Shams University.

Plant viruses cause serious disease of crop plants reducing both quality and quantity of final produce. Serological tests are used all over the world in laboratory and field based detection of plant viruses and they are of great indispensable importance in agricultural production; virus certification programs; agricultural quarantine and production of virus-free crops grown for processing or fresh market. Cross reaction between viruses and their strains antisera limits serological differentiation of viruses and their strains by enzyme-linked immunosorbent assay (ELISA). This study aims to characterize the antigenic property of Tomato mosaic virus ToMV coat protein by using some bioinformatic tools to analyze its gene. Therefore, new methods in antibody production could be used as equivalent to Mabs in its high specificity. ToMV isolate was confirmed by Transmission electron microscope and differential hosts and propagated on N. tabacum cv. Samson. Systemic infection was developed on N. tabacum cv. Samson and local infection on Datora metel; D. stramonium; N. glutinosa; Chenopodium amaranticolor; C. quinoa. ToMV was purified and used as immunizing agent for antiserum production. TEM showed rod shaped particles with 300 x 18 nm dimensions. The titer of produced antiserum was 1:1024 evaluated by microprecipitin test and indirect-ELISA. Coat protein gene was amplified by RT-PCR with expected size of (Approx. 500 bp). The PCR product was sequenced then the generated nucleotide sequence was translated into 160 amino acids. The amino acid sequence of Five B-cell epitopes, of 14 amino acid residues each, were predicted. Identifying Bcell epitopes play an important role in vaccine design, immunodiagnostic tests, and antibody production. Therefore, computational tools for reliably predicting B-cell epitopes are studied.

Cucumber mosaic virus (CMV, cucumovirus) is the most important virus infecting cucurbit crops in Egypt and worldwide causing significant loss in yield quality and quantity. The main target of the present work was to establish a simple controlling system for an Egyptian isolate of such virus (belonging to the subgroup I) via production of tobacco transgenic plants expressing viral coat protein (CP). Coat protein gene (cp) was isolated and amplified using immunocapture-reverse transcriptase-polymerase chain reaction (IC-RT-PCR) and primers with add-on restriction sites for SmaI and SacI enzymes. The genes were cloned in pBI121 vector plasmid between the CaMV 35S promoter and the nos terminator after removing the Gus gene by restriction enzymes digestion. The new construct was used for Agrobacterium tumefaciens transformation, which was then used for tobacco transformation. Evaluation of transformation success and CP expression degree were confirmed using indirect enzyme-linked immunosorbent assay (I-ELISA) and dot blot immuno-binding assay (DBIA). PCR and RT-PCR were used to study the integration of cp within genetic plant system and to what extent this gene was transcript. It was concluded that in spite of integration success some transformed plants can transcript the gene more than the others do. Plants resistance was tested by challenging with CMV under study and remarkable success was obtained in plants with higher gene transcription and translation degree.