Molecular characterization of a cucumber mosaic cucumovirus isolated from lettuce in Egypt

Abstract

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<inf>260/280</inf> 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.