1957-2010 ) Title Studies on the Coat Protein Genes of Four Isolates of Citrus Tristeza Closterovirus from India : Cloning , Sequencing and Expression Permalink

Four isolates of citrus tristeza closterovirus (CTV) from India causing stem pitting and vein flecking symptoms in lime, lemon, citron, sweet orange and grapefruit were used in this study. Two isolates caused severe stem pitting in mandarins, rough lemon and Cleopatra mandarin. The CTV coat protein gene (CPG) was selectively amplified by polymerase chain reaction from infected tissue extracts, cloned into the pUC 118 phagemid vector and sequenced. There was more than 90% similarity at the nucleotide level and above 95% at the deduced amino acid level among the sequences of these isolates. When compared to T36, a severe isolate from Florida, the same amino acid changes were found consistently at seven positions in all four Indian isolates. The coding region of the CPG of isolate B227 was expressed in E. coli BL21 (DE3) pLysS using the pETH3a vector. The identity of the E. coli expressed coat protein (ECP) was confirmed by Western blot analysis using polyclonal antibodies and MCA-13, a monoclonal antibody. Polyclonal antibodies raised against partially purified ECP expressed at 29 C reacted well with ECP and with CTV-infected tissue. The ECP can be produced in large quantities and can serve as a good source of antigen for production of antibodies and for use as a positive control in serological reactions. Citrus tristeza virus (CTV) causes one of the most destructive disease syndromes of citrus. Quick decline on sour orange rootstock and stem pitting are two major components of the disease. Many new destructive isolates continue to be reported (12). The virus is spread by many aphid spp. , Toxoptera citricida (Kirk.) being the most efficient (1). The virus is phloem-limited and the particles are approximately 2000-nm long flexuous rods consisting of a single type of coat protein with M, of 26,000 (5,8,14) and agenome consisting of a single, positive strand RNA of about 20,000 nucleotides (1). The CPGs of several biologicallydistinct CTV isolates have been sequenced (8,14). Information on amino acid and nucleotide sequences of different CTV isolates will be useful in developing strain specific antibodies and nucleic acid probes and in understanding the different host reactions caused by various isolates of CTV. Four severe Mention of a trademark, warranty, property product, or vendor does not constitute a guarantee by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products or vendors that may also be suitable. isolates of CTV from India used in this study caused vein flecking and stem pitting on many tolerant varieties such as mandarin, rough lemon and cleopatra mandarin (6). Rapid detection techniques are important in prevention of the disease by the use of disease free budwood and in the eradication of the disease. Enzyme linked immunosorbent assay (ELISA) is being used widely for detection of the virus in large citrus areas worldwide (3). Production of polyclonal antibodies to various isolates of CTV is often limited by various factors including production of sufficient quantities of infected tissue, international quarantine, low yields of virus, contamination by host proteins etc. To circumvent these obstacles, the CPG was cloned in an expression vector and the protein was expressed in E. coli cells. Antibodies were raised against the purified protein and tested for their efficacy in detecting the virus in the in-