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Cloning and Sequencing of the Coat Protein Gene of a New Isolate of Citrus Tristeza Virus from Mexico

Citrus tristeza virus (CTV) causes one of the most important citrus diseases. CTV strains cause a wide range of symptoms in infected citrus worldwide. Although it has not yet affected Mexico's citrus industry, CTV constitutes a threat since one of its most efficient vectors, Toxoptera citricida, is migrating north from South America and is now in the Caribbean region. Efforts have been made to prevent spread of the virus, through early detection with serological methods (3). Use of the polymerase chain reaction (PCR), not yet a competitor of immunological methods in field diagnosis of CTV, offers a quick detection alternative. As a first step toward using PCR in early diagnosis and characterization of CTV, we searched for the CTV coat protein gene in experimentally infected leaves of Citrus aurantifolia grown at a government research station (INIFAP) at General Terán, NL. Researchers at General Terán had collected the isolates during a random sampling of orange orchards in the Gulf state of Veracruz. The original orange trees had no apparent symptoms and had been examined as a preventive measure. Several of the original orange trees were CTV positive in their initial tests with polyclonal antibody and in subsequent tests with CTV-specific monoclonal antibody MCA-13. Characteristic CTV-like particles had also been observed by electron microscopy. The only symptom induced in indicator plants was yellowing of the leaf veins. In our laboratory, reverse transcription of RNA from one of the indicator plants, coupled with PCR (RT-PCR) (1), was used to clone and sequence one of the isolates to confirm the CTV identification and establish PCR methods. Oligonucleotide primers were derived from published sequences, but unique restriction enzyme sites (EcoRI and XbaI) were added to their 5′ ends to facilitate cloning. To exclude artifacts, nucleotide sequences were obtained from both strands after cloning in M13 vectors. Although the expected RT-PCR product of 700 bp was obtained, an unexpected EcoRI site was found at position 678 of the coat protein gene. A phenylalanine residue was found at position 124, as in severe strains of CTV from various regions of the world (2). Similarities between our CT sequence (U32116) and those of other GenBank accessions are as follows: 91.17% for M76485; 90.14% for L12175; and 89.32% for S67800.

References: (1) M. D. Jones and N. S. Foulkes. Nucleic Acids Res. 17:8387, 1989. (2) H. Pappu et al. Proc. Natl. Acad. Sci. USA. 90:3641, 1993. (3) C. Vela et al. J. Gen. Virol. 67:91, 1986.