A Short Note on Reflections and Publications on Citrus tristeza virus (CTV) Methodologies.

My PhD thesis work of Citrus tristeza virus (CTV) purification was aimed to develop a rapid serological assay to replace biological indexing. The task turned difficult and was achieved after a lengthy struggle, rewarded by allowing (1) the rapid diagnosis of the first incidences of natural spread of a severe CTV-VT strain in our region and (2) finding that the CTV particle isolation protocol, with some modifications, was also useful for Beet yellows virus (BYV) particles, leading to their assignment in the Closterovirus group, the first group of elongated plant viruses with different modal lengths. Later, following the introduction of ELISA for large-scale diagnosis of tristeza-infected citrus trees, the CTV infection rates through the coastal citrus production areas were continually increasing, with many ELISA-positive samples appearing symptomless, prompting the need to develop strain-specific assays. Using CTV-VT cDNA fragments, as hybridization probes, the genetic diversity among local CTV isolates was demonstrated. With the emergence of the PCR technology, we developed a CTV-dsRNA cloning method based on the ligation of known oligonucleotide molecules to dsRNA ends and the use of complementary oligonucleotides for cDNA synthesis and PCR amplification. The method allowed the cloning of a cDNA molecule complementary to a defective dsRNA of 2.4 kb with intact 5 and 3 ends of the CTV-VT genome. A list of publications, resulting from continuous collaborative work with local and foreign associates and students on the development and adaptation of novel CTV methodologies, is present.