Citrus Virologists Conference Proceedings ( 1957-2010 ) Title Application of Bi-Directional PCR to Citrus Tristeza Virus : Detection and Strain Differentiation Permalink

The monoclonal antibody MCA13 reacts predominantly with severe strains of citrus tristeza virus (CTV) occurring in many regions of the world. Its specificity is based on a single nucleotide (AIT) difference a t position 371 of the capsid protein gene (CPG). Based on this single nucleotide difference, we designed two internal primers, one specific for mild strains and the other specific for severe strains of CTV. These primers, along with two terminal primers for the ends of the CPG, were used to develop a bi-directional, reverse transcription/polymerase chain reaction (BDPCR) to differentiate mild and severe strains of CTV. Under our standard PCR conditions, a full length CPG (-700 bp) and a 400 bp DNA fragment were produced with mild strains, and a 700 and a 300 bp DNA fragment were produced with severe strains. With a mixture of mild and severe strains, the 700,400 and 300 bp DNA fragments were all produced. Reducing both the terminal primer concentrations and extension times eliminated the 700 bp fragment. Thus, this method can be manipulated for different purposes. Tests of BDPCR with samples from naturallyinfected trees indicated that BDPCR were more sensitive than ELISA, because BDPCR amplified a 300 bp fragment from some samples which were MCA13 negative by ELISA tests. Citrus tristeza virus (CTV), the causal agent of tristeza disease of citrus, is the most destructive and economically important virus of commercial citrus worldwide (2,lO). It is a phloem limited closterovirus whose virions are thread-like filamentous particles, about 2000 x 11 nm in size (1, 3). The molecular weight of the positive sense, singlestranded RNA (ssRNA) genome of CTV was estimated to be about 6.5 x lo6 Daltons (3). The recently completed sequence of strain T36 from Florida revealed the genome size to be 19,296 nucleotides (9). There are many strains of CTV and they cause distinct symptoms on different hosts. In Florida, mild strains cause no noticeable symptoms on commercially grown varieties, whereas the severe strains cause decline on trees grafted on sour orange rootstock. Some severe strains may also induce a seedling yellow reaction and lor stem pitting on some grapefruit or sweet orange scions. The monoclonal antibody MCA13 reacts predominantly with severe (decline inducing andlor stem pitting) strains of CTV from many regions of the world. However, some severe strains from Spain, Israel and California and most mild strains generally fail to react with MCA13. Thus, MCA13 (16) is now commonly used as a first step for differentiation of mild and severe strains of CTV in Florida, South America and the Caribbean region. The molecular basis for the MCA13 structural epitope has been described (12, 13, 14). Analysis of capsid protein (CP) amino acid sequences of a number of mild and severe strains of CTV revealed a consistent difference at amino acid position 124. The amino acid was always tyrosine (Y) in mild strains, while it was always phenylalanine (F) in severe strains. When the Y in a mild strain and the F in a severe strain were changed to F and Y, respectively, by site-directed oligonucleotide mutagenesis of the capsid protein gene (CPG), the mutant mild strain protein reacted with MCA13, whereas the mutant severe strain protein did not react with MCA13. The reactivity of MCAl3 is thus controlled by a single nucleotide (A-T) change at position 371 of CTV CPG