Nucleotide Sequence Assessment of Four ORFs of Citrus Tristeza Virus: Evidence of Recombination

Citrus Tristeza Virus (CTV), usually occurs in nature as a mixture of genotypes. Six naturally infected citrus(Citrus sinensis) trees grafted on sour orange rootstock were collected from three citrus growing governoratesin Egypt (Sharqia, Qalyubia and Garbia). In this study, RT-PCR, Single-Strand Conformation Polymorphism(SSCP) and nucleotide sequence analysis were used for four independent CTV genomic regions (p65, p18,p20, and p23) to detect and assess the sequence and genetic variabilities among CTV Egyptian isolates. RTPCR products (650 bp) for the CTV p23 gene obtained from the selected isolates were used for the SSCP analysisand DNA sequencing. SSCP patterns of p23 gene for individual isolates yielded different complex haplotype patterns. Nucleotide sequence analysis of p23 region amplified from six isolates under study revealed that p23 sharedhigh nucleotide identity 98.7% with T36 isolate from USA, Florida. Phylogenetic analysis of p23 gene indicated aclose evolutionary relationship between all examined isolates and Qaha isolate (T36 isolate group), suggesting thatthey may have originated from closely related ancestors. Nucleotide sequence analysis of the three genes locatedon CTV 3′-coterminal overhang, p18, p20 and p65, amplified from isolate A3, Sharqia governorate, revealed thatthe p18, p65, and p20 genes were related to the T3-KB isolate from South Africa with 99%–100% sequencehomology. Phylogenetic relationship analysis for p65, p18 and p20 ORFs clustered the current A3 isolate withT3 genotype group. The recombination analysis identified three of six isolates from Sharqia, and Garbia as potential recombinant for p23 gene. The isolates T36 and T3 were identified as major donors for recombination eventsin isolate A3. Our results concluded that p23 ORF likely to be as a hotspot region for recombination and originated through recombination event. The current study indicated that recombination is an important factor for theorigin of CTV strains in Egypt.