Nucleotide heterogeneity at the terminal ends of the genomes of two California Citrus tristeza virus strains and their complete genome sequence analysis

The terminal nts of the viral genomes were identified by sequencing cDNA clones of the plus- and/or minus-strand of the viral double-stranded (ds) RNAs generated using 5′ and 3′ rapid amplification of cDNA ends. Cloned cDNAs corresponding to the complete genome sequences of both viruses were generated using reverse transcription-polymerase chain reactions, sequenced, and subjected to phylogenetic analysis.

Among the predominant terminal nts identified, some were identical to the consensus sequences in GenBank, while others were different or unique. Remarkably, one of the predominant 5′ nt variants of T36-CA contained the consensus nts “AATTTCAAA” in which a highly conserved cytidylate, seen in all other full-length T36 sequences, was absent. As expected, but never systematically verified before, unique variants with additional nt (s) incorporated upstream of the 5′ terminal consensus nts of T36-CA and T30-CA were also identified. In contrast to the extreme 5′ terminal nts, those at the extreme 3′ termini of T36-CA and T30-CA were more conserved compared to the reference sequences, although nt variants were also found. Notably, an additional thymidylate at the extreme 3′ end was identified in many T36-CA sequences. Finally, based on pairwise comparisons and phylogenetic analysis with multiple reference sequences, the complete sequences of both viruses were found to be highly conserved with those of the respective genotypes.

The extreme terminal nts in the T36-CA and T30-CA genomes were identified, revealing new insights on the heterogeneity of these CTV genomic regions. T36-CA and T30-CA were the first and the second genotypes, respectively, of CTV originating from California to be completely sequenced and analyzed.