Sequences of Citrus tristeza virus separated in time and space are essentially identical

Abstract

The first Citrus tristeza virus (CTV) genomes completely sequenced (19.3-kb positive-sense RNA), from four biologically distinct isolates, are unexpectedly divergent in nucleotide sequence (up to 60% divergence). Understanding of whether these large sequence differences resulted from recent evolution is important for the design of disease management strategies, particularly the use of genetically engineered mild (essentially symptomless)-strain cross protection and RNA-mediated transgenic resistance. The complete sequence of a mild isolate (T30) which has been endemic in Florida for about a century was found to be nearly identical to the genomic sequence of a mild isolate (T385) from Spain. Moreover, samples of sequences of other isolates from distinct geographic locations, maintained in different citrus hosts and also separated in time (B252 from Taiwan, B272 from Colombia, and B354 from California), were nearly identical to the T30 sequence. The sequence differences between these isolates were within or near the range of variability of the T30 population. A possible explanation for these results is that the parents of isolates T30, T385, B252, B272, and B354 have a common origin, probably Asia, and have changed little since they were dispersed throughout the world by the movement of citrus. Considering that the nucleotide divergence among the other known CTV genomes is much greater than those expected for strains of the same virus, the remarkable similarity of these five isolates indicates a high degree of evolutionary stasis in some CTV populations.

FIG. 1

cDNA cloning strategy for mild CTV isolate sequences. (A) Diagram of the organization of the CTV T30 genome (19,259 nt). PRO, papain protease-like domain; MT, methyltransferase-like domain; HEL, helicase-like domain. (B, top panel) Schematic representation of the localization of the oligo(dT)-primed random T30 cDNA clones, the naturally occurring dRNA in isolate T30 (DI) (M. Mawassi et al., unpublished data), and the cDNA clone T308 (20) used to design T30-specific primers. (B, bottom panel) Schematic representation of the localization and size of the eight overlapping RT-PCR T30 cDNA clones used to determine the T30 genomic sequence. (C) Schematic representation of the localization of RT-PCR CTV clones A and B and locations (solid boxes) of the four hypervariable regions sequenced for comparing CTV isolates B252, B272, and B354 with T30 and T385. These CTV cDNA clones were obtained as indicated in Material and Methods.

FIG. 2

Nucleotide sequence alignments of CTV hypervariable genomic regions (ClustalW program alignments; see Materials and Methods). (A) Alignment of variable region I (5′ NTR, 108 nt) sequences. (B) Alignment of variable region II (located from nt 109 to nt 324 of the T30 genome in the 5′-proximal region of the replicase gene) sequences. (C) Alignment of variable region III (located from nt 3324 to nt 3706 of the T30 genome in the putative methyltransferase domain) sequences. (D) Alignment of variable region IV (located from nt 8265 to nt 8627 of the T30 genome in the putative helicase domain of the replicase ORF) sequences. In all panels, nucleotide differences in aligned sequences are showed in boldface. +, nucleotide differences between T30 and the mild CTV isolates T385, B252, B272, and B354; ∗, nucleotide differences between T30 and severe isolates T36 and VT.

FIG. 2

Nucleotide sequence alignments of CTV hypervariable genomic regions (ClustalW program alignments; see Materials and Methods). (A) Alignment of variable region I (5′ NTR, 108 nt) sequences. (B) Alignment of variable region II (located from nt 109 to nt 324 of the T30 genome in the 5′-proximal region of the replicase gene) sequences. (C) Alignment of variable region III (located from nt 3324 to nt 3706 of the T30 genome in the putative methyltransferase domain) sequences. (D) Alignment of variable region IV (located from nt 8265 to nt 8627 of the T30 genome in the putative helicase domain of the replicase ORF) sequences. In all panels, nucleotide differences in aligned sequences are showed in boldface. +, nucleotide differences between T30 and the mild CTV isolates T385, B252, B272, and B354; ∗, nucleotide differences between T30 and severe isolates T36 and VT.

FIG. 2

Nucleotide sequence alignments of CTV hypervariable genomic regions (ClustalW program alignments; see Materials and Methods). (A) Alignment of variable region I (5′ NTR, 108 nt) sequences. (B) Alignment of variable region II (located from nt 109 to nt 324 of the T30 genome in the 5′-proximal region of the replicase gene) sequences. (C) Alignment of variable region III (located from nt 3324 to nt 3706 of the T30 genome in the putative methyltransferase domain) sequences. (D) Alignment of variable region IV (located from nt 8265 to nt 8627 of the T30 genome in the putative helicase domain of the replicase ORF) sequences. In all panels, nucleotide differences in aligned sequences are showed in boldface. +, nucleotide differences between T30 and the mild CTV isolates T385, B252, B272, and B354; ∗, nucleotide differences between T30 and severe isolates T36 and VT.

FIG. 2

Nucleotide sequence alignments of CTV hypervariable genomic regions (ClustalW program alignments; see Materials and Methods). (A) Alignment of variable region I (5′ NTR, 108 nt) sequences. (B) Alignment of variable region II (located from nt 109 to nt 324 of the T30 genome in the 5′-proximal region of the replicase gene) sequences. (C) Alignment of variable region III (located from nt 3324 to nt 3706 of the T30 genome in the putative methyltransferase domain) sequences. (D) Alignment of variable region IV (located from nt 8265 to nt 8627 of the T30 genome in the putative helicase domain of the replicase ORF) sequences. In all panels, nucleotide differences in aligned sequences are showed in boldface. +, nucleotide differences between T30 and the mild CTV isolates T385, B252, B272, and B354; ∗, nucleotide differences between T30 and severe isolates T36 and VT.