**3.** *Citrus tristeza virus* **sequence diversity**

Sequencing the complete genome of CTV was the first breakthrough towards the study of CTV genetics [2]. Actually, there are twenty CTV genomic sequences available. These are T36 and T30 from Florida [2, 27]; VT from Israel [28]; SY568R from California [29, 30]; T385 and T318A from Spain [31, 32]; NuagA from Japan [33]; Qaha (AY340974) from Egypt; Mexican isolate (DQ272579); B165 form India [34]; NZ-M16, NZ-B18, NZRB-TH28, NZRB-TH30, NZRB-M12, NZRB-M17 and NZRB-G9 from New Zealand [35, 36]; HA16-5 and HA18-9 from Hawaii [37] and Kpg3 from China [38]. Genetic comparison of these CTV genomes revealed an extreme genomic divergence for genotypes of the same viral species (Figure 3). Nevertheless, these divergent CTV genotypes retained the same genomic organization [3].

Phylogenetic analysis classified the twenty CTV genomic sequences in seven main genotypes [35, 37, 38]. Six of them induce severe syndromes: (1) T36-like (T36, Qaha and Mexican); (2) the RB group plus HA18-9; (3) the VT-like (VT, NUagA, T318A, SY568 and Kpg3); (4) HA16-5; (5) B165 and NZ-B18; and the (6) NZ-M16 genomic sequences [35, 37, 38]. The group 7 consisted in the T30-like asymptomatic or mild genotypes (T30, T385). Sequence comparison of complete CTV genomes yielded nucleotide identities from 79.9%, between Qaha (a T36-like strain) and VT, to 99.3% (between T30 and T385) (Figure 3) [37]. The most conserved sequences were located in the 3´NTR region, which is almost identical in most of the cases (Figure 3). The nucleotide divergence was mostly concentrated at the 5´ half of the CTV genome and increased towards the 5´NTR region to raise, in some cases, nucleotide identities as low as 42% [27, 37, 39] (Figure 3). This pattern of genomic divergence was more evident between the T36-like genotypes and close relatives (groups 1 and 2) and the other five CTV groups [36]. However, two paths of sequence divergence were observed [39]. The sequence divergence between CTV genotype groups 3 to 7, although slightly increased in the 5´NTR region, was relatively constant in proportion and distribution along the genome [37, 39]. On the other hand, the T36 like genotypes and close relatives showed considerable genetic distance to the other five main CTV genotypes [36]. Actually, the comparison of the genomic sequences of T30 and T36 diverged from 5% in the 3´ NTR to as high as 58% in the 5´NTR (Figure 3) [27]. Based in these two paths of sequence divergence detected between CTV genomic sequences [39], it was speculate that the T36 genotype and relatives evolved from a recombinant of a CTV genome and an unknown virus millions of years ago in Asia [28].

In relation to host-plant interactions, CTV is a virus with a large genome and complex genetics, while the citrus host includes many species, varieties, and intergenic hybrids with which the virus could interact causing a range of physiological and biochemical responses. In fact, CTV evolved ending up with three proteins, CP, p20 and p23, which are suppressors of the plant RNA silencing mechanism in *N. benthamiana* and *N. tabacum* plants [18]. Unexpectedly, the ORFs that encode proteins p33, p18 and p13 are not required either for replication or assembly [6, 13] or for systemic infection of Mexican lime [*C. aurantifolia* (Christm.) Swing.] and *C. macrophylla* Wester plants [14]. Nevertheless, they are involved in CTV infection and move‐

4 Current Issues in Molecular Virology - Viral Genetics and Biotechnological Applications

Furthermore, several CTV genomic regions have been found to be related with viral symptom development in citrus hosts. The symptomatology determinant of SY syndrome was located at the 3´ region composed by p23 ORF and the 3´NTR [20]. Nevertheless, the p33, p18 and p13 are involved in the SP syndrome development [21], although the participation in this process of other CTV regions, undetected until the moment, has not been discarded. Mild strain cross protection has been widely applied for millions of citrus trees in Australia, Brazil and South Africa [4, 5] to protect against SP economic losses. The mechanism of this type of viral superinfection exclusion is mainly a mystery. Recently, it has been found that the lack of the functional CTV p33 protein completely eliminated the ability of the virus to exclude superin‐

Sequencing the complete genome of CTV was the first breakthrough towards the study of CTV genetics [2]. Actually, there are twenty CTV genomic sequences available. These are T36 and T30 from Florida [2, 27]; VT from Israel [28]; SY568R from California [29, 30]; T385 and T318A from Spain [31, 32]; NuagA from Japan [33]; Qaha (AY340974) from Egypt; Mexican isolate (DQ272579); B165 form India [34]; NZ-M16, NZ-B18, NZRB-TH28, NZRB-TH30, NZRB-M12, NZRB-M17 and NZRB-G9 from New Zealand [35, 36]; HA16-5 and HA18-9 from Hawaii [37] and Kpg3 from China [38]. Genetic comparison of these CTV genomes revealed an extreme genomic divergence for genotypes of the same viral species (Figure 3). Nevertheless, these

Phylogenetic analysis classified the twenty CTV genomic sequences in seven main genotypes [35, 37, 38]. Six of them induce severe syndromes: (1) T36-like (T36, Qaha and Mexican); (2) the RB group plus HA18-9; (3) the VT-like (VT, NUagA, T318A, SY568 and Kpg3); (4) HA16-5; (5) B165 and NZ-B18; and the (6) NZ-M16 genomic sequences [35, 37, 38]. The group 7 consisted in the T30-like asymptomatic or mild genotypes (T30, T385). Sequence comparison of complete CTV genomes yielded nucleotide identities from 79.9%, between Qaha (a T36-like strain) and VT, to 99.3% (between T30 and T385) (Figure 3) [37]. The most conserved sequences were located in the 3´NTR region, which is almost identical in most of the cases (Figure 3). The nucleotide divergence was mostly concentrated at the 5´ half of the CTV genome and increased towards the 5´NTR region to raise, in some cases, nucleotide identities as low as 42% [27, 37,

ment in other citrus hosts [19].

fection by the same or closely related virus [22].

**3.** *Citrus tristeza virus* **sequence diversity**

divergent CTV genotypes retained the same genomic organization [3].

**Figure 3.** Graphic of the nucleotide identity along the CTV genome when comparing T30 genomic sequence with the sequences of (A) T385 (B) T36 and (C) VT genomes. From Albiach-Marti et al. [27].

Comparison of each of the CTV regions pointed to an unevenly distributed sequence variation along the CTV genome, likely reflecting different selective pressures along the genomic RNA [26, 37, 39]. Analysis of nucleotide diversity in some coding regions between CTV strains yield values higher than 0.13. However, most of the nucleotide exchanges were reported at the third codon position, indicating the preservation of the protein sequence among divergent geno‐ types. Actually, the ratio between non-synonymous and synonymous substitutions assessed for CTV coding regions was below the value 1, thus suggesting selective pressure for amino acid conservation [40]. In addition, analysis of the CTV genomic and D-RNAs sequences indicate homologous and non-homologous recombination events among different genotypes [30, 32, 41, 42, 43], possibly as a result of mixed infections on trees that are recurrently inocu‐ lated by aphid transmission.

Conversely, in spite of this genetic variability, sequence comparisons of some CTV ge‐ nomes revealed a remarkable viral genetic stasis as the genomes of some CTV strains, separated geographically and in time, were found essentially identical [27]. This genetic stability has been explained as a consequence of strong selection and competition between the mutants that arise in each replication cycle, which creates equilibrium in the viral quasispecies distribution [27]. In this context, there is a hypothesis to explain the high sequence variability found in the wild CTV isolates [3, 27]. In a fist stage, each of the main genotypes evolved separately in different *Citrus* species at their point of origin in Asia. This was followed by the dispersal of the main CTV genotypes to different environments around the world with the advent of the modern citrus industry in the XIX century. After that, RNA virus muta‐ tion, due the error-prone nature of RNA-dependent RNA polymerases, in addition to recombination events between diverged sequence variants, plus selection, genetic drift and gene flow could have been promoted rapid evolution [3, 5, 27].
