**1. Introduction**

The 2000 x 11 nm long bipolar flexuous filamentous particles of *Citrus tristeza virus* (CTV) (genus *Closterovirus*, family *Closteroviridae*) (Figure 1) contain a single-stranded positive-sense RNA genome of 19.3 kb, which is encapsidated in two different capsid proteins that coat the opposite ends of the virions [1, 2]. CTV is the largest identified RNA virus infecting plants and the second largest worldwide after the animal *Coronaviruses*. The virus is phloem limited and it is transmitted by aphids (*Hemiptera: Aphididae*) (Figure 1), and mechanically by graft propagation of virus-infected plant tissues. CTV isolates from different hosts and areas display great variability either biologically or genetically. There are wild CTV isolates that consist basically of a main genotype and its quasispecies, but others could contain a mixture of strains (groups of viral variants with similar sequence) that differ in symptomology and in viral transmission efficiency by aphids. These CTV strains could bear divergent CTV genotypes. Additionally, wild isolates are also composed by a population of defective RNAs (D-RNAs) that could change by aphid or graft transmission or by host passage [3].

The *Tristeza* syndrome, induced by CTV, has devastated entire commercial citrus industries around the world, since it has caused the death of hundred million trees worldwide. In point of fact, this virus is present in most of the citrus producing areas infecting nearly all species, cultivars and hybrids of *Citrus* spp. and related genera. Phenotypically, CTV induces differ‐ ent grade and wide range of symptoms in *Citrus* species. In effect, depending on the virus isolate and the variety/rootstock combination, CTV strains can cause different syndromes in the field like 'decline' (QD) or 'stem pitting' (SP). Some CTV isolates induce a third syndrome, in glasshouse conditions, that is referred as 'seedling yellows' (SY). Furthermore, CTV causes a myriad of different symptom combinations in indicator plants depending on the CTV strain, or the mixture of strains, present in the plant host indexed. Remarkably, there are mild CTV strains that cause a complete lack of symptoms in almost all species and varieties of citrus, including those present in the citrus orchards, even though these mild viruses multiply to high titers [4, 5].

© 2013 Albiach-Marti; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

The study of the CTV genetics and the virus-host interactions have been hampered during long time as a consequence of the difficulties of experimenting with a virus with a large RNA genome, assembled in fragile particles and present in reduced amounts in a tree, where CTV could take long time to colonize the entire plant and to induce symptomatology. For that reason, CTV was for decades a virus complicated to isolate and characterize. Moreover, the elevated diversity of CTV populations impeded the separation of the sequence variants, composing a specific isolate, to analyze each of the genotypes independently in order to understand every aspect of viral infection. Likewise, the myriad of diseases induced by CTV, depending on the *Citrus* host, viral strain and environmental conditions, challenged the study of the host-plant interactions. In the last century, the study of CTV genetics was focused in generating molecular techniques to improve CTV detection and genotype differentiation [5]. However, in a decade, a remarkable progress has been achieved in developing the genetic engineering tools to overcome the challenges of examining CTV genetics. A cDNA clone (*T36- CTV9*) of the Florida isolate T36 was generated and an *in vitro* genetic system was developed to analyze CTV genotypes, D-RNAs, mutants and self-replicating constructs in *Nicotiana benthamiana* protoplasts or indexing plants [6, 7, 8, 9, 10]. The last advances in CTV genetics and the different biotechnological approaches used to study CTV are discussed in this chapter.

NTR of around 107 nt contains the sequences necessary for both replication and particle assembly [6, 23, 24]. Remarkably, the CTV 5´NTR predicted secondary structure is similar even for divergent genotypes and folded in two stem-loops separated by a short spacer region [23, 25]. The 3´NTR (273 nt) lacks a poly-A tract and does not appear to fold in a tRNA-like structure [2] but instead consists a predicted secondary structure of minimum energy of 10 stem-loop

The Complex Genetics of *Citrus tristeza* virus http://dx.doi.org/10.5772/56122 3

The CTV genome maintains the two characteristic clusters of genes of the family *Closteroviri‐ dae* (Figure 2) [11]. The *replication gene block,* which is also conserved in the supergroup of sindbis-like viruses, comprises ORF 1a and 1b and makes up the 5´ half of the viral genome [2] (Figure 2). The ORF1a encodes a 349 kDa polyprotein with two papain-like protease domains, a type I methyltransferase-like domain, and a helicase-like domain bearing the motifs of the superfamily I helicases. The ORF1b encodes a 54 kDa protein with RNA-dependent RNA polymerase (RdRp) domains. When ORF 1 is are directly translated from the positive-strand gRNA yield a 400 kDa polyprotein [2]. The conserved *quintuple gene block* (Figure 2) is related with virion assembly and trafficking in the plant [11]. This consists of the major coat protein (CP) of 25kDa, the minor coat protein (CPm) of 27kDa and other three proteins, p61, HSP70h and p6. HSP70h is a 65 kDa protein homologue of the HSP70 plant heat-shock proteins [2], a family of plant chaperones involved in protein-protein interactions, translocation into organelles, and intracellular trafficking [12]. The p6 gene encodes a small hydrophobic protein that belongs to the single-span transmembrane proteins [2]. While CP, CPm, p61 and HSP70h are necessary for proper particle assembly, p6 is required for systemic invasion of host plant [13, 14]. The additional five ORFs located at the 3´ half of the genome (Figure 2) are the p20, an homologue of p21 of *Beet yellows virus* (BYV) (genus *Closterovirus*), and four genes encoding proteins with no homologue in other closteroviruses (p33, p18, p13 and p23) (Figure 2) [11]. The p20 protein is the main component of the CTV-induced amorphous inclusion bodies [15] and it is essential for systemic infection [14]. The multifunctional protein p23 contains a Zn finger domain that binds cooperatively both single-stranded (ss) and double-stranded (ds) RNA molecules in a non-sequence specific manner [16]. In addition, p23 controls asymmetrical accumulation of positive and negative RNA strands during viral replication, ensuring the presence of enough quantity of positive genomic RNA (gRNA) ready for virion assembly [17].

**Figure 2.** Scheme of CTV genome structure and organization. CTV ORFs are delimited by boxes. The acronyms PRO, MT, HEL and RdRp indicate protein domains of papain-like protease, methyltranferase, helicase and RNA-dependent RNA polymerase, respectively. HSP70h, CPm and CP indicate ORFs encoding a homologue of heat shock protein 70,

the minor and the major coat proteins, respectively. From Karasev et al. [2].

(SL) structures [26].

**Figure 1.** Left: Viral particles from *Swinglea glutinosa (Blanco) Merr.* protoplasts, transfected with CTV isolate T36, col‐ lected at 4 dpi and examined by SSEM electron microscopy. The bar indicates 200 nm. From Albiach-Marti et al. [72]. Top right: colony of *Toxoptera citricida* (Photo: Dr. A. Urbaneja). Bottom right: *Aphis gossiipi* (Photo: Dr. A. Hermoso de Mendoza)
