Genetic Variation of Citrus Tristeza Virus Isolates from California and Spain: Evidence for Mixed Infections and Recombination

Abstract

We examined the population structure and genetic variation of four genomic regions within and between 30 Citrus tristeza virus (CTV) isolates from Spain and California. Our analyses showed that most isolates contained a population of sequence variants, with one being predominant. Four isolates showed two major sequence variants in some genomic regions. The two major variants of three of these isolates showed very low nucleotide identity to each other but were very similar to those of other isolates, suggesting the possibility of mixed infections with two divergent isolates. Incongruencies of phylogenetic relationships in the different genomic regions and statistical analyses suggested that the genomes of some CTV sequence variants originated by recombination events between diverged sequence variants. No correlation was observed between geographic origin and nucleotide distance, and thus from a genetic view, the Spanish and Californian isolates analyzed here could be considered members of the same population.Citrus tristeza virus (CTV) is distributed worldwide and is the causal agent of one of the most economically important diseases of citrus. CTV, a member of the genus Closterovirus within the family Closteroviridae, is phloem limited and is transmitted by aphids in a semipersistent manner. CTV virions are filamentous flexuous particles about 2,000 nm long, with two coat proteins (CP and CPm) covering 95 and 5% of the particle length, respectively (8). The CTV genome is a single-stranded, positive-sense RNA of 19,226 to 19,296 nucleotides (nt) (18,27,48,51) organized in 12 open reading frames encoding at least 19 proteins. These include two papain-like proteases, replication-associated proteins (RNA polymerase, helicase, and methyltransferase), a homologue of the HSP70 protein, two coat proteins (CP and CPm), RNA-binding protein p23 (23), a p20 protein that accumulates in the amorphous inclusion bodies (14), and other proteins of so far unknown function (p61, p13, and p18) (Fig. 1). CTV-infected plants contain, in addition to the genomic RNA, 3Ј-coterminal subgenomic RNAs (15) and defective RNAs (D RNAs), the latter resulting from extensive internal deletions of the genomic RNA (2,26,28,50).CTV isolates differing in the type and intensity of symptoms induced in different citrus species and cultivars and in their aphid transmissibility have been reported worldwide (38). In the last two decades, efforts have been taken to develop molecular techniques for rapid differentiation of CTV isolates and identification of molecular markers related to CTV-induced symptoms. Variation in serological reactivity, peptide maps of the coat protein, double-stranded RNA (dsRNA) patterns, hybridization with cDNA probes, restriction fragment length polymorphism, and single-strand conformation polymorphism (SSCP) have been described in attempts to differentiate CTV isolates (29).Nucleotide sequence analysis is the most accurate procedure for CTV differentiation and estimation of molecular or genetic variation. To date, the complete g...

Genetic diversity of the coat protein of olive latent virus 1 isolates

“…No relationship was found among isolates according to collection time or spatial separation. These results agree with those of other authors who have reported a high spatial and temporal genetic stability for several RNA viruses, suggesting that selective pressures to preserve biological functions are stronger than speciation due to geographic separation [14,23,32,34,40].…”

“…However, selective pressures imposed by certain aspects of viral biology (e.g., host type and range) may limit genetic diversity [31,33,34,40,41].…”

Comparison of multiple viral population characterization methods on a candidate cross-protection Citrus tristeza virus (CTV) source

“…The Rubio et al (2001) primers was used which was designed based on the T30 genotype, but has been shown to also amplify strains from the VT, T3, B165, HA16-5 and RB genotypes (Read and Pietersen, 2015). Amplicons were purified using the Promega Wizard ® SV Gel and PCR Cleanup System (Promega, Madison, WI) according to the manufacturer"s instructions and ligated into the p-GEM ® -T Easy vector.…”

“…The acknowledged genotypes of CTV are continually being expanded, and thus far include T36, T30, T3, VT, B165, HA16-5, T68 and RB (Albiach-Marti et al, 2000;Harper, Dawson, and Pearson, 2010;Harper, 2013;Karasev et al, 1995;Mawassi et al, 1996;Melzer et al, 2010;. The CTV population of a single host may contain many different genotypes as the longevity of citrus plants allows for repeated infections of various CTV sources through aphid transmission (Rubio et al, 2001). CTV can be controlled through mild strain cross-protection.…”

“…This approach also only provides information on the specific region amplified. Since recombination is common within the CTV genome Rubio et al, 2001;Vives et al, 2005), targeting only a specific region may provide distorted identification of the genotype as recombination events that occurred elsewhere in the genome would not be detected. This approach is however warranted when targeting a gene important for a specific biological function (Cook et al, 2015) for example the p33 gene which has been shown to be involved in the genotype specificity of the super-infection exclusion mechanism of CTV (Folimonova, 2012).…”