Citrus Tristeza Virus Genotypes Research Articles

Distribution, genetic diversity and recombination analysis of Citrus tristeza virus of India

Citrus tristeza virus (CTV) isolates representing all the citrus-growing geographical zones of India were analyzed for nucleotide sequence of the 5'ORF1a fragments of the partial LProI domain and for the coat protein (CP) gene. The nucleotide sequences were compared with previously reported Indian and CTV genotypes from GenBank. The Indian isolates had 80-99 % sequence identity for the 5'ORF1a and 89-99 % identity for the CP genes. In phylogenetic tree analysis, all the Indian and previously reported isolates segregated into eight clades or groups for the 5'ORF1a region. Indian CTV isolates were clustered in all the clades, four of which, D13, K5, BAN-1, and B165, consisted of only Indian isolates. Phylogenetic tree analysis of the CP genes resulted in seven clades. Indian CTV isolates clustered in six of them, and clades I and VI consisted of only Indian isolates. In the phylogenetic tree the Indian CTV isolates clustered in different groups regardless their geographical origin. Diversities in CTV isolates within individual citrus farms were highlighted. Because incongruent phylogenetic relationships were observed for both of the genomic regions, 5'ORF1a and CP gene, recombination analysis was performed using program RDP3. This analysis detected potential recombination events among the CTV isolates which involved exchange of sequences between divergent CTV variants. The SplitsTree analysis showed evidence of phylogenetic conflicts in evolutionary relationships among CTV isolates.

Development and Application of a Multiplex Reverse-Transcription Polymerase Chain Reaction Assay for Screening a Global Collection of Citrus tristeza virus Isolates

The emerging diversity of Citrus tristeza virus (CTV) genotypes has complicated detection and diagnostic measures and prompted the search for new differentiation methods. To simplify the identification and differentiation of CTV genotypes, a multiplex reverse-transcription polymerase chain reaction (RT-PCR) technique for the screening of CTV isolates was developed. Variable regions within the open reading frame (ORF)-1a of diverse CTV genotypes were identified to develop first a simplex (S) and then a hexaplex (H) RT-PCR. CTV isolates have been grouped previously into five genotypes (namely, T3, T30, T36, VT, and B165) based on the nucleotide sequence comparisons and phylogenetic analyses. Nucleotide sequences from GenBank were used to design species and genotype-specific primers (GSPs). The GSPs were initially used for reliable detection of all CTV genotypes using S-RT-PCR. Furthermore, detection of all five recognized CTV genotypes was established using the H-RT-PCR. Six amplicons, one generic to all CTV isolates and one for each of the five recognized genotypes, were identified on the basis of their size and were confirmed by sequence analysis. In all, 175 CTV isolates from 29 citrus-growing countries were successfully analyzed by S- and H-RT-PCR. Of these, 97 isolates contained T36 genotypes, 95 contained T3 genotypes, 76 contained T30 genotypes, 71 contained VT genotypes, and 24 contained B165 genotype isolates. In total, 126 isolates contained mixed infections of 2 to 5 of the known CTV genotypes. Two of the CTV isolates could not be assigned to a known genotype. H-RT-PCR provides a sensitive, specific, reliable, and rapid way to screen for CTV genotypes compared with other methods for CTV genotype detection. Efficient identification of CTV genotypes will facilitate a better understanding of CTV isolates, including the possible interaction of different genotypes in causing or preventing diseases. The methods described can also be used in virus-free citrus propagation programs and in the development of CTV-resistant cultivars.

Genome analysis of an orange stem pitting citrus tristeza virus isolate reveals a novel recombinant genotype

An orange stem pitting citrus tristeza virus (CTV) isolate CTV-B165 was found to be symptomatically similar to other known CTV-VT isolates however molecular methods failed to classify it as an identifiable CTV genotype. The sequence variation of the Indian CTV-B165 isolate was compared to the three well known CTV genotypes, T36, T30, and VT. The genome of the predominant component of CTV-B165 was 19,247 nt in length with 12 open reading frames (ORFs) and was structurally identical to the other CTV isolates. All the completely sequenced CTV isolates except the VT isolate were 2–55 nt longer than the CTV-B165. In comparison to the other fully sequenced T36, T30 and VT genotypic isolates, CTV-B165 had nucleotide identity of 72–86% in ORF1 and 92–99% in ORFs 2–11. Sequence data of independent overlapping clones from the CTV-B165 genome showed highly divergent sequences of the overlapping region of 5′-UTR and ORF1a, the inter-domain region of ORF1a and the partial regions of ORF2. Phylogenetic analysis of five domains of ORF1a, ORF1b, and ORF2 revealed that CTV-B165 isolate distinctly segregates from the existing three genotypes in the dendrograms and was supported by high bootstrap values and robust tree topology. The PHYLPRO graphical analysis showed multiple recombination signals with significant correlation values. The precise detection of recombination sites for different genomic regions in CTV sequences was supported by several recombination-detecting methods. Collectively, the phylogenetic and recombination analyses suggest that the observed CTV-B165 genotype variation is an outcome of inter-genotype recombination. To determine the presence of the CTV-B165 genotype a pair of genome specific primers was designed and standardized for reliable detection of the novel CTV genotype by reverse-transcription polymerase chain reaction.

Genetic diversity and evidence for recent modular recombination in Hawaiian Citrus tristeza virus

The Hawaiian Islands are home to a widespread and diverse population of Citrus tristeza virus (CTV), an economically important pathogen of citrus. In this study, we quantified the genetic diversity of two CTV genes and determined the complete genomic sequence for two strains of Hawaiian CTV. The nucleotide diversity was estimated to be 0.0565 + or - 0.0022 for the coat protein (CP) gene (n = 137) and 0.0822 + or - 0.0033 for the p23 gene (n = 30). The genome size and organization of CTV strains HA18-9 and HA16-5 were similar to other fully sequenced strains of CTV. The 3'-terminal halves of their genomes were nearly identical (98.5% nucleotide identity), whereas the 5'-terminal halves were more distantly related (72.3% nucleotide identity), suggesting a possible recombination event. Closer examination of strain HA16-5 indicated that it arose through recent recombination between the movement module of an HA18-9 genotype, and the replication module of an undescribed CTV genotype.

Persistent Infection and Promiscuous Recombination of Multiple Genotypes of an RNA Virus within a Single Host Generate Extensive Diversity

Recombination and reassortment of viral genomes are major processes contributing to the creation of new, emerging viruses. These processes are especially significant in long-term persistent infections where multiple viral genotypes co-replicate in a single host, generating abundant genotypic variants, some of which may possess novel host-colonizing and pathogenicity traits. In some plants, successive vegetative propagation of infected tissues and introduction of new genotypes of a virus by vector transmission allows for viral populations to increase in complexity for hundreds of years allowing co-replication and subsequent recombination of the multiple viral genotypes. Using a resequencing microarray, we examined a persistent infection by a Citrus tristeza virus (CTV) complex in citrus, a vegetatively propagated, globally important fruit crop, and found that the complex comprised three major and a number of minor genotypes. Subsequent deep sequencing analysis of the viral population confirmed the presence of the three major CTV genotypes and, in addition, revealed that the minor genotypes consisted of an extraordinarily large number of genetic variants generated by promiscuous recombination between the major genotypes. Further analysis provided evidence that some of the recombinants underwent subsequent divergence, further increasing the genotypic complexity. These data demonstrate that persistent infection of multiple viral genotypes within a host organism is sufficient to drive the large-scale production of viral genetic variants that may evolve into new and emerging viruses.

Genetic Marker Analysis of a Global Collection of Isolates of Citrus tristeza virus: Characterization and Distribution of CTV Genotypes and Association with Symptoms

ABSTRACT Genetic markers amplified from three noncontiguous regions by sequence specific primers designed from the partial or complete genome sequences of Citrus tristeza virus (CTV) isolates T3, T30, T36, and VT were used to assess genetic relatedness of 372 isolates in an international collection. Eighty-five isolates were judged similar to the T3 isolate, 81 to T30, 11 to T36, and 89 to VT. Fifty-one isolates were mixed infections by two or more identifiable viral genotypes, and 55 isolates could not be assigned unequivocally to a group defined by marker patterns. Maximum parsimony analysis of aligned marker sequences supported the grouping of isolates on the basis of marker patterns only. Specific disease symptoms induced in select citrus host plants were shared across molecular groups, although symptoms were least severe among isolates grouped by markers with the T30 isolate and were most severe among isolates grouped by markers with the T3 isolate. Isolates assigned the same genotype showed variable symptoms and symptom severity. A classification strategy for CTV isolates is proposed that combines genetic marker patterns and nucleotide sequence data.

Evaluation of the Transmission of Different Field Sources of Citrus Tristeza Virus and the Separation of Different Genotypes by Single Brown Citrus Aphids

Four field sources of citrus tristeza virus (CTV) (Y3, Y6, Y7 and Y23) collected from grapefruit trees at groves in Fort Pierce, Florida, and isolate T36 were used to evaluate the transmission and separation of different virus genotypes by single brown citrus aphids (BrCA). Analysis of the field sources of CTV by inoculation to indicator plants, ELISA and RT-PCR showed that Y6 was a decline-inducing isolate and Y23 a nondecline-inducing isolate. Assays of genotype by RT-PCR indicated that Y6 contained the T36 genotype while Y23 contained the T30 genotype. Both Y3 and Y7 were a mixture of decline-inducing and nondecline-inducing CTV isolates and were a mixture of T36 and T30 genotypes. When Y6 and Y23 were the acquisition host for single BrCA, only the T36 or T30 genotypes, respectively, were detected by RT-PCR in `Mexican' Lime receptor plants. Only the T36 genotype was transmitted to receptor plants from infected Y3 and Y7 plants although these acquisition plants contained more than one genotype. No T3 or VT genotypes were detected in any acquisition or receptor plants. CTV genotype mixtures in the various field sources were separated by single BrCA transmission and that the T36 genotype in T36/T30 mixtures was more easily transmitted than the T30 genotype when the acquisition plant was `Duncan' grapefruit and the receptor plant was `Mexican' lime.