Differentiation of citrus tristeza virus isolates by serological analysis of p25 coat protein peptide maps

A procedure was developed to purify rapidly and easily a sufficient quantity of native p25 coat protein (CP) to allow comparison of five isolates of citrus tristeza virus (CTV) by serological analysis of peptide maps, using monoclonal and polyclonal antibodies. CTV particles were concentrated by centrifugation and purified by agarose gel electrophoresis. The CP was extracted from gel slices riched in virions and protein yields were about three times greater than those obtained previously and of comparable purity. The purified CP was partially digested with either V8 or papain endo-protease, and the peptides generated were separated and electroblotted to a membrane. Protein blots were tested with four monoclonal antibodies and one source of polyclonal antibodies. The serological maps generated by papain allowed differentiation of all the isolates examined, and those generated by V8 endoprotease allowed discrimination of four of the five isolates tested. Some of these isolates had been indistinguishable based on their reactivity in DASI-ELISA, dsRNA pattern and biological characterization. Serological analysis of peptide maps, as described below, allowed accurate comparison of CTV isolates with minimum amounts of p25 CP and proved superior to other techniques for discriminating CTV isolates.     

Grouping and comparison of Indian citrus tristeza virus isolates based on coat protein gene sequences and restriction analysis patterns

Summary.  Citrus tristeza virus (CTV) is an aphid-transmitted closterovirus, which causes one of the most important citrus diseases worldwide. Isolates of CTV differ widely in their biological properties. CTV-infected samples were collected from four locations in India: Bangalore (CTV-B), Delhi (CTV-D), Nagpur (CTV-N), and Pune (CTV-P), and were maintained by grafting into Kagzi lime (Citrus aurantifolia (Christm. Swing.). All isolates produced typical vein clearing and flecking symptoms 6–8 weeks after grafting. In addition, CTV-B and CTV-P isolates produced stem-pitting symptoms after 8–10 months. The CTV coat protein gene (CPG) was amplified by RT-PCR using CPG specific primers, yielding an amplicon of 672 bp for all the isolates. Sequence analysis of the CPG amplicon of all the four Indian isolates showed 93–94% nucleotide sequence homology to the Californian CTV severe stem pitting isolate SY568 and 92–93% homology to the Japanese seedling yellows isolate NUagA and Israeli VT p346 isolates. In phylogenetic tree analysis, Indian CTV isolates appeared far different from other isolates as they formed a separate branch. Comparison among the Indian isolates was carried out by restriction analysis and restriction fragment length polymorphism (RFLP). Specific primers to various genome segments of well-characterized CTV isolates were used to further classify the Indian CTV isolates. Received September 13, 2002; accepted October 30, 2002     

Deep sequencing analysis of viral short RNAs from an infected Pinot Noir grapevine

Virus-derived short interfering RNAs (vsiRNAs) isolated from grapevine V. vinifera Pinot Noir clone ENTAV 115 were analyzed by high-throughput sequencing using the Illumina Solexa platform. We identified and characterized vsiRNAs derived from grapevine field plants naturally infected with different viruses belonging to the genera Foveavirus, Maculavirus, Marafivirus and Nepovirus. These vsiRNAs were mainly of 21 and 22 nucleotides (nt) in size and were discontinuously distributed throughout Grapevine rupestris stem-pitting associated virus (GRSPaV) and Grapevine fleck virus (GFkV) genomic RNAs. Among the studied viruses, GRSPaV and GFkV vsiRNAs had a 5′ terminal nucleotide bias, which differed from that described for experimental viral infections in Arabidopsis thaliana. VsiRNAs were found to originate from both genomic and antigenomic GRSPaV RNA strands, whereas with the grapevine tymoviruses GFkV and Grapevine Red Globe associated virus (GRGV), the large majority derived from the antigenomic viral strand, a feature never observed in other plant-virus interactions.     

The genotypes of citrus tristeza virus isolates from China revealed by sequence analysis of multiple molecular markers

The genotypes of ten citrus tristeza virus (CTV) isolates from central China were determined by examining multiple molecular markers (MMMs) using 11 primer pairs. The results revealed that one isolate contained a single T30 genotype, two isolates contained a single VT genotype, and the other seven isolates were mixtures of two or more genotypes. Sequence analysis of amplified MMMs showed a high genetic diversity in Chinese CTV populations. The genotypes resembling T36, RB and B165 were identified from Chinese CTV isolates for the first time. Our results suggest that genotype assignment of CTV cannot be based solely on the amplification profiles of MMMs, and sequencing of MMMs is required.     

Progress on strain differentiation of Citrus tristeza virus and its application to the epidemiology of citrus tristeza disease

Citrus tristeza virus (CTV) occurs in most citrus producing regions of the world, and it is the most serious viral pathogen of citrus. With the recent establishment of the brown citrus aphid, Toxoptera citricida, its most efficient vector, on Madeira Island (Portugal) and in Florida (USA) and the countries of the Caribbean Basin, the impact of CTV is likely to increase in these regions. Since there are many strains of CTV and CTV infections frequently occur as mixtures of several strains, it is necessary to be able to distinguish the strains for regulatory purposes, disease management and epidemiology. We describe the evolution of techniques developed to detect CTV and to differentiate the individual strains, and present the results of tests using these latest methods on CTV isolates from mainland Portugal, Madeira Island and Florida. Mild and decline-inducing strains of CTV were detected in mainland Portugal and mild, decline-inducing and severe stem pitting strains on Madeira Island. In Florida we demonstrated the presence of infections that reacted with probes made against stem pitting strains not previously detected there. It is concluded that CTV presents a significant threat to citrus production in mainland Portugal, on Madeira Island and in the neighbouring countries of the Mediterranean Basin, as well as in Florida, elsewhere in the USA and throughout the Caribbean Basin, especially following the widespread establishment of T. citricida throughout the region.     

Mutational analysis of the replication signals in the 3'-nontranslated region of citrus tristeza virus

Citrus tristeza virus (CTV), a member of the Closteroviridae, has a 19.3-kb messenger-sense RNA genome consisting of 12 open reading frames with nontranslated regions (NTR) at the 5' and 3' termini. The 273 nucleotide (nt) 3'-NTR is highly conserved ( approximately 95%) among the sequenced CTV isolates in contrast to the highly diverse 5'-NTR sequences. The 3' replication signals were mapped to the 3' 234 nts within the NTR. This region of CTV does not contain a poly-A tract nor does it appear to fold as a tRNA-mimic. Instead, a computer-predicted thermodynamically stable secondary structure comprised of 10 stem-and-loop (SL) structures, referred to as SL1 to SL10 (5' to 3'), was common to all CTV isolates. This putative structure was used as a guide to examine the 3' requirements for replication in vivo. The resulting data suggest that a complex 3' structure is required for those functions that provide for efficient replication of CTV in vivo such as minus-strand initiation, regulation of strand asymmetry, effective translation of the myriad of viral mRNAs, or stability of RNAs. Deletions into the 3'-NTR, up to 66 nts from the 5' direction and 11 nts from the 3' direction, deleting or disrupting putative SL1, SL2 and SL3, or SL10, resulted in continued replication, suggesting that these sequences are not essential for basal-level replication, but are required for efficient replication. Predicted stem loops 3 through 10 were examined by mutations designed to alter the primary structures while preserving the secondary structures. Mutations designed to disrupt the predicted stems of SL3, SL5, SL7, SL9, or SL10 resulted in substantially reduced levels of replication, while compensatory mutations resulted in partial restorations of replication, suggesting that these predicted secondary structures are involved in replication. Also, the putative loop sequences of SL5, SL6, SL7, and SL9 tolerated mutagenesis with continued but reduced levels of replication. In contrast, all mutations introduced into putative SL4, SL8, and the stem of SL6 prevented replication, suggesting that the primary structure of these regions make up the core of the 3' replication signal. The 3' triplet, CCA, was shown to be necessary for efficient replication, but deletion of eleven nts to expose an internal CCA resulted in continued replication.     

Nucleotide sequence of the coat protein gene of citrus tristeza virus: Comparison of biologically diverse isolates collected in Israel

The sequences of the coat protein genes of four seedling yellows (SY) and four non-SY (NSY) of citrus tristeza virus (CTV) isolates, which were collected in Israel over a period of 30 years, were analyzed. Pairwise comparisons showed extensive similarities in the nucleotide and amino acid sequences of six isolates designated the VT group. This group consists of three NSY isolates that cause a very mild CTV reaction on the sensitive combination of sweet orange (SwO) grafted on sour orange (SO), and three SY isolates that cause severe SY and SwO/SO reactions. MT, a CTV isolate that is consistently nontransmitted byAphis gossypii, was found to be different in two amino acids (Val 103 and Glu 113) from each of theA. gossypii transmissible CTV isolates. Sequencing of the cDNA clones obtained from ST, a variably transmitted CTV isolate, showed extensive sequence variation among the tested clones. The sequence information indicates that the current CTV epidemics in Israel are caused by at least two CTV subspecies (VT and HT) displaying extensive differences in their coat protein genes.     

Evolutionary analysis of genetic variation observed in citrus tristeza virus (CTV) after host passage

Summary. We have studied the genetic variability in two genes (p18 and p20) from two groups of Citrus tristeza virus (CTV) isolates. One group (isolates T385, T317, T318, and T305) was derived from a Spanish source by successive host passages while the other (isolates T388 and T390) was obtained after aphid transmission from a Japanese source. A total of 274 sequences were obtained for gene p18 and 451 for p20. In the corresponding phylogenetic trees, sequences derived from the severe isolates (T318, T305, and T388) clustered together and separately from those derived from mild or moderate isolates (T385, T317, and T390), regardless of their geographic origin. Hierarchical analyses of molecular variance showed that up to 53% of the total genetic variability in p18 and up to 87% of the variation in p20 could be explained by differences in the pathogenicity features of the isolates. Neutrality tests revealed that different selection forces had been acting between isolates and between genes, with purifying selection being suggested for p18 from isolates T385 and T390 and for p20 from isolates T385, T317, and T388, and balancing selection for p18 from isolates T318, T305, and T388 and for p20 from isolates T318 and T390. Furthermore, several models of codon selection were observed, with purifying selection being the most notable one, compatible with low effective population size of the virus populations resulting from transmission bottlenecks. We found no evidence of recombination playing a significant role during p18 and p20 evolution in these isolates. These results suggest that hosts can be an important evolutionary factor for CTV isolates.     

Deep sequencing of small RNAs from human skin reveals major alterations in the psoriasis miRNAome

Psoriasis is a chronic and complex inflammatory skin disease with lesions displaying dramatically altered mRNA expression profiles. However, much less is known about the expression of small RNAs. Here, we describe a comprehensive analysis of the normal and psoriatic skin miRNAome with next-generation sequencing in a large patient cohort. We generated 6.7 × 10(8) small RNA reads representing 717 known and 284 putative novel microRNAs (miRNAs). We also observed widespread expression of isomiRs and miRNA*s derived from known and novel miRNA loci, and a low frequency of miRNA editing in normal and psoriatic skin. The expression and processing of selected novel miRNAs were confirmed with qRT-PCR in skin and other human tissues or cell lines. Eighty known and 18 novel miRNAs were 2-42-fold differentially expressed in psoriatic skin. Of particular significance was the 2.7-fold upregulation of a validated novel miRNA derived from the antisense strand of the miR-203 locus, which plays a role in epithelial differentiation. Other differentially expressed miRNAs included hematopoietic-specific miRNAs such as miR-142-3p and miR-223/223*, and angiogenic miRNAs such as miR-21, miR-378, miR-100 and miR-31, which was the most highly upregulated miRNA in psoriatic skin. The functions of these miRNAs are consistent with the inflammatory and hyperproliferative phenotype of psoriatic lesions. In situ hybridization of differentially expressed miRNAs revealed stratified epidermal expression of an uncharacterized keratinocyte-derived miRNA, miR-135b, as well as the epidermal infiltration of the hematopoietic-specific miRNA, miR-142-3p, in psoriatic lesions. This study lays a critical framework for functional characterization of miRNAs in healthy and diseased skin.     

Deep sequencing analysis of RNAs from a grapevine showing Syrah decline symptoms reveals a multiple virus infection that includes a novel virus

In a search for viruses associated with decline symptoms of Syrah grapevines, we have undertaken an analysis of total plant RNA sequences using Life Sciences 454 high-throughput sequencing. 67.5 megabases of sequence data were derived from reverse-transcribed cDNA fragments, and screened for sequences of viral or viroid origin. The data revealed that a vine showing decline symptoms supported a mixed infection that included seven different RNA genomes. Fragments identified as derived from viruses or viroids spanned a ∼ ten thousand fold range in relative prevalence, from 48,278 fragments derived from Rupestris stem pitting-associated virus to 4 fragments from Australian grapevine viroid. 1527 fragments were identified as derived from an unknown marafivirus. Its complete genome was sequenced and characterized, and an RT-PCR test was developed to analyze its field distribution and to demonstrate its presence in leafhoppers (vector for marafiviruses) collected from diseased vines. Initial surveys detected a limited presence of the virus in grape-growing regions of California.     

Characterization of a novel citrus tristeza virus genotype within three cross-protecting source GFMS12 sub-isolates in South Africa by means of Illumina sequencing

Tristeza disease (caused by citrus tristeza virus, CTV) is currently controlled in South Africa by means of cross-protection. In this study, we characterized the CTV populations of three grapefruit mild strain 12 (GFMS12) single-aphid-transmission-derived sub-isolates at the whole-genome level using Illumina sequencing technology. A novel South African isolate (CT-ZA3, of the T68 genotype) was shown to be the dominant genotype in all GFMS12 sub-isolates tested, along with reads unique to various other genotypes occurring as minor components. Uncertainty remains as to the significance of these minor components.     

The fitness of citrus tristeza virus defective RNAs is affected by the lengths of their 5'- and 3'-termini and by the coding capacity

Populations of the Closterovirus Citrus tristeza virus (CTV) generally contain defective RNAs (dRNAs) that vary in size, abundance, and sequence. The variation in abundance of the different dRNAs in a population suggests selection for those of higher fitness. To examine factors affecting fitness of dRNAs, we investigated a series of in vitro constructed dRNAs for their ability to be amplified in protoplasts by an efficiently replicated CTV deletion mutant. The minimal sequences required for accumulation of the dRNAs were within the genomic 5' proximal approximately 1 kb and the 3' 270 nucleotides. However, other factors were involved, because a dRNA with only the minimal sequences failed to be replicated. Rescue of a nonviable dRNA by insertion of nonviral sequences between the termini suggested that "spacing" between terminal cis-acting signals influenced fitness. A continuous open reading frame (ORF) through most of the sequences derived from the 5' of the genome was a requirement for dRNA amplification. In general, insertions, deletions, or nucleotide substitutions were tolerated in the dRNAs as long as an ORF was retained, whereas dRNAs with mutations that prematurely terminated the ORF were not viable. To discriminate between a requirement for an essential protein and ribosomal travel, perhaps to present replication signals to the replicase complex, mutations were made to modify the potential protein but still maintain an ORF. Deletions, insertions of nonviral sequences, or switching of reading frames that altered the amino acid sequence of the protein, except the N-terminal 161 amino acids, did not destroy the fitness of the dRNAs. Yet termination of the ORF in the middle of nonviral sequences did destroy the ability of the dRNAs to be amplified. These results suggest that even though a continuous ORF was needed for fitness, its protein product did not affect the amplification of the dRNAs.     

A new grapevine virus discovered by deep sequencing of virus- and viroid-derived small RNAs in Cv Pinot gris

Field symptoms of chlorotic mottling and leaf deformations were observed on the cv Pinot gris (PG) in the Trentino region (Italy). Extensive assays excluded the presence of widely distributed nepo-, ampelo- and vitiviruses. An analysis of small RNA populations from two PG grapevines showing or not symptoms was carried out by Illumina high throughput sequencing. The study disclosed the virus and viroids contents of the two vines that was composed by Grapevine rupestris stem pitting-associated virus (GRSPaV), two viroids Hop stunt viroid (HSVd) and Grapevine yellow speckle viroid 1 (GYSVd1), the marafiviruses Grapevine rupestris vein feathering virus (GRVFV) and Grapevine Syrah virus 1 (GSyV-1), and a hitherto unrecorded virus. This virus had a genome organization identical to that of Grapevine berry inner necrosis virus (GINV), a trichovirus reported only from Japan, with which it grouped in phylogenetic trees constructed with sequences of the RdRp domain and the coat protein gene. However, molecular differences with GINV are wide enough to warrant classification of the virus in question as a new species, for which the provisional name of Grapevine Pinot gris virus (GPGV) is proposed. A limited field survey for the presence of GPGV in diseased and symptomless plants from three different cultivars did not allow to clearly associating the virus to the observed symptoms.