Seed Transmission of Citrus leaf botch virus: Implications in Quarantine and Certification Programs.

Citrus leaf blotch virus (CLBV) was purified and characterized from a Nagami kumquat (Fortunella margarita (Lour.) Swingle), showing bud union crease when propagated on Troyer citrange (Citrus sinensis (L.) Osbeck × Poncirus trifoliata (L.) Raf.) (2). The complete nucleotide sequence of its genomic RNA was determined (4), and biological and molecular diagnosis methods were developed (1,3). CLBV, detected in several citrus cultivars from Australia, the United States (Florida and California), Japan, and Spain is usually associated with bud union crease on citrange or citumelo (C. paradisi (Macfad.) × P. trifoliata). The economic importance of CLBV for the citrus industry is presently unknown since its incidence in different citrus areas has not been evaluated, and its actual involvement in causing bud union crease on trifoliate rootstocks has not yet been proved. To assess seed transmissibility of this virus, 120 to 210 seeds from CLBV-infected Troyer citrange, Nagami kumquat, or sour orange (Citrus aurantium L.) plants were grown in a greenhouse. Individual 4-month-old seedlings were analyzed for CLBV by reverse transcriptase polymerase chain reaction, and infection was confirmed by biological indexing on Dweet tangor (C. tangerina Hort. ex Tanaka × C. sinensis (L.) Osbeck) when seedlings were approximately 18 months old. Seed transmission was found in 2.50, 2.52, and 2.46% of the citrange, kumquat, and sour orange seedlings, respectively. This finding indicates that control of CLBV spread during citrus propagation will require, not only virus-free buds, but also rootstock seedlings that originate from CLBV-free seed source trees. Because CLBV is seed transmissible, regulations of citrus certification programs may need to be changed to include increased control of seed source trees. Also, international regulations for citrus seed movement likely will have to be augmented to include a phytosanitary certification indicating that seeds have been collected from CLBV-free trees. References: (1) L. Galipienso et al. Plant Pathol. 49:308, 2000. (2) L. Galipienso et al. Arch. Virol. 146:357, 2001. (3) L. Galipienso et al. Eur. J. Plant Pathol. 110:175,2004. (4) M. C. Vives et al. Virology 287:225, 2001.

Carotid intima-media thickness is slightly increased over time in HIV-1-infected patients.

OBJECTIVES: HIV-infected patients are at risk of atherosclerosis and cardiovascular diseases. In a 12-month follow-up study, we aimed to investigate changes in carotid intima-media thickness (IMT), a surrogate marker of atherosclerosis, and its determinants in HIV-1-infected patients. METHODS: Our multicentre prospective longitudinal cohort study included 346 HIV-infected patients, for each of whom two IMT measurements were taken by B-mode ultrasonography at baseline (M0) and 1 year later (M12). RESULTS: We observed a significant but moderate increase in the common carotid artery (CCA) median IMT, from 0.54 to 0.56 mm (P<10(-4)), i.e. an increase of 0.020 mm (95% confidence interval 0.012-0.029). There was a significant association between cross-sectional CCA IMT measures at M12 and conventional cardiovascular risk factors (higher CCA IMT with older age, P<10(-4); male gender, P=0.02; tobacco consumption, P=0.05), as well as higher CD4 cell count at M12 (>median 455 cells/microL, P=0.01). Only CD4 cell count at M0 was strongly and positively associated with the variation in IMT between M0 and M12 (P=4 x 10(-3)). IMT progression was +0.0020 mm for the lowest quartile of CD4 cell count distribution at M0, i.e. 3-253 cells/microL, +0.010 mm for 253-402 cells/microL, +0.043 mm for 402-590 cells/microL, and +0.028 mm for 590-2270 cells/microL. No association was found with type or duration of antiretroviral exposure. CONCLUSIONS: Conventional cardiovascular risk factors are major determinants of IMT evolution. The link between immunological status and carotid IMT requires further study.

The nucleotide sequence and genomic organization of Citrus leaf blotch virus: candidate type species for a new virus genus.

The complete nucleotide sequence of Citrus leaf blotch virus (CLBV) was determined. CLBV genomic RNA (gRNA) has 8747 nt, excluding the 3'-terminal poly(A) tail, and contains three open reading frames (ORFs) and untranslated regions (UTR) of 73 and 541 nucleotides at the 5' and 3' termini, respectively. ORF1 potentially encodes a 227.4-kDa polypeptide, which has methyltransferase, papain-like protease, helicase, and RNA-dependent RNA polymerase motifs. ORF2 encodes a 40.2-kDa polypeptide containing a motif characteristic of cell-to-cell movement proteins. The 40.7-kDa polypeptide encoded by ORF3 was identified as the coat protein. The genome organization of CLBV resembles that of viruses in the genus Trichovirus, but they differ in various aspects: (i) in trichoviruses ORF2 overlaps ORFs 1 and 3, whereas in CLBV, ORFs 2 and 3 are separated and ORFs 1 and 2 overlap in one nucleotide; (ii) CLBV gRNA and CP are larger than those of trichoviruses; and (iii) the CLBV 3' UTR is larger than that of trichoviruses. Phylogenetic comparisons based on CP amino acid signatures clearly separates CLBV from trichoviruses. Also contrasting with trichoviruses, CLBV could not be transmitted to Chenopodium quinoa Willd. Considering these singularities, we propose that CLBV should be included in a new virus genus.

Partial characterisation of citrus leaf blotch virus, a new virus from Nagami kumquat.

Citrus leaf blotch virus (CLBV) was purified from leaves of Nagami kumquat SRA-153 that showed bud union crease when propagated on Troyer citrange. Virions were filamentous particles (960 x 14 nm) containing a 42 kDa protein and a single-stranded RNA (ssRNA) of about 9,000 nt (Mr 3 x 10(6)). Infected tissue contained three species of double-stranded RNA (dsRNA) of Mr 6, 4.5 and 3.4 x 10(6). The nucleotide sequence of several complementary DNA (cDNA) clones showed significant similarities with replication-related proteins from plant filamentous viruses in several genera. A digoxigenin-labelled probe from one of these cDNA clones hybridised in Northern blots with ssRNA from virions and with the three dsRNA species, suggesting that the ssRNA is the genomic RNA of the virus, the largest dsRNA is its replicative form, and the two smaller dsRNAs probably replicative forms of 5' co-terminal subgenomic RNAs. CLBV was also detected in several citrus cultivars from Spain and Japan including Navelina sweet orange field trees propagated on Troyer citrange showing bud union crease; however, no virus could be detected in other citrus trees with similar symptoms. This indicates that CLBV is not restricted to kumquat SRA-153, but its involvement in causing the bud union disorder remains unclear.

The complete genome sequence of the major component of a mild citrus tristeza virus isolate.

The genome of the Spanish mild isolate T385 of citrus tristeza virus (CTV) was completely sequenced and compared with the genomes of the severe isolates T36 (Florida), VT (Israel) and SY568 (California). The genome of T385 was 19,259 nt in length, 37 nt shorter than the genome of T36, and 33 and 10 nt longer than those of VT and SY568, respectively, but their organization was identical. T385 had mean nucleotide identities of 81.3, 89.3 and 94% with T36, VT and SY568, respectively. The 3' UTR had over 97% identity in all isolates, whereas the 5' UTR of T385 had 67% identity with VT, 66.3% with SY568 and only 42.5% with T36. In the coding regions, the nucleotide differences between T385 and VT were evenly distributed along the genome (around 90% identity); this was not observed between T385 and the other isolates. T385 and T36 had nucleotide identities around 90% in the eight 3'-terminal ORFs of the genome, but only 72.3% in ORF 1a, a divergence pattern similar to that reported previously for T36 and VT. T385 and SY568 had nucleotide identities close to 90% in the 5'- and 3'-terminal regions of the genome, whereas the central region had over 99% identity. Our data suggest that the central region in the SY568 genome results from RNA recombination between two CTV genomes, one of which was almost identical to T385.

Sequences of Citrus tristeza virus separated in time and space are essentially identical.

The first Citrus tristeza virus (CTV) genomes completely sequenced (19.3-kb positive-sense RNA), from four biologically distinct isolates, are unexpectedly divergent in nucleotide sequence (up to 60% divergence). Understanding of whether these large sequence differences resulted from recent evolution is important for the design of disease management strategies, particularly the use of genetically engineered mild (essentially symptomless)-strain cross protection and RNA-mediated transgenic resistance. The complete sequence of a mild isolate (T30) which has been endemic in Florida for about a century was found to be nearly identical to the genomic sequence of a mild isolate (T385) from Spain. Moreover, samples of sequences of other isolates from distinct geographic locations, maintained in different citrus hosts and also separated in time (B252 from Taiwan, B272 from Colombia, and B354 from California), were nearly identical to the T30 sequence. The sequence differences between these isolates were within or near the range of variability of the T30 population. A possible explanation for these results is that the parents of isolates T30, T385, B252, B272, and B354 have a common origin, probably Asia, and have changed little since they were dispersed throughout the world by the movement of citrus. Considering that the nucleotide divergence among the other known CTV genomes is much greater than those expected for strains of the same virus, the remarkable similarity of these five isolates indicates a high degree of evolutionary stasis in some CTV populations.