Molecular characterization of grapevine yellow speckle viroid-2 (GYSVd-2)

Grapevine yellow speckle viroid-2 (GYSVd-2) is a viroid found only in grapevines in China and Australia. Here, we report the molecular characterization of GYSVd-2 isolated from three grapevine varieties in China. A total of 90 cDNA clones were sequenced including 30 cDNA clones obtained from each of the Black Olympia, Zaoyu, and Thomson Seedless isolates. Sequencing analysis identified 20, 18, and 12 different sequence variants from the 3 isolates, respectively. Furthermore, each of the isolates included one predominant sequence variant. Compared to the Australian variant of GYSVd-2 (Accession number: NC_003612), the Black Olympia variant was identical and the Zaoyu variant contained one substitution. In contrast, the Thomson Seedless isolate significantly varied from the Australian variant with three substitutions, two insertions, and four deletions. In silico structure analysis predicted that the variations were clustered in the terminal left, the pathogenicity, and the variable region of the predicted secondary structure of GYSVd-2.     

Occurrence of grapevine leafroll-associated virus 5 in Portugal: genetic variability and population structure in field-grown grapevines

A comparison of 15 field isolates of grapevine leafroll-associated virus 5 (GLRaV-5) was conducted, based on the analysis of nucleotide sequences of two viral ORFs: the coat protein (CP) and the heat shock protein 90 homolog (HSP90h). After amplification and cloning, the population of viral sequences was analyzed for each isolate, revealing the within-isolate presence of sequence variants for both genes, with one or more major CP variants. Phylogenetic analysis showed the gene sequence variants detected to be exclusive for each isolate. These data, together with estimates of genetic diversity and positive selection, did not reveal evidence of vector-mediated transfer of GLRaV-5. Conversely, a strong effect of host vegetative propagation on divergence dynamics of GLRaV-5 variants was suggested by the isolates from this work The phylogeny of the CP gene further revealed clustering of GLRaV-5 isolates into eight lineages, four of which were detected in our study, revealing a higher diversity than previously described. The information gathered also contributes to firmly establishing GLRaV-5 as a cohesive taxonomic group within the ampeloviruses.     

Sequence analysis of grapevine isolates of Raspberry ringspot nepovirus

Summary. The nucleotide sequences of RNAs 1 and 2 of a German isolate of Raspberry ringspot virus (RpRSV) infecting grapevine (RpRSV-Grapevine), as well as partial sequences of another grapevine isolate from Switzerland (RAC815) were determined. The sequences of the protease-polymerase region encoded by RNA1, and the movement protein and coat protein genes encoded by RNA 2, of these isolates were compared with those of other isolates available in databases. The coat proteins of the grapevine isolates formed a sister group to all those from other RpRSV isolates, but whether this resulted from divergence or recombination was uncertain.     

Complete nucleotide sequence of a new strain of grapevine leafroll-associated virus 3 in South Africa

The complete genome sequences of two newly identified genetic variants of grapevine leafroll-associated virus 3 (GLRaV-3) are described. Two isolates, GH11 (18671 nt) and GH30 (18576 nt) were sequenced and found to be less than 70?% similar to other South African GLRaV-3 variants. The genome organization of GH11 and GH30 is similar to that of previously described GLRaV-3 isolates. However, no corresponding open reading frame 2 (ORF2) could be identified. Phylogenetic analysis indicates that GH11 and GH30 cluster in a subgroup (group VI) that is separate from the previously described GLRaV-3 isolates and should be regarded as a different strain of GLRaV-3.     

Identification and characterization of a new vitivirus from grapevine

New virus-like sequences, TvAQ7 and TvP15, were found in a Japanese grapevine accession of OKY-AQ7 (cv. Aki Queen) and of OKY-P15 (cv. Pione) by nested RT-PCR to simultaneously amplify a segment of the RNA-dependent RNA polymerase (RdRp) gene from members of the genera Vitivirus and Foveavirus. The TvAQ7 genome, except for an exact 5′ terminus, is composed of about 7.6 kb containing five potential open reading frames. The genomic organization resembles those of grapevine virus A and other known vitiviruses. The 199-amino-acid sequence deduced from the ORF4 of TvAQ7 has 38.5–54.0% identity with the coat protein (CP) of known grapevine vitiviruses and 86.9% identity with TvP15. Phylogenetic analysis based on amino acid sequences of the CP showed that TvAQ7 and TvP15 were closely related to the vitiviruses. In addition, we confirmed that TvAQ7 and TvP15 were transmitted from infected grapevine to healthy seedlings by the mealybug Pseudococcus comstocki Kuwanae. On the basis of our findings, TvAQ7 and TvP15 should be considered isolates of a new species of the genus Vitivirus, and both isolates are probably genetic variants of the new species. We propose to name this virus grapevine virus E (GVE).     

Genetic diversity and phylogenetic analysis of Australian Grapevine Viroid (AGVd) isolated from different grapevines in China

Australian grapevine viroid (AGVd) is found in only three countries in the world. Here, the genetic diversity and phylogenetic relationships of AGVd isolates from three different grape varieties (Thomson Seedless, Jingchuan and Zaoyu) in China were studied. A hundred of independent cDNA clones from each of the three isolates, in total of 300, were sequenced. We identified 29 sequence variants including two predominant ones in Thomson Seedless, and 48 each including a unique predominant one in Jingchuan and Zaoyu. In silico structure analysis revealed that base changes were clustered in the left terminal domain of the predicted secondary structure in all three isolates. Further, these changes were shown to affect their secondary structures to varying degrees. Genetic diversity and phylogenetic analysis of four predominant sequence variants from this study, plus four others from Australia and Tunisia, revealed obvious regional disparity and variety-specificity in AGVd.     

Molecular characterisation of two divergent variants of grapevine leafroll-associated virus 3 in New Zealand

Partial genomic sequences of two divergent grapevine leafroll-associated virus 3 (GLRaV-3) variants, NZ1-B and NZ2, from New Zealand were determined and analysed (11,827 nt and 7,612 nt, respectively). At the nucleotide level, both variants are more than 20 % different from the previously published GLRaV-3 sequences, from phylogenetic groups 1 to 5. Phylogenetic analysis indicated that NZ1-B is a variant of the previously identified divergent NZ-1, while NZ2 is a novel sequence with only 76 % nucleotide sequence identity to GLRaV-3 variants NZ-1, GH11, and GH30. Therefore, NZ2 is a new variant of GLRaV-3. Amino acid sequence analysis of the NZ1-B and NZ2 coat proteins indicated significant substitutions that are predicted to alter the coat protein structure, which potentially leads to the observed reduced immunological reactivity of both variants to the Bioreba anti-GLRaV-3 conjugated monoclonal antibody.     

Population genetic analysis of grapevine fanleaf virus

Population genetic analysis of grapevine fanleaf virus (GFLV) was done on the basis of the virus movement protein (MP) gene sequences from the isolates detected and identified in this study and those of all previously reported GFLV strains/isolates. These revealed that the GFLV populations of Iran and Slovenia were highly distinct, whereas those of France, Germany, Italy and the USA were composed of multiple lineages. All populations were significantly differentiated from each other. However, two GFLV isolates from Tunisia, the only recorded GFLVs from that country, were not statistically distinct from the French, German and Italian populations. The ratio of non-synonymous nucleotide diversity to synonymous nucleotide diversity (Pi(a)/Pi(s)) was less than 1, suggesting that the MP gene has been under purifying selection. The neutrality tests were indicative of a balancing selection that is operating within Iranian and USA GFLV isolates, but they show a purifying selection within the other populations. Eleven recombination events were detected in a total of 50 isolates from France, Germany, Iran, Italy, Slovenia and the USA. The results from the recombination analysis were in agreement with those of the phylogenetic analysis. This study suggests that diversity among GFLV geographical populations resulted from possible host adaptation, recombination and founder effects.     

Variation of the antimicrobial susceptibility profiles of <Emphasis Type="Italic">Burkholderia cepacia</Emphasis> complex clonal isolates obtained from chronically infected cystic fibrosis patients: a five-year survey in the major Portuguese treatment center

The treatment of cystic fibrosis (CF) patients chronically infected with Burkholderia cepacia complex (Bcc) bacteria requires extensive and aggressive antibiotics therapy, exposing these bacteria to prolonged antibiotics-selective pressure. In the present study, we have compared the susceptibility patterns to 13 antimicrobials of 94 Bcc isolates obtained from 15 Portuguese CF patients in the course of chronic infection during a five-year survey. These isolates were previously genotyped and represent 11 different strains of the species B. cenocepacia (subgroups A and B), B. cepacia, B. multivorans, and B. stabilis. The results are consistent with the notion that CF Bcc isolates are resistant to the most clinically relevant antimicrobials and suggest an uneven distribution of resistance rates among the different species, with B. cenocepacia subgroup A isolates being the most resistant. Phenotypic variants exhibiting differences in the antimicrobial susceptibility patterns were obtained from the sputum samples of clinically deteriorated CF patients during chronic lung infection. The isolation of resistant variants coincided with periods of pulmonary exacerbation and antibiotics therapy.     

Rupestris stem pitting associated virus isolates are composed by mixtures of genomic variants which share a highly conserved coat protein

Summary. Broad spectrum primers were used to amplify a fragment comprising the CP gene and putative ORF6 by RT-PCR from ds-RNA templates originating from 46 Portuguese varieties, totalling 190 samples, including some wild Vitis ssp sylvestris vines, and 2 vines from Slovenia. SSCP analysis was used as a preliminary screen to avoid cloning and sequencing very similar variants. Four groups of variants were recognized. In pair wise comparisons between nucleotide sequences the minimal homology found was 81%. In case of the cultivated varieties, no relationship could be seen between the phylogenetic groups and geographic origin or grape variety. Several isolates were found harbouring mixed infections with genomic variants from different groups, but the mixing did not lead to an extensive recombination between them. The deduced amino-acid sequences revealed a conserved CP subjected to strong purifying selection pressure. Analysis of the selection pressure operating on the putative ORF6 suggests that this ORF does not exist. Previously produced polyclonal antiserum raised against the recombinant CP of RSPaV expressed in Escherichia coli was shown to be able to detect all four groups of variants of RSPaV included in this study, which might enable the diagnosis of the virus on a serological basis.     

Diversity within natural progenies of the grapevine dieback fungus Eutypa lata

The diversity within 16 natural progenies of the grapevine dieback fungus, Eutypa lata, was investigated by sampling single-ascospore isolates mainly in France and using random amplified polymorphic DNA (RAPD) markers, vegetative compatibility (VC), and pathogenicity testing. The combination of RAPD and VC data identified each isolate as a unique genotype within each progeny. Only three RAPD haplotypes did not cluster within the expected groups, i.e. the ascospore families. Within each set of clustering haplotypes, Mendelian 1:1 ratios for absence and presence were observed for RAPD markers, indicating that each progeny was the result of a biparental cross. Only one mycelium was obtained when isolation was performed from the discolored wood sustaining the perithecial stroma. This mycelium was identified as a likely parent of the corresponding progeny by RAPD analysis. The level of diversity measured by the average distance between haplotypes calculated from RAPD data, the percentage of vegetatively compatible pairs and the range of pathogenicity appeared similar between all but one progeny, indicating that crosses occurred within a random-mating population. All the results were consistent with the hypothesis that E. lata is a random-mating species having a high degree of genetic diversity. Received: 18 December 1998 / 7 July 1999     

Phylogenetic Analysis of Hop and Grapevine Isolates of Hop Stunt Viroid Supports a Grapevine Origin for Hop Stunt Disease

We have examined sequence variability among nine isolates of hop stunt viroid (HSVd) collected from hop gardens in Tohoku district in Japan, the only area in the world where hop stunt disease is endemic. Six different consensus and one-consensus sequences as well as 12 sequence variants were detected in the nine HSVd-hop isolates, which suggested the sequence of HSVd-hop was remarkably variable. A neighbor-joining analysis was carried out on the new HSVd-hop sequences together with 44 previously described variants of HSVd isolated from hop and other species. All the HSVd-hop sequences recovered from hops cultivated in the Tohoku district of Japan as well as the type isolate and two Korean isolates form a cluster with the HSVd-g subtype 1 commonly recovered from grapevine. This close relationship between HSVd-hop and -grapevine isolates strongly supports the grapevine origin for hop stunt disease.     

Variations in Two Gene Sequences of Citrus Tristeza Virus after Host Passage

We estimated genetic variation in two groups of Citrus tristeza virus (CTV) isolates: one of them (isolates T385, T317, T318 and T305) derived from a Spanish source by successive host passages, and the other (isolates T388 and T390), obtained after aphid transmission of a Japanese source. The population structure of these isolates had been characterized by single-strand conformation polymorphism analysis of genes p18 and p20. The nucleotide sequences of representative haplotypes of each isolate and gene were used to estimate genetic diversity within and between isolates and to evaluate genetic differentiation between populations. Phylogenetic analysis of p18 and p20 sequence variants showed two main groups: one them included variants predominant in the severe isolates (T318, T305 and T388), and the other comprised variants present in both mild (T385, T317) and severe isolates. Most sequence variants of isolate T390 were not associated to these groups. In some isolates, within-isolate diversity was higher than diversity with other isolates because their population contained distantly related sequence variants, some of which were genetically close to variants predominant in the second isolate. Isolates T388 and T390 were genetically different for the two genes, as estimated by the F statistic. Furthermore, genetic differentiation between T385 and T317, T318 and T305 increased after each host passage. Our results suggest that aphid transmission and host passage may significantly alter the composition of CTV populations and thus be an important factor in their evolution.     

Phylogenetic analysis of five Portuguese strains of FIV

Summary.  Feline immunodeficiency virus (FIV) was isolated from five Portuguese cats. The five strains were named RP1, PP2, TLP3, FP4 and CP5. The LTR, the CA region of the gag gene, and the V3-V5 region of the env gene were amplified by PCR, cloned and sequenced. The phylogenetic relationships among these three regions and other previously published sequences revealed an independent clustering of the Portuguese isolates in the LTR and CA protein. Based on the V3-V5 region the Portuguese isolates were classified as Subtype B, although they appear to be a subcluster within Subtype B. The study of these new FIV isolates showed the presence of Subtype B in Portugal and could provide a contribution for the understanding of FIV’s genetic diversity. Received May 3, 2001; accepted October 25, 2001     

Characterisation of two citrus apscaviroids isolated in Spain

Summary.  Sequence variability in the PCR amplified cDNAs from two citrus apscaviroid isolates CVd-Ia and CVd-IIId from Spain, was analysed. CVd-IIId sequence was shown to be identical to previously described CVd-III sequences and no important variability was encountered within the viroid population. Conversely, CVd-Ia displayed population heterogeneity as shown by SSCP analysis, Sal I restriction site polymorphism and sequences of 27 CVd-Ia cloned DNAs. The CVd-Ia genomic heterogeneity is characterised by two major subpopulations with the most divergent sequences, and by the presence of individual variants, making a sequence continuum between the two major groups. Most sequence variations are clustered in the left part of the viroid molecule. Received October 1, 1999/Accepted April 3, 2000     

A sensitive one-step real-time RT-PCR method for detecting Grapevine leafroll-associated virus 2 variants in grapevine

Grapevine leafroll syndrome is caused by a complex of up to nine different Grapevine leafroll-associated viruses (GLRaV-1-9) with GLRaV-2 being reported as one of the most variable species of this group. Many methods, including indexing, serological and molecular procedures, have been developed for the detection of GLRaV-2. However, due to the low concentration of the virus in plants and the high variability of GLRaV-2, a method with improved sensitivity and with the capacity to detect of all known variants is required. Such improvement is essential for grapevine rootstocks, as these are suspected to harbour frequent GLRaV-2 infections difficult to detect, thus contributing to the spread of the leafroll disease. The development of new universal primers is described using a target sequence located in the 3' end of the virus genome. These primers were combined with a one-step SYBR Green real-time RT-PCR assay to achieve quantitative detection. All 43 GLRaV-2 isolates tested in this study were identified readily and reproducibly, regardless of their geographical origin or variety of grapevine. Using the procedure developed in this study, the sensitivity was increased 125 times compared to a conventional single-tube RT-PCR. This real-time method opens new perspectives for the sanitary selection of grapevine and in leafroll 2 disease monitoring.     

Evidence for three groups of sequence variants of beet necrotic yellow vein virus RNA 5

Summary.  About half of Japanese isolates of beet necrotic yellow vein virus (BNYVV) were found to contain RNA 5 molecules, which were also detected in virus isolates from China and France. Sequence comparisons of RNA 5 (nucleotides 327 to 1171) in 25 isolates showed that there are up to 8% sequence differences, and that RNA 5 variants fall into three groups: group I contains most of the Japanese and Chinese isolates, group II two Japanese isolates, and group III four French isolates. The group I isolates fall into three small clusters. In the 26 kDa coding region of RNA 5, there was a maximum of 1.5% nucleotide sequence differences (6 amino acid changes) within the group and 8.4% nucleotide sequence differences (17 amino acid changes) between the groups. Comparisons of the coat protein gene of RNA 2 revealed that most of the Japanese and Chinese isolates belonged to the A type strain, but some isolates were of the B type. The French isolates (P type) were closely related to those of the A type. Mixed infections of the two types of virus and the two groups of RNA 5 were detected in a small area of Hokkaido. BNYVV might have been introduced into Japan and China by a similar route from at least two origins. These results, together with other evidence, suggest that the three groups of RNA 5 variants separated from an original population a long time ago and, thereafter, the group I population diverged further into three clusters, which may have been associated with the A type strain rather than the B type. Received November 3, 1998 Accepted January 19, 1999     

Stem pitting and seedling yellows symptoms of Citrus tristeza virus infection may be determined by minor sequence variants

The isolates of Citrus tristeza virus (CTV), the most destructive viral pathogen of citrus, display a high level of variability. As a result of genetic bottleneck induced by the bud-inoculation of CTV-infected material, inoculated seedlings of Citrus wilsonii Tanaka displayed different symptoms. All successfully grafted plants showed severe symptoms of stem pitting and seedling yellows, while plants in which inoculated buds died displayed mild symptoms. Since complex CTV population structure was detected in the parental host, the aim of this work was to investigate how it changed after the virus transmission, and to correlate it with observed symptoms. The coat protein gene sequence of the predominant genotype was identical in parental and grafted plants and clustered to the phylogenetic group 5 encompassing severe reference isolates. In seedlings displaying severe symptoms, the low-frequency variants clustering to other phylogenetic groups were detected, as well. Indicator plants were inoculated with buds taken from unsuccessfully grafted C. wilsonii seedlings. Surprisingly, they displayed no severe symptoms despite the presence of phylogenetic group 5 genomic variants. The results suggest that the appearance of severe symptoms in this case is probably induced by a complex CTV population structure found in seedlings displaying severe symptoms, and not directly by the predominant genomic variant.     

Three genetic grapevine leafroll-associated virus 3 variants identified from South African vineyards show high variability in their 5′UTR

Three genetic variants of grapevine leafroll-associated virus 3 (GLRaV-3) were identified in vineyards of the Western Cape, South Africa. The GLRaV-3 variants were identified by single-strand conformation polymorphism (SSCP) profiles generated from a region amplified in ORF5. ORF5 sequence data confirmed the three genetic variant groups, and a specific SSCP profile was assigned to each variant group. The results of SSCP analysis of this region in ORF5 showed that this method gives a fast and reliable indication of the GLRaV-3 variant status of a plant, which in many instances showed mixed infections. The full genome sequence of one representative of each variant group i.e. isolates 621 (group I), 623 (group II) and PL-20 (group III), was determined by sequencing overlapping cloned fragments of these isolates. The sequences of genomic 5′ ends of these isolates were determined by RLM-RACE. Sequence alignment of the 5′UTRs indicated significant sequence and length variation in this region between the three South African variant groups. Alignment of the Hsp70h and CP gene regions of these isolates with those of isolates from elsewhere in the world, followed by phylogenetic analysis, further supported the presence of three variants of GLRaV-3 in South Africa and the presence of two or three additional variant groups elsewhere in the world.