A distinct tymovirus infecting Cassia hoffmannseggii in Brazil

Leaves of Cassia hoffmannseggii, a wild fabaceous species found in the Atlantic Forest, with a severe mosaic symptom were collected in Pernambuco State, Brazil. By transmission electron microscopy, two types of virus particles were found: the first was recognized as particles of a potyvirus, which was later identified as Cowpea aphid-borne mosaic virus; and the second was isometric and present in high concentration. The observation of vesicles at the periphery of chloroplasts suggested a tymovirus infection, which was confirmed by subsequent assays. A serological assay against several tymovirus antisera resulted in positive reaction of this tymo-like virus with an antiserum of Passion fruit yellow mosaic virus. By means of RT-PCR and using degenerated primers for the conserved region of RNA-dependent RNA polymerase (RdRp) gene of tymoviruses, a specific DNA fragment was amplified and sequenced. Based on this sequence, a specific forward primer was synthesized and successfully used to amplify the 3' terminal genome region, containing the partial RdRp gene and the complete coat protein (CP) sequences. The CP was 188 amino acids (aa) long, and the highest CP aa identity was observed with Kennedya yellow mosaic virus (61 %). Based on the current ICTV demarcation criterion, this isolate was considered as a distinct tymovirus and tentatively named as Cassia yellow mosaic-associated virus.     

Characterization of tomato yellow vein streak virus,a begomovirus from Brazil

Tomato yellow vein streak virus (ToYVSV) is a tentative begomovirus (Family Geminiviridae) species that seriously affects tomato and potato production in Brazil. Here, we have determined the genomic and biological characteristics of a ToYVSV isolate (Ba3) from a potato plant sampled in Rio Grande do Sul State, Brazil. The DNA-A nucleotide sequence of Ba3 and another previously reported ToYVSV isolate share 89.7% sequence identity. These ToYVSV isolates should be classified as a new species in that they are most closely related to Soybean blistering mosaic virus with which they share only ~80% identity. Cloned constructs containing 1.5 mer copies of the ToYVSV genomic components were found, by biolistic bombardment, to be infectious in at least 11 plant species in 2 families (Solanaceae and Malvaceae). Symptoms on tomato and potato plants were identical to those originally observed on field-infected plants. ToYVSV was also sap-transmissible from Nicotiana benthamiana to N. benthamiana and tomato, but not to potato plants.     

Six novel begomoviruses infecting tomato and associated weeds in Southeastern Brazil

The incidence of tomato-infecting begomoviruses has sharply increased in Brazil following the introduction of the B biotype of the whitefly vector in the early 1990s. Five definitive species and six tentative species have been described since then. Here, we report the detection of members of an additional six novel species, three in tomato and three infecting weeds that are commonly associated with tomato fields: Blainvillea rhomboidea, Sida rhombifolia and Sida micrantha. Tomato and weed samples were collected in two major tomato-growing regions of southeastern Brazil in 2005 and 2007. Two of the novel viruses were present in tomato plants collected in Paty do Alferes, Rio de Janeiro state. Three novel viruses were present in weed samples collected in Coimbra, Minas Gerais state. One virus was present in tomato samples collected at both locations. Genome features indicate that all six species are typical New World, bipartite begomoviruses. However, the viruses belonging to two of the novel species did not cluster with the Brazilian viruses in a phylogenetic tree. These species could represent a distinct lineage of New World begomoviruses, found in Brazil for the first time. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. The sequences reported in this paper have been deposited in the GenBank under the accession numbers EU710749–EU710757.     

Characterization of small RNAs derived from Citrus exocortis viroid (CEVd) in infected tomato plants

In plants, RNA silencing provides an adaptive immune system that inactivates pathogenic nucleic acids, guided by 21-24-mer RNAs of pathogen origin. The characterization of pathogen-related small RNAs (sRNAs) is relevant to uncovering the strategies used by pathogens to evade host defense responses. Several groups have reported the detection of viroid-derived sRNAs during infections, although the origin of these sRNAs and their chemical characteristics were poorly understood. Here, we describe the in vivo cleavage of Citrus exocortis viroid (CEVd) RNA into sRNAs of 21-22 nt, that are phosphorylated at their 5'-end and methylated at 3'. Our studies suggested that the CEVd genomic RNA might be the predominant in vivo substrate for cleavage by Dicer-like enzyme(s), which preferentially targeted residues mainly located within the right-end domain of the viroid. Further analysis on the accumulation levels of specific miRNAs controlling major regulators of leaf development and the miRNA pathway and the levels of their target mRNAs provided evidence that the endogenous tomato miRNA pathway was not affected by CEVd infection.     

Molecular characterization of a new tymovirus from <Emphasis Type="Italic">Diascia</Emphasis> ornamental plants

Two tymoviruses were identified in plants of Diascia x hybrida 'Sun Chimes Coral' that exhibited chlorotic mottling and reduced growth. A strain of Nemesia ring necrosis virus (NeRNV) designated NeRNV-WA was detected in symptomatic plants; the deduced amino acid sequence is virtually identical to that of the previously reported NeRNV-Nf from Nemesia fruticosa. Sequence analysis also revealed the presence of a new tymovirus, and the entire genomic sequence of this virus was determined. The genome of 6,290 nucleotides was organized into three potential open reading frames (ORFs) typical of viruses in the genus Tymovirus. Based on sequence identity to tymovirus sequences, ORFs I to III encoded the replicase, movement protein and coat protein, respectively. Amino acid sequence identities to those of NeRNV-Nf were 84.8, 50.3 and 94.8%, respectively. The 5'-untranslated region could potentially form four hairpin structures. Secondary structure analysis of the 3'-terminus showed that the RNA can form a transfer-RNA-like structure that has an anticodon specific for histidine. Only 77.9% nucleotide identity was found when complete genomic sequences of this tymovirus from diascia and NeRNV-Nf were compared. The name Diascia yellow mottle virus (DiaYMV) is proposed for this new tymovirus.     

Characterization of tomato zonate spot virus,a new tospovirus in China

An isolate of a new tospovirus species, causing concentric zoned ringspots on fruits and necrotic lesions on leaves of infected plants, was characterised based on particle morphology, host range and serological properties. The complete nucleotide sequences of large (L), medium (M), and small (S) RNAs of this virus were found to contain 8919, 4945, and 3279 nts respectively. The L RNA encoded the RNA-dependent RNA polymerase (RdRp) (2885 aa, 332.7 kDa). The M RNA encoded a non-structural (NSm) protein (309 aa, 34.4 kDa) and a viral glycoprotein precursor (Gn/Gc) (1122 aa, 127.4 kDa). The S RNA encoded a non-structural protein (NSs) (459 aa, 51.9 kDa) and the nucleocapsid (N) protein (278 aa, 30.6 kDa). This N protein shared amino acid identities of 80.9% with those of calla lily chlorotic spot virus. Our results suggest that the virus studied here belongs to a new tospovirus species, for which the name tomato zonate spot virus is proposed. Jia-Hong Dong and Xiao-Fei Cheng: making equal contributions to this paper. The nucleotide sequence data reported in this paper are available in the GenBank database under accession numbers: EF552433 (S RNA), EF552434 (M RNA), EF552435 (L RNA).     

Characterization of a member of a new Potyvirus species infecting arracacha in Brazil

An arracacha (Arracacia xanthorrhiza) plant collected in Brazil was found to be infected by a new virus. This viral isolate (named C17) systemically infected Nicotiana benthamiana and Apium graveolens. A polyclonal antibody was raised, and analysis of our arracacha germplasm collection showed a high infection rate of C17-like viruses (93% infection). Sequencing of the ca. 1.7 kb 3′-terminal genomic region revealed a typical potyvirus genome organization. It shared less than 70% nucleotide identity with any other potyvirus sequence, which thus indicated that it is possibly a member of a new Potyvirus species tentatively named Arracacha mottle virus (AMoV).     

Reduced allergenicity of tomato fruits harvested from Lyc e 1-silenced transgenic tomato plants

BACKGROUND: Profilin is a small actin-binding protein that contributes to the allergenic potency of many fruits and vegetables, including tomato. Two highly similar genes encoding tomato profilin have been isolated and designated as allergen Lyc e 1.01 and Lyc e 1.02. OBJECTIVE: The aim of the study was to generate profilin-reduced hypoallergenic tomato fruits by silencing of both genes in transgenic tomato plants by means of RNA interference (RNAi). METHODS: The efficiency of gene silencing was documented by means of Northern blotting, immunoblotting, and skin prick testing. RESULTS: Quantification of the remaining protein revealed that profilin accumulation in transgenic fruits was decreased 10-fold compared with that seen in untransformed controls. This decrease was sufficient to cause a reduced allergenic reactivity in patients with tomato allergy, as determined with skin prick tests. Because most patients with tomato allergy are not monosensitized to profilin, the IgE reactivity to the profilin-silenced tomato fruits in vivo varied widely between individuals tested. CONCLUSION: We could demonstrate the efficient silencing of both profilin genes in transgenic tomato plants using RNAi. This resulted in Lyc e 1-diminished tomato fruits, providing proof of concept and demonstrating that RNAi can be used to design allergen-reduced food. However, simultaneous silencing of multiple allergens will be required to design hypoallergenic tomatoes. CLINICAL IMPLICATIONS: Our findings demonstrate the feasibility of creating low-allergenic food by using RNAi. This concept constitutes a novel approach to allergen avoidance.     

Genetic variation in populations of kennedya yellow mosaic tymovirus

Summary Kennedya yellow mosaic tymovirus (KYMV) occurs along the eastern Australian seaboard in the perennial legumesDesmodium triflorum andD. scorpiurus in the north, andKennedya rubicunda in the south. The genetic variation of more than 100 isolates of KYMV, most of them from the north, has been studied using an RNA hybrid mismatch polymorphism (RHMP) method. The method clearly separated the isolates into two groups; all the northernDesmodium isolates formed one group and all theKennedya isolates from the south another. These sub-populations were themselves variable and theDesmodium population alone was more variable than that of the related turnip yellow mosaic tymovirus in the Kosciusko alpine area.     

Soybean chlorotic spot virus, a novel begomovirus infecting soybean in Brazil

A novel soybean-infecting begomovirus from Brazil was identified in Jaíba, in the state of Minas Gerais, and molecularly characterized. By using rolling-circle amplification-based cloning of viral DNAs, three DNA-A variants and a cognate DNA-B were isolated from infected samples. The DNA variants share more than 98 % sequence identity but have less than 89 % identity to other reported begomovirus, the limit for demarcation of new species. In a phylogenetic analysis, both DNA-A and DNA-B clustered with other Brazilian begomoviruses. Infectious cloned DNA-A and DNA-B components induced distinct symptoms in Solanaceae and Fabaceae species by biolistic inoculation. In soybean, the virus induced mild symptoms, i.e., chlorotic spots on the leaves, from which the name soybean chlorotic spot virus (SoCSV) was proposed. The most severe symptoms were displayed by common beans, which exhibited leaf distortion, blistering, interveinal chlorosis, mosaic and golden mosaic. The possibility that SoCSV may become a threat to bean production in Brazil is discussed.     

Characterization of a new anulavirus isolated from Amazon lily plants

A quasi-spherical virus was isolated from a cultivated Amazon lily plant (Eucharis grandiflora) that could be mechanically transmitted to healthy E. grandiflora plants, subsequently producing mild mosaic or mottle symptoms on the leaves. The purified virus consisted of three quasi-spherical particles about 20 nm wide and 70, 40 and 30 nm in length, containing three segmented genomes of 3,169, 2,507 and 2,530 nucleotides, respectively. Sequence analysis showed that the newly isolated virus is related to pelargonium zonate spot virus, a member of the genus Anulavirus. We propose that the virus should be designated as Amazon lily mild mottle virus (ALiMMV).     

Characterization of a novel Taenia solium oncosphere antigen

Infections due to Taenia solium in humans (taeniasis/cysticercosis) remain a complex health problem, particularly in developing countries. We identified two oncosphere proteins that might protect the porcine intermediate host against cysticercosis and therefore help prevent disease in humans. One of these proteins was further identified by two-dimensional gel electrophoresis and micro-sequencing. The gene encoding this protective protein was also identified, cloned and characterized. The native 31.5 kDa protein Tso31 has four variants at the cDNA level. The longest sequence from which the others seem to derive, encodes a 253 amino acid peptide. The predicted protein has a molecular weight of 25.1 kDa, one putative N-glycosylation site, two fibronectin type III domains, and one C terminal transmembrane domain. The gene structure of the protein consists of four exons and three introns. The finding of one gene and four different cDNAs for Tso31 suggests the existence of a possible mechanism of differential splicing in this parasite. The Tso31 protein is exclusive to T. solium oncospheres with a putative protein structure of an extra-cellular receptor-like protein. The Tso31 protein was expressed as a recombinant protein fused to GST and tested in a vaccine to determine its effectiveness in protecting pigs against cysticercosis. Only two pigs out of eight vaccinated were protected and although the total median number of cyst decreased in vaccinated pigs compared to controls this decrease was not statistically significant (P = 0.09).     

Characterization of a novel single-stranded RNA mycovirus in Pleurotus ostreatus

A mycovirus, named oyster mushroom spherical virus (OMSV), was isolated from cultivated oyster mushrooms with a severe epidemic of oyster mushroom Die-back disease. OMSV was a 27-nm spherical virus encapsidating a single-stranded RNA (ssRNA) of 5.784 kb with a coat protein of approximately 28.5 kDa. The nucleotide sequence of the virus revealed that its genomic RNA was positive strand, containing 5784 bases with seven open reading frames (ORF). ORF1 had the motifs of RNA-dependent RNA polymerases (RdRp) and helicase. ORF2 encoded a coat protein. ORF3 to 7 could encode putative polypeptides of approximately 12, 12.5, 21, 14.5, and 23 kDa, respectively, but none of them showed significant similarity to any other known polypeptides. The 5' end of the viral RNA was uncapped and the 3' end was polyadenylated with 74 bases. Genomic structure and organization and the derived amino acid sequence of RdRp and helicase domain were similar to those of tymoviruses, a plant virus group.     

Characterization of a novel KRAS mutation identified in Noonan syndrome

Noonan syndrome (NS) is the most common non-chromosomal syndrome seen in children and is characterized by short stature, dysmorphic facial features, chest deformity, a wide range of congenital heart defects and developmental delay of variable degree. Mutations in the Ras/mitogen-activated protein kinase (MAPK) signaling pathways cause about 70% of NS cases with a KRAS mutation present in about 2%. In a cohort of 65 clinically confirmed NS patients of Japanese origin, we screened for mutations in the RAS genes by direct sequencing. We found a novel mutation in KRAS with an amino acid substitution of asparagine to serine at codon 116 (N116S). We analyzed the biological activity of this mutant by ectopic expression of wild-type or mutant KRAS. NS-associated KRAS mutation resulted in Erk activation and active Ras-GTP levels, and exhibited mild cell proliferation. In addition, kras-targeted morpholino knocked-down zebrafish embryos caused heart and craniofacial malformations, while the expression of mutated kras resulted in maldevelopment of the heart. Our findings implicate that N116S change in KRAS is a hyperactive mutation which is a causative agent of NS through maldevelopment of the heart.     

Characterization of a novel allele HLA-B*3549

A novel HLA-B allele, B*3549, was identified in a bone marrow transplantation candidate. B*3549 differs from B*3525 by two nucleotides at exon 2, position 142 (T to G) and 165 (G to C). The difference at position 142 resulted in an amino acid difference (serine to alanine). However, the difference at position 165 did not cause any amino acid change. This novel allele was found on a haplotype with A*3101, B*3549, Cw*0401, DRB1*0407, and DQB1*0302.     

RNA hybrid mismatch polymorphisms in Australian populations of turnip yellow mosaic tymovirus

Summary In the Mt. Kosciusko alpine area of Australia there are three well-separated populations ofCardamine lilacina, an endemic sward-forming perennial brassica, and these are infected with turnip yellow mosaic tymovirus. The genetic variation in these viral populations has been assessed by an RNA hybrid mismatch polymorphism method. About 100 isolates were examined; the genomic RNA of each isolate was prepared from a shoot of a single wildC. lilacina plant. RNA hybrid mismatch polymorphisms (RHMPs) were assessed in six regions of the genomes using labelled negative-strand probes transcribed from selected portions of a cloned TYMV genome. The probed region at the 3 end of the genome showed little variation and over 95% of the isolates gave the same pattern. However, other parts of the genome, including the 5 non-coding region, were much more variable. There was no significant correlation between groupings based on the RHMP patterns, and the location from which the isolates were collected, nor with the symptom type or severity shown by their host plants. The patterns of variation suggested that all three populations of the virus are a single quasi-species; at most one tenth of the isolates gave similar RHMP patterns, those of the master copy.