Molecular variability of Tobacco vein banding mosaic virus populations

The incidence of Tobacco vein banding mosaic virus (TVBMV) on tobacco increases dramatically in China recently and it has caused great economic losses. To gain insights into the evolutionary mechanisms of TVBMV, a total of 40 TVBMV isolates were collected from different tobacco production regions in China and their genomic regions encoding helper component-proteinase (HC-Pro), the third protein (P3), the first 6K protein (6K1) and coat protein (CP) were sequenced. Phylogenetic analyses revealed that TVBMV isolates can be divided into two evolutionary divergent groups based on P3, the frame-shifting pipo and 6K1 genes, and three groups on HC-Pro and CP genes. The populations from most parts of mainland China (MC) showed frequent gene flow; those from Yunnan province in south western China always formed a separate group (YN) and also had frequent within-group gene flow. However, the gene flow between groups MC and YN was uncommon. Our results revealed that all the tested TVBMV genes were under negative selection and the HC-Pro gene was under the strongest constraints. Recombination events were identified in 13 of the 42 analyzed isolates. This study suggested that negative selection, gene flow and recombination were important evolutionary factors driving the genetic diversification of TVBMV.     

Characterization of 10 tobacco vein banding mosaic virus isolates from China

The complete RNA genome sequences of 10 tobacco vein banding mosaic virus (TVBMV) isolates from China were determined using five overlapping cDNA clones. TVBMV was divided into three groups for HC-Pro and CP and into two groups for P3 and 6K1. With more isolate sequences analyzed, the phylogenetic results suggest that TVBMV could be divided into four subgroups based on HC-Pro and CP and into three subgroups with P3 and 6K1. Nucleotide sequence diversity analysis showed geographical differentiation among the TVBMV isolates. Three of the 10 isolates were found to have undergone recombination and new recombination sites were identified in the TVBMV genome. All coding genes were under negative selection and the population appeared to have remained stable over a long period. This study also provides preliminary data on the 3′-untranslated region as potentially the best genome sequence for developing transgenic tobacco.     

Complete genome sequence of a tobacco isolate of the tobacco vein banding mosaic virus strain prevailing in China

Tobacco vein banding mosaic virus (TVBMV) is a species of the largest plant virus genus Potyvirus. Its incidence has been increasing in Chinese tobacco-growing area. TVBMV isolates can be clustered into three genetic groups that are corresponding with their geographical origin. We have reported the complete genomic sequence of TVBMV isolate YND with unique NIb/CP cleavage site. Here, we determined and analyzed the complete genomic sequence of isolate HN39, which was collected from tobacco in Henan Province and represented Chinese prevalent strain of TVBMV. HN39 has similar host range with YND, but induce mild vein banding symptom in Nicotiana tabacum cv. Samsun. The genome of TVBMV-HN39 is composed of 9,570 nucleotides, excluding the poly(A) tail. It contains a large ORF of 9,240 nucleotides and encode a polyprotein of 3,079 amino acids. The putative NIa-Pro cleavage site for NIb/CP is Q/G. The identities between the complete genomes of isolates HN39 and YND were 90.0% at nucleotide level and 95.4% at amino acid level. As for other potyviruses, HN39 shared the highest identity with wild tomato mosaic virus (WTMV) at complete genomic level, while different genes shared the highest identities with different potyviruses. This is the second complete genomic sequence of TVBMV reported.     

Molecular analysis of gooseberry vein banding associated virus

The intraspecies variability of gooseberry vein banding associated virus (GVBaV) was analyzed by using 5 complete and 9 partial sequences and compared with other badnavirus species. GVBaV was recognized to be a?monophyletic and very homogeneous species with up to 94% identities in distinct proteins. Analysis of non-synonymous and synonymous substitution ratios (dNS/dS) revealed higher values for ORF4 in comparison with other genes. This could reflect different evolutionary pressure upon this ORF. A?highly variable region with possible diagnostic value has been localized in the intergenic region of the virus. Keywords: badnavirus; complete genome; phylogeny.     

Molecular cloning of DNA complementary to tobacco vein mottling virus RNA

RNA isolated from tobacco vein mottling virus (TVMV) was used as a template for avian myeloblastosis virus RNA-dependent DNA polymerase, primed with oligo(dT). The largest single-stranded cDNA synthesized was 10 kb, the same as the viral RNA. This material was converted to double-stranded cDNA with Escherichia coli DNA polymerase I and digested with restriction endonuclease HindIII. The cDNA fragments were ligated to HindIII-digested plasmid pBR322 and the product used to transform E. coli strain DG-75. Clones containing recombinant plasmids were selected by ampicillin resistance, and those containing TVMV RNA sequences were selected by colony hybridization with a single-stranded cDNA probe. Four different sizes of recombinant plasmid were reproducibly observed. The inserted DNA portion could be excised in each case with HindIII. The lengths of inserted DNA were 3.0, 1.85, 1.1, and 0.72 kb. A similar procedure was used with PstI-digested cDNA and pBR322. A single type of recombinant plasmid, containing a DNA insertion of 1.85 kb, was reproducibly observed. Hybridization with TVMV RNA confirmed that the five inserted DNA segments were derived from the viral RNA. Hybridization of each recombinant plasmid with the others established that each of the cloned HindIII fragments was unique and that one of them overlapped the cloned PstI fragment. The cloned cDNA fragments were ordered by establishing a restriction map of the cDNA. Together the cloned cDNA fragments account for over 80% of the viral genome.     

Differential pulse-polarographic analysis of tobacco mosaic virus

The tobacco mosaic virus (TMV) strain vulgare and its mutant TMV 483 (with glutamine-9 replaced by histidine) and the denatured protein of TMV vulgare were analysed by direct current (d. c.) and differential (derivative) pulse polarography (DPP) in the basic electrolyte composed of 0.001 M Co(NH3)6Cl3, 0.1 M NH4Cl and 0.1 M NH3 at 0 degrees C. The DPP method gave a substantially better resolution of the polarographic catalytic maxima A and B, but a much lower resolution of the maxima B and C, as compared with the d. c. polarographic method. The clear differentiation of the maximum A from maximum B by DPP permitted to study the variation of maximum A in the course of alkaline degradation of TMV. But for the study of TMV protein denaturation the d. c. polarography is preferable, because the denaturation is accompanied by the appearance and rise of maximum C which can be clearly differentiated from maximum B by d. c. polarography rather than by DPP. The DPP method was more, sensitive than d. c. polarography. The denatured TMV protein can be determined by DPP at concentrations around 0.1 microgram/ml.     

Molecular characterization and population structure of blackberry vein banding associated virus,new ampelovirus associated with yellow vein disease

Blackberry yellow vein disease is the most important viral disease of blackberry in the United States. Experiments were conducted to characterize a new virus identified in symptomatic plants. Molecular analysis revealed a genome organization resembling Grapevine leafroll-associated virus 3, the type species of the genus Ampelovirus in the family Closteroviridae. The genome of the virus, provisionally named blackberry vein banding associated virus (BVBaV), consists of 18,643 nucleotides and contains 10 open reading frames (ORFs). These ORFs encode closterovirid signature replication-associated and quintuple gene block proteins, as well as four additional proteins of unknown function. Phylogenetic analyses of taxonomically relevant products consistently placed BVBaV in the same cluster with GLRaV-3 and other members of the subgroup I of the genus Ampelovirus. The virus population structure in the U.S. was studied using the replication associated polyprotein 1a, heat shock 70 homolog and minor coat proteins of 25 isolates. This study revealed significant intra-species variation without any clustering among isolates based on their geographic origin. Further analyses indicated that these proteins are under stringent purifying selections. High genetic variability and incongruent clustering of isolates suggested the possible involvement of recombination in the evolution of BVBaV.     

Infection of tobacco leaf epidermal protoplasts with tobacco mosaic virus

Protoplasts from the epidermal cells of tobacco leaves were prepared and inoculated with tobacco mosaic virus. Up to 50% of the protoplasts became infected and, within 3 days, contained an average of 5 x 10(5) virus particles per protoplast. Hexagonal crystalline inclusions were observed in many of the infected protoplasts.     

Retention and dissociation of tobacco mosaic virus by tobacco protoplasts

When tobacco protoplasts were inoculated with radioactive tobacco mosaic virus (TMV) at a concentration of 1 μg/ml, about 15% of the particles (approximately 8 × 10³ particles per protoplast) were retained. When poly-l-ornithine was not included in the inoculum, the amount of virus retained was reduced 6- to 30-fold. Maximum retention was observed 20–30 min after the addition of virus to the protoplasts.Thirty minutes after mixing virus and protoplasts, about 5% of the protein of the retained TMV had been removed and some of the virus particles appeared to be partially dissociated. A considerable amount of the retained viral material in extracts of inoculated protoplasts was found in the pellets after sucrose gradient centrifugation. This abnormally rapid sedimentation was correlated with the presence of poly-l-ornithine in the inoculum.     

Cation binding by tobacco mosaic virus.

Hydrogen ion titration curves of tobacco mosaic virus and its protein, alone or in the presence of various multivalent ions, have been measured. Three groups titrating near neutral pH in the virion have significant metal ion binding, but the tightest of these binding sites, significantly specific for Ca²⁺ over Mg²⁺ appears to be absent from the RNA-free protein. A low Ca″ concentration appears to be a necessary but not a sufficient condition for TMV disassembly.