[Show abstract][Hide abstract] ABSTRACT: Background
Beet necrotic yellow vein virus (BNYVV) is the infectious agent of sugar beet rhizomania, which consists of four or five plus-sense RNAs. RNA4 of BNYVV is not essential for virus propagation in Nicotiana benthamiana but has a major effect on symptom expression. Early reports showed that RNA4-encoded P31 was associated with severe symptoms, such as curling and dwarfing, in N. benthamiana.
We discovered that the pathogenesis-related protein 10 (PR-10) gene can be up-regulated in BNYVV-infected N. benthamiana in the presence of RNA4 and that it had a close link with symptom development. Our frame-shift, deletion and substitution analysis showed that only the entire P31 could induce PR-10 up-regulation during BNYVV infection and that all the tryptophans and six cysteines (C174, C183, C186, C190, C197 and C199) in the cysteine-rich P31 had significant effects on PR-10 expression. However, P31 could not interact directly with PR-10 in yeast.
Our data demonstrated that only integrated P31 specifically induced PR-10 transcription, which coincided closely with the appearance of severe symptoms in BNYVV-infected N. benthamiana, although they could not interact directly with each other in yeast.
[Show abstract][Hide abstract] ABSTRACT: For brassica yellows virus (BrYV), proposed to be a member of a new polerovirus species, two clearly distinct genotypes (BrYV-A and BrYV-B) have been described. In this study, the complete nucleotide sequences of two BrYV isolates from radish and Chinese cabbage were determined. Sequence analysis suggested that these isolates represent a new genotype, referred to here as BrYV-C. The full-length sequences of the two BrYV-C isolates shared 93.4-94.8 % identity with BrYV-A and BrYV-B. Further phylogenetic analysis showed that the BrYV-C isolates formed a subgroup that was distinct from the BrYV-A and BrYV-B isolates based on all of the proteins except P5.
No preview · Article · Mar 2014 · Archives of Virology
[Show abstract][Hide abstract] ABSTRACT: A new carlavirus, tentatively named Potato virus H (PVH), was found on potato plants with mild symptoms in Hohhot, Inner Mongolia Autonomous Region, China. PVH was confirmed by genome sequencing, serological reactions, electron microscopy, and host index assays. The PVH particles were filamentous and slightly curved, with a modal length of 570 nm. Complete RNA genomic sequences of two isolates of PVH were determined using reverse transcription-PCR (RT-PCR) and the 5' rapid amplification of cDNA ends (5' RACE) method. Sequence analysis revealed that PVH had the typical genomic organization of members of the genus Carlavirus, with a positive-sense single-stranded genome of 8410 nt. It shared coat protein (CP) and replicase amino acid sequence identities of 17.9-56.7% with those of reported carlaviruses. Phylogenetic analyses based on the protein-coding sequences of replicase and CP showed that PVH formed a distinct branch, which was related only distantly to other carlaviruses. Western blotting assays showed that PVH was not related serologically to other potato carlaviruses (Potato virus S, Potato virus M, and Potato latent virus). PVH systemically infected Nicotianaglutinosa but not Nicotiana tabacum, Nicotianabenthamiana, or Chenopodiumquinoa, which is in contrast with the other potato carlaviruses. These results support the classification of PVH as a novel species in the genus Carlavirus. Preliminary results also indicated that a cysteine-rich protein encoded by the smallest ORF located in the 3' proximal region of the genome suppressed local RNA silencing and enhanced the pathogenicity of the recombinant PVX.
[Show abstract][Hide abstract] ABSTRACT: We determined the genome sequence of a new polerovirus that infects field pea and faba bean in China. Its entire nucleotide sequence (6021 nt) was most closely related (83.3% identity) to that of an Ethiopian isolate of chickpea chlorotic stunt virus (CpCSV-Eth). With the exception of the coat protein (encoded by ORF3), amino acid sequence identities of all gene products of this virus to those of CpCSV-Eth and other poleroviruses were <90%. This suggests that it is a new member of the genus Polerovirus, and the name pea mild chlorosis virus is proposed.
No preview · Article · Apr 2012 · Archives of Virology
[Show abstract][Hide abstract] ABSTRACT: The complete genome sequence of a cucurbit-infecting fabavirus was determined. Sequence analysis revealed that it had a genomic organization typical of fabaviruses, with genome segment sizes of 5870 nt (RNA-1) and 3294 nt (RNA-2). It shared CP and Pro-Pol amino acid sequence identities of 52.0-58.9% with those of reported fabaviruses. ELISA and western blots gave no cross-reactions between this cucurbit virus and broad bean wilt viruses 1 and 2. Based on molecular and serological criteria for species demarcation in the genus Fabavirus, the virus represents a distinct species, for which the species name Cucurbit mild mosaic virus (CuMMV) is proposed.
No preview · Article · Dec 2011 · Archives of Virology
[Show abstract][Hide abstract] ABSTRACT: For the detection of wheat yellow mosaic virus (WYMV), we established a reverse transcription loop-mediated isothermal amplification (RT-LAMP) method. Using Primer Explorer software, four sets of primers were designed and RT-LAMP assay reaction conditions were optimized. The RT-LAMP was performed at different times by four primer sets. Agarose gel analysis showed that WYMV could be detected after 30 min with the primer set III and after 45 min with the other three primer sets, both under the 80-min reaction time. RT-LAMP had the same results with the four primer sets, thus primer set III and 65°C for 80 min reaction were selected for virus detection. There was no significant different when avian myeloblastosis virus (AMV) and moloney murine leukemia virus (M-MLV) RT-LAMP with the four primer sets and M-MLV was chosen due to its relatively cheap price. The result on specificity showed that the assay could amplify WYMV specifically, and the sensitivity comparison showed that the RT-LAMP was 100 times more sensitive than conventional reverse-transcriptase-polymerase chain reaction (RT-PCR). Overall, RT-LAMP was found to be a simple, specific, sensitive, convenient and time-saving method for WYMV detection.
[Show abstract][Hide abstract] ABSTRACT: A series of recombinant viruses were engineered by gene replacements or gene insertions into an infectious cDNA clone of Tobacco necrosis virus A Chinese isolate (TNV-AC). TNV-AC-based virus induced gene silencing (VIGS) containing different structural arrangements of sequences targeting specific genes and variations in the positions of exogenous fragment insertions were evaluated along with temperature effects on inoculated plants. Replacement of the coat protein (CP) gene by exogenous gene fragments could not be applied to TNV-AC because the chimeric TNV-AC constructs were unable to induce silencing of corresponding endogenous gene and derivatives lacking the CP could not move systematically in Nicotiana benthamiana. We found that the optimal position for insertion of exogenous gene fragments was the region immediately behind the stop codon of the CP gene, and the maximum lengths of fragments that could be accommodated within virions was around 120 nt. An NbPDS fragment inserted into TNV-AC as an inverted repeat produced a VIGS derivative that could silence endogenous NbPDS in N. benthamiana with extremely high efficiency. We also found that temperature could significantly affect gene silencing efficiency of inoculated plants and the stability of heterologous gene fragments. Plants grown at 18? exhibited substantially higher gene silencing efficiency compared with 24?, and the duration of silencing persisted for more than 110 days. The TNV-AC-based VIGS vectors were also able to induce efficient silencing of endogenous Su and ChlH genes in N. benthamiana. Taken together, our results show that TNV-AC has the potential for development into a novel VIGS vector suitable for functional genomics of N. benthamiana.
No preview · Article · Oct 2011 · Progress in Biochemistry and Biophysics
[Show abstract][Hide abstract] ABSTRACT: The genomic RNA sequences of two genotypes of a brassica-infecting polerovirus from China were determined. Sequence analysis revealed that the virus was closely related to but significantly different from turnip yellows virus (TuYV). This virus and other poleroviruses, including TuYV, had less than 90% amino acid sequence identity in all gene products except the coat protein. Based on the molecular criterion (>10% amino acid sequence difference) for species demarcation in the genus Polerovirus, the virus represents a distinct species for which the name Brassica yellows virus (BrYV) is proposed. Interestingly, there were two genotypes of BrYV, which mainly differed in the 5'-terminal half of the genome.
No preview · Article · Aug 2011 · Archives of Virology
[Show abstract][Hide abstract] ABSTRACT: The complete genomic sequences of two distinct Beet western yellows virus (BWYV) genotypes infecting sugar beet in Beijing, named as BWYV-BJ(A) and BWYV-BJ(B) (GenBank accession number HM804471, HM804472, respectively), were determined by RT-PCR sub-cloning approach. BWYV-BJ(A) and BWYV-BJ(B) were 5674 and 5626nt in length, respectively. BWYV-BJ(B) was 48nt shorter than BWYV-BJ(A) in the regions 1589-1615 and 1629-1649nt. Sequence alignment analysis showed that the full length of BWYV-BJ(A) and BWYV-BJ(B) shared 93% nucleotide sequence identity, with relatively high variability within ORFs 0, 1, 2 (at the nucleotide level was 86.3-88.8%) and high conservation within ORFs 3, 4, 5 (at the nucleotide level was 99.3-99.5%). The complete nucleotide sequences of BWYV-BJ(A) and BWYV-BJ(B) were most related to BWYV-US (80.6 and 79.0%, respectively). ORFs 1, 2 of BWYV-BJ(A) and BWYV-BJ(B) shared the highest homology with BWYV-US (nucleotide identity 91.2-93.3, 86.7-89.5%, respectively) and their ORFs 3, 4 were more closely related to BWYV-IM. However, their ORF5 were more closely related to that of Cucurbit aphid-borne yellows virus China strain (CABYV-CHN), with 68.1 and 68.5% nucleotide identity, respectively. Based on the sequence and phylogenetic analysis, we proposed that BWYV-BJ was at least a novel strain of BWYV, and BWYV-BJ(A), BWYV-BJ(B) were two distinct genotypes of BWYV-BJ. In addition, phylogenetic analysis and recombination analysis suggested that BWYV-BJ(A) and BWYV-BJ(B) might be recombinant viruses.
[Show abstract][Hide abstract] ABSTRACT: Melon aphid-borne yellows virus (MABYV) is a newly identified polerovirus occurring in China. Here, we demonstrate that the MABYV encoded P0 (P0(MA)) protein is a strong suppressor of post-transcriptional gene silencing (PTGS) with activity comparable to tobacco etch virus (TEV) HC-Pro. In addition we have shown that the LP F-box motif present at the N-terminus of P0(MA) is required for suppressor activity. Detailed mutational analyses on P0(MA) revealed that changing the conserved Trp 212 with non-ring structured amino acids altered silencing suppressor functions. Ala substitutions at positions 12 and 211 for Phe had no effect on P0 suppression-activity, whereas Arg and Glu substitutions had greatly decreased suppressor activity. Furthermore, substitutions targeting Phe at position 30 also resulted in reduced P0 suppression-activity. Altogether, these results suggest that ring structured Trp/Phe residues in P0 have important roles in suppressor activity.
[Show abstract][Hide abstract] ABSTRACT: A new method for the fast discrimination of varieties of transgene wheat by means of near infrared spectroscopy and biomimetic pattern recognition (BPR) was proposed and the recognition models of seven varieties of transgene wheat and two varieties of acceptor wheat were built. The experiment adopted 225 samples, which were acquired from nine varieties of wheat. Firstly, a field spectroradiometer was used for collecting spectra in the wave number range from 4 000 to 12 000 cm(-1). Secondly, the original spectral data were pretreated in order to eliminate noise and improve the efficiency of models. Thirdly, principal component analysis (PCA) was used to compress spectral data into several variables, and the cumulate reliabilities of the first ten components were more than 97.28%. Finally, the recognition models were established based on BPR For the every 25 samples in each variety, 15 samples were randomly selected as the training set. The remaining 10 samples of the same variety were used as the first testing set, and all the 200 samples of the other varieties were used as the second testing set. As the 96.7% samples in the second set were correctly rejected, the average correct recognition rate of first testing set was 94.3%. The experimental results demonstrated that the recognition models were effective and efficient. In short, it is feasible to discriminate varieties of transgene wheat based on near infrared spectroscopy and BPR.
No preview · Article · Apr 2010 · Guang pu xue yu guang pu fen xi = Guang pu
[Show abstract][Hide abstract] ABSTRACT: Beet western yellows virus (BWYV) has previously been reported as an agent of sugar beet yellowing disease in China. In this article, the complete genomic RNA sequences of two Chinese BWYV isolates infecting beet from Inner Mongolia (BWYV-IM) and Gansu (BWYV-GS) were determined and compared with three beet poleroviruses (BMYV, BChV and BWYV-US) and other non-beet-infecting poleroviruses. The genomes of the two isolates were 5,668 nt in length, and had almost the same genomic organization and characteristics as BWYV-US. The full length of BWYV-IM shared nucleotide sequence identities of 97.4, 86.6, 64.4 and 70.8% with BWYV-GS, BWYV-US, BChV and BMYV, respectively. Further sequence analysis indicated that the Chinese BWYV isolates were more closely related to BWYV-US; however, the identity of any gene product between the Chinese isolates and BWYV-US was <90%. Therefore, on the basis of genome sequence, we propose that these Chinese isolates are a distinct strain of BWYV that infect sugar beet. In addition, recombinant detection analysis revealed that BWYV-IM might be a recombinant virus.
[Show abstract][Hide abstract] ABSTRACT: Cucurbit aphid-borne yellows virus (CABYV) and Melon aphid-borne yellows virus (MABYV) have been found to be associated with cucurbit yellowing disease in China. Our report identifies for the first time a third distinct polerovirus, tentatively named Suakwa aphid-borne yellows virus (SABYV), infecting Suakwa vegetable sponge. To better understand the distribution and molecular diversity of these three poleroviruses infecting cucurbits, a total of 214 cucurbitaceous crop samples were collected from 25 provinces in China, and were investigated by RT-PCR and sequencing. Of these, 108 samples tested positive for CABYV, while 40 samples from five provinces were positive for MABYV, and SABYV was detected in only 4 samples which were collected in the southern part of China. Forty-one PCR-amplified fragments containing a portion of the RdRp gene, intergenic NCR and CP gene were cloned and sequenced. Sequence comparisons showed that CABYV isolates shared 78.0-79.2% nucleotide sequence identity with MABYV isolates, and 69.7-70.8% with SABYV. Sequence identity between MABYV and SABYV was 73.3-76.5%. In contrast, the nucleotide identities within each species were 93.2-98.7% (CABYV), 98.1-99.9% (MABYV), and 96.1-98.6% (SABYV). Phylogenetic analyses revealed that the polerovirus isolates fit into three distinct groups, corresponding to the three species. The CABYV group could be further divided into two subgroups: the Asia subgroup and the Mediterranean subgroup, based on CP gene and partial RdRp gene sequences. Recombination analysis suggested that MABYV may be a recombinant virus.
[Show abstract][Hide abstract] ABSTRACT: The complete RNA genomes of a Chinese isolate of cucurbit aphid-borne yellows virus (CABYV-CHN) and a new polerovirus tentatively referred to as melon aphid-borne yellows virus (MABYV) were determined. The entire genome of CABYV-CHN shared 89.0% nucleotide sequence identity with the French CABYV isolate. In contrast, nucleotide sequence identities between MABYV and CABYV and other poleroviruses were in the range of 50.7-74.2%, with amino acid sequence identities ranging from 24.8 to 82.9% for individual gene products. We propose that CABYV-CHN is a strain of CABYV and that MABYV is a member of a tentative distinct species within the genus Polerovirus.
Preview · Article · Feb 2008 · Archives of Virology
[Show abstract][Hide abstract] ABSTRACT: The Rice black-streaked dwarf virus (RBSDV) virion is composed of two layers of capsid proteins and 10 segments of double-stranded genomic RNA (S1-S10). Due to the fragility of the RBSDV outer capsid, it is very difficult to obtain intact virus particles for preparation of antiserum needed in virus detection. In this work, the major outer capsid protein (P10) encoded by S10 of RBSDV was expressed in Escherichia coli cells as a glutathione-S-transferase (GST) fusion protein. After purification of GST-P10 through affinity chromatography, P10 was released from the fusion protein by thrombin digestion and the purified P10 protein was used to immunize rabbits. The resulting polyclonal antiserum specifically recognized RBSDV from infected plant tissue and a planthopper vector in Western blotting assays. To facilitate screening of large numbers of samples, an indirect enzyme-linked immunosorbent assay (ID-ELISA) protocol capable of detecting RBSDV in very dilute wheat leaf extracts was developed. Based on the results, we conclude that efficient and economic detection of RBSDV can be performed routinely using polyclonal antiserum against P10 expressed in prokaryotic cells.
No preview · Article · Jul 2006 · Journal of Virological Methods
[Show abstract][Hide abstract] ABSTRACT: Wheat (Triticum aestivum L.) yellow mosaic virus (WYMV) is transmitted by a fungal vector through soil and causes serious wheat yield losses due to yellow mosaic disease, with yellow-streaked leaves and stunted plants. In the present study, the amplified fragment length polymorphisms (AFLP) and simple sequence repeat (SSR) were used to identify the molecular linkages with the resistance gene against WYMV. Bulked segregant analysis was performed with an F2 population derived from the cross of cultivar Ningmai 9 (resistant) × cultivar Yangmai 10 (susceptible). By screening among the resistant or susceptible parents, the F2 pools and the individuals in the F2 population with 64 combined selective AFLP primers (EcoRI/MseI) or 290 reported SSR primers, a polymorphic DNA segment (approximately 120 bp) was amplified using the primer pair E2/M5, and an SSR marker (approximately 180 bp) was located on wheat chromosome 2 A using the primer Xgwm328. Analysis with MAPMAKER/Exp Version 3.0b (Whitehead institute for Biomedical Research, Cambridge, MA, USA) indicated that these two markers were dominantly associated with the resistance gene at distances of 5.4 cM or 17.6 cM, respectively. The resistance gene to WYMV derived from Ningmai 9, is temporarily named YmNM, and was mapped to wheat chromosome 2A.
(Managing editor: Li-Hui ZHAO)
No preview · Article · Sep 2005 · Journal of Integrative Plant Biology
[Show abstract][Hide abstract] ABSTRACT: The full-length sequence of a satellite RNA (sat-RNA) of Beet black scorch virus isolate X (BBSV-X) was determined. This agent is 615 nucleotides long and lacks extensive sequence homology with its helper virus or with other reported viruses. Purified virus particles contained abundant single-stranded plus-sense monomers and smaller amounts of dimers. Single-stranded RNAs from total plant RNA extracts also included primarily monomers and smaller amounts of dimers that could be revealed by hybridization, and preparations of purified double-stranded RNAs also contained monomers and dimers. Coinoculation of in vitro transcripts of sat-RNA to Chenopodium amaranticolor with BBSV RNAs was used to assess the replication and accumulation of various forms of sat-RNA, including monomers, dimers, and tetramers. Dimeric sat-RNAs with 5- or 10-base deletions or 15-base insertions within the junction regions accumulated preferentially. In contrast, the replication of monomeric sat-RNA was severely inhibited by five-nucleotide deletions in either the 5' or the 3' termini. Therefore, sequences at both the 5' and the 3' ends of the monomers or the presence of intact juxtaposed multimers is essential for the replication of sat-RNA and for the predomination of monomeric progeny. Comparisons of the time courses of replication initiated by in vitro-synthesized monomeric or multimeric sat-RNAs raised the possibility that the dimeric form has an intermediate role in replication. We propose that replication primarily involves multimers, possibly as dimeric forms. These forms may revert to monomers by a termination of replication at 5' end sequences and/or by internal initiation at the 3' ends of multimeric junctions.
Preview · Article · Apr 2005 · Journal of Virology