Potyviral Sequences Research Articles

Basella rugose mosaic virus, a new potyvirus infecting Basella rubra

Plant PathologyVolume 55, Issue 6 p. 819-819 Free Access Basella rugose mosaic virus, a new potyvirus infecting Basella rubra C.-H. Huang, C.-H. Huang Department of Plant Pathology and Microbiology, National Taiwan University, Taipei, Taiwan 10617, Republic of ChinaSearch for more papers by this authorY.-C. Chang, Corresponding Author Y.-C. Chang *E-mail: ycchang@ntu.edu.tw.Search for more papers by this author C.-H. Huang, C.-H. Huang Department of Plant Pathology and Microbiology, National Taiwan University, Taipei, Taiwan 10617, Republic of ChinaSearch for more papers by this authorY.-C. Chang, Corresponding Author Y.-C. Chang *E-mail: ycchang@ntu.edu.tw.Search for more papers by this author First published: 09 November 2006 https://doi.org/10.1111/j.1365-3059.2006.01477.xCitations: 7AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Basella rubra and B. alba (Malabar spinach) are plants of the family Basellaceae, native to tropical Asia. They were introduced into Taiwan during the 1660s and are grown as vegetables and herbs. In 2001, mosaic and rugose symptoms were observed on leaves of B. rubra in the Taipei area. Extracts from these plants caused chlorotic lesions on inoculated leaves of Chenopodium amaranticolor and C. quinoa; the latter became systemically infected. A virus isolate, BR, was obtained after three successive single lesion isolations from C. amaranticolor. Following mechanical inoculation, seedlings of B. rubra and B. alba produced symptoms similar to field diseased plants and indicator plants Nicotiana benthamiana and N. clevelandii displayed mosaic symptoms. Transmission electron microscopy of negatively-stained sap extracts showed the presence of flexuous filaments c. 680 nm in length. Using indirect ELISA, plants of B. rubra and C. quinoa with symptoms tested positive for potyvirus using an anti-potyvirus group monoclonal antibody (Agdia Inc.). The only potyvirus previously reported to infect a member of the Basellaceae (Ullucus tuberosus) is Ullucus mosaic virus (Brunt et al., 1982). However this virus differs from isolate BR, as it causes local lesions in C. quinoa. The 3′-terminal region of the BR isolate was amplified by RT-PCR from total RNA of diseased plants, using a potyvirus-specific degenerate primer (PNIbF1) and an oligo-dT primer (Huang & Chang, 2005). A 1·8 kb fragment, including partial NIb, the CP and 3′ UTR, was obtained and cloned. Two independent clones were sequenced and a BLAST search was performed against the NCBI database. Sequence analysis of the putative capsid protein gene revealed that the highest amino acid and nucleotide sequence identities were 70% and 68% respectively, between the BR isolate (DQ394891) and all other searched potyvirus sequences. According to the species demarcation criteria for the Potyviridae (Adams et al., 2005) the BR isolate is a new potyvirus and is herein designated as Basella rugose mosaic virus (BaRMV). References Adams MJ, Antoniw JF, Fauquet CM, 2005. Molecular criteria for genus and species discrimination within the family Potyviridae. Archives of Virology 150, 459– 79CrossrefCASPubMedWeb of Science®Google Scholar Brunt AA, Phillips S, Jones RAC, Kenten RH, 1982. Viruses detected in Ullucus tuberosus (Basellaceae) from Peru and Bolivia. Annals of Applied Biology 101, 65– 71. Wiley Online LibraryWeb of Science®Google Scholar Huang CH, Chang YC, 2005. Identification and molecular characterization of Zantedeschia mild mosaic virus, a new calla lily-infecting potyvirus. Archives of Virology 150, 1221– 30. CrossrefCASPubMedWeb of Science®Google Scholar Citing Literature Volume55, Issue6December 2006Pages 819-819 ReferencesRelatedInformation

DETECTION AND CHARACTERIZATION OF TWO PREVIOUSLY UNDESCRIBED POTYVIRUSES IN THE TERRESTRIAL ORCHID SPIRANTHES CERNUA

Spiranthes cernua is a terrestrial orchid that grows in moist areas in the southeastern US. Cultivated varieties are now also being used as flowering perennials. We have previously reported Dasheen mosaic virus (DsMV-Sc) infection in commercially-grown plants exhibiting chlorotic blotching and/or mosaic symptoms. Other similarly symptomatic plants were, however, ELISA negative using DsMV polyclonal antisera, yet gave a positive reaction with the broad­ spectrum potyvirus monoclonal antibody PTY 1, an antibody specific for a highly conserved site in the potyviral coat protein. Total RNA extracts from infected leaves were used in RT-PCR assays with generic potyvirus-specific primers which amplify various highly conserved 335, c.700, or c.1600bp fragments from the 3' terminus of most potyviruses which includes the 3' non-coding region (3'NCR) and the potyviral coat protein (CP). The PCR amplified fragments were subsequently cloned and sequenced and compared to the corresponding regions of other potyviruses. This analysis revealed the presence of two unique sets of potyviral sequences, neither of which had any significant identity with each other (56% CP identity and 39% 3'NCR identity), nor with DsMV (49-530/0 CP, 33-430/0 3'NCR), nor with any other potyvirus sequence «64% CP identity and <57% 3'NCR identity) currently in the GenBank database, including other orchid-infecting potyviruses. Pairwise, alignments and Clustal W analysis of the coat protein amino acid sequences and 3' NCR regions confirm the classification of these two viruses as new potyvirus members. We have tentatively named these two new potyviruses Spiranthes mosaic virus (SpiMV) 2 and SpiMV 3. This is only the second report of any virus infection in the genus Spiranthes and the first report of two new potyviruses infecting a member of the Orchidaceae.

Identification of potyviruses infecting vanilla by direct sequencing of a short RT‐PCR amplicon

A simple one‐tube one‐step RT‐PCR assay with degenerate primers followed by direct sequencing of a 327 bp coat protein gene fragment was used to identify the potyviruses infecting vanilla. With this technique, unambiguous species allocation was achieved for 34 potyvirus‐infected vanilla samples collected in the Indian Ocean and the Pacific areas between 1997 and 2005. Virus identification relied on blast homology and nucleotide identities of 162 to 327 nt fragments with known potyvirus sequences. Species allocation was confirmed by neighbour‐joining of the 149 nt common to 32 vanilla sequences and 51 known potyviruses. Subject to further identification, these data revealed four additional Potyvirus species that may infect vanilla: Bean yellow mosaic virus, Cowpea aphid‐borne mosaic virus, Ornithogalum mosaic virus and Wisteria vein mosaic virus. The procedure was rapid, cost‐effective, easy to use and showed a good taxonomic discriminating value. It also enabled the identification of potyviruses in adjacent weeds and should thus aid the understanding of outbreaks of potyviruses occurring in varied epidemiological circumstances.

Identification of a Potyvirus Causing Latent Infection in Calla Lilies.

A new potyvirus designated as Calla lily latent virus (CLLV) was isolated from apparently healthy calla lilies (Zantedeschia spp.) collected from nurseries in Taichung County, Taiwan. Different from most calla lily-infecting potyviruses, CLLV infects Chenopodium quinoa and develops local lesions on inoculated leaves (3). Typical potyvirus particles approximately 780 nm long were detected from CLLV-induced C. quinoa local lesions. CLLV was transmitted readily to and established in C. quinoa. Attempts to establish CLLV infection in calla lilies from extracts of C. quinoa lesions were not successful. The virus was transmitted from infected to healthy calla lilies with difficulty. A 1.3-kb cDNA product was amplified by reverse transcription-polymerase chain reaction (RT-PCR) from CLLV-infected calla lilies and C. quinoa using potyvirus degenerate primers (2). The PCR product was cloned and sequenced. It was found to consist of 1,339 nucleotides (nt) (GenBank Accession No. AF469171) corresponding to the genome organization of the 3'terminal region of potyviruses. The deduced amino acid sequence contains 362 residues encoding the 3'terminal region of the nuclear inclusion b gene (80 residues) and the complete coat protein (CP) gene (282 residues). A 253-nt noncoding region (NCR) was found at the 3'terminal region of the cDNA. By comparing with known sequences of potyviruses, CLLV was identified as a new species of Potyvirus based on the uniqueness in the CP gene and 3' NCR. Soybean mosaic virus and Watermelon mosaic virus 2 are the potyviruses most similar to CLLV, but they share only approximately 80% nucleotide identity with CLLV in the CP and NCR regions. Attempts to purify sufficient CLLV from C. quinoa for antiserum preparation were not successful. Alternatively, polyclonal antibodies were produced using E. coli-expressed CLLV CP (1). The antibodies were useful for detection of CLLV and its CP in calla lilies using enzyme-linked immunosorbent assay, sodium dodecyl sulfate-immunodiffusion, immuno-specific electron microscopy, and western blot. Field surveys showed that calla lily plants found positive for CLLV by serological methods always remained symptomless throughout the six-month growing season. Occasionally, CLLV was detected in symptomatic calla lilies, but these plants were consistently confirmed dually infected by other viruses (Dasheen mosaic virus and Konjak mosaic virus found most commonly). Infection of CLLV alone in calla lilies may not have a direct impact on the production and marketing of the crop. Synergism is not currently known when calla lilies are coinfected with other viruses. CLLV is spread by vegetative propagation through infected rhizomes or tubers.

Sequence Variability of Helper Component Protein of Potato Virus Y Identified by Thermodynamic Methods

An extent of helper component protein (HC-Pro) sequence variability within virus population of single Czech isolate of potato virus YNTN (PVYNTN) Nicola was identified by temperature-gradient gel electrophoresis (TGGE) and heteroduplex analysis. HC-Pro region was approximated with 6 pairs of primers derived from Hungarian PVYNTN isolate (sequence AC M95491). Immunocapture reverse transcription - polymerase chain reaction (RT-PCR) was used to obtain six mixtures of individual overlapping polymerase chain reaction (PCR) products. cDNA libraries were prepared by cloning of purified PCR products in pCR-Script vector and screened by heteroduplex analyses. 15 different subfragments within the HC-Pro region were isolated and sequenced. In comparison to AC M95491, 19 nucleotide changes were identified, 13 led to amino acid (aa) changes. In comparison to available post-transcriptional gene silencing (PTGS) specific suppressor HC-Pro sequences of potyviruses we found out 4 aa changes in conserved regions.

Molecular Characterization of Tuberose mild mosaic virus and Preparation of Its Antiserum to the Coat Protein Expressed in Bacteria

A 2-kb DNA product was amplified from purified Tuberose mild mosaic virus (TMMV) virions as well as from infected tissues of tuberose by the use of degenerate primers for potyvirus. The PCR product was subsequently cloned and its sequence analyzed. It was found comprised of 1947 nucleotides (nts) corresponding to the 3′-terminal region of potyviruses. The deduced amino acid sequence contained 598 residues encoding part of the 3′-terminal region of NIb gene (319 residues) and the complete sequence of coat protein (CP) gene (279 residues). A 136 nts of non-coding region (NCR1 was found located at the 3′-terminal region of the DNA. A genetic code for aphid transmissibility of potyviruses, DAG triplet, was found at the 19-2 1 residues from the N-terminus of CP gene. Compared to the known sequences of potyviruses, the percent of nucleotide identities of the CP gene and the NCR were less than 62% and 39%, respectively. Similarly, percent identities of TMMV’s CP amino acid sequence to those of other known potyviruses were all below 58%, confirming our previous finding that TMMV is a new species of Polyvirus. Using directional cloning technology, a 39-kDa fusion protein containing a complete CP sequence of TMMV and a partial sequence encoded by the expression vector plasmid (pET-30b, Novagene) was highly expressed and purified from E. coli cell cultures. The antigenicity of the fusion protein was determined to be indistinguishable from the viral CP. Antiserum prepared against this fusion protein showed comparable reactivities in the serological detection of TMMV with the conventional antibodies against purified virus particles.

Molecular characterization and coat protein serology of watermelon leaf mottle virus (Potyvirus).

A cDNA library was generated from purified RNA of watermelon leaf mottle virus (WLMV) (Genus Potyvirus). Two overlapping clones totaling 2,316 nucleotides at the 3' terminus of the virus were identified by immunoscreening with coat protein antiserum. The sequence analyses of the clones indicated an open reading frame (ORF) of 2,050 nucleotides which encoded part of the replicase and the coat protein, a 243-nucleotide non-coding region (3'UTR), and 23 adenine residues of the poly (A) tail. The taxonomic status of WLMV was determined by comparisons of the sequence of the cloned coat protein gene and 3'UTR with potyvirus sequences obtained from GenBank. The nucleotide sequence identities of WLMV compared with 17 other potyviruses ranged from 55.6 to 63.5% for the coat protein, and from 37.2 to 48.3% for the 3'UTR. Phylogenetic analyses of the coat protein region and the 3'UTR indicated that WLMV did not cluster with other potyviruses in a clade with high bootstrap support. The coat protein gene was expressed in Escherichia coli and a polyclonal antiserum was prepared to the expressed coat protein. In immunodiffusion tests, WLMV was found to be serologically distinct from papaya ringspot virus type W, watermelon mosaic virus 2, zucchini yellow mosaic virus, and Moroccan watermelon mosaic virus. In Western blots and ELISA, serological cross-reactivity with other cucurbit potyviruses was observed. Serological and sequence comparisons indicated that watermelon leaf mottle virus is a distinct member of the Potyvirus genus.

Characterization of petunia flower mottle virus (PetFMV), a new potyvirus infecting Petunia x hybrida.

With the introduction of cutting-grown Petunia x hybrida plants on the European market, a new potyvirus which showed no serological reaction with antisera against any other potyviruses infecting petunias was discovered. Infected leaves contained flexuous rod-shaped virus particles of 750-800 nm in length and inclusion bodies (pinwheel structures) typical for potyviruses in ultrathin leaf sections. The purified coat protein with a Mr of approximately 36 kDa could be detected in Western immunoblots with a specific antibody to the coat protein of the petunia-infecting virus. The 3' end of the viral genome encompassing the 3' non-coding region, the coat protein gene, and part of the NIb gene was amplified from infected leaf material by IC/PCR using degenerate and specific primers. Sequences of PCR-generated cDNA clones were compared to other known sequences of potyviruses. Maximum homology of 56% was found in the 3' non-coding region between the petunia isolate and other potyviruses. A maximum homology of 69% was found between the amino acid sequence of the coat protein of the petunia isolate and corresponding sequences of other potyviruses. These data indicate that the petunia-infecting virus is a previously undescribed potyvirus and the name petunia flower mottle virus (PetFMV) is suggested.

Characterization of a Potyvirus Causing Mild Mosaic on Tuberose.

A virus inducing mild mosaic symptoms on the leaves and peduncles of tuberose (Polianthes tuberosa) was isolated and partially characterized. The isolate, designated Tbr1, could be transmitted mechanically and by green peach aphids (Myzus persicae) in a non-persistent manner to tuberose seedlings but not to 16 common assay species. Flexuous rod-shaped particles with a mean length of 750 nm could be easily seen in infected leaf dips and in purified samples. Cytoplasmic cylindrical inclusions, pinwheel and laminated aggregates similar to those assigned to potyviral cylindrical inclusion type II, were observed in infected tuberose leaves. The purified capsid contained a single species of protein monomer with an estimated relative mass of 38 kDa. In reciprocal sodium dodecyl sulfate-immunodiffusion tests, antiserum against Tbr1 reacted only with its homologous antigen but not with 22 different known potyviruses. Using primer pairs designed for potyvirus sequence amplification, a 2-kb DNA product equivalent to the estimated size for potyviruses was consistently amplified from purified Tbr1 virions or from crude infected tissue by reverse transcription polymerase chain reaction. On the basis of these results, Tbr1 was recognized as a unique species in the genus Potyvirus and hence designated as tuberose mild mosaic potyvirus (TMMV).

5′ Proximal potyviral sequences mediate potato virus X/potyviral synergistic disease in transgenic tobacco

The interaction of potato virus X (PVX) and potato virus Y (PVY) in tobacco causes a synergistic disease characterizedby a dramatic increase in symptom severity, a change in the regulation of PVX RNA replication, and an increase in accumulation of PVX. In this study we demonstrate that PVX also interacts synergistically with three other members of the potyvirus group of plant viruses, tobacco vein mottling virus (TVMV), tobacco etch virus (TEV), and pepper mottle virus. These synergisms resemble the classic PVX/PVY synergism with respect to both the increase in host response and the change in PVX replication. To determine if the induction of PVX/potyviral synergism requires potyviral genome replication per se or if the response is mediated by expression of one or more potyviral genes, we used tobacco plants stably transformed with various subsets of the TVMV genome. PVX infections of transgenic plants expressing the 5′-proximal region of the TVMV genome, including the protease-1, helper component protease, and protein-3 genes, result in symptoms resembling those of PVX/potyviral synergism. A similar synergistic-like response occurs when transgenic tobacco plants expressing the analogous but smaller region from the 5′-proximal region of the TEV genome were infected with PVX. Replication of PVX RNA is altered in transgenic plants expressing 5′-proximal sequences of either TVMV or TEV, and in a manner similar to that observed in double infections. These results indicate that replication of the potyviral genome is not required for PVX/potyviral synergism and that the response is mediated by expression of potyviral sequences which have been localized to the 5′-proximal third of the genomic RNAs of both TVMV and TEV.

Evolutionary relationships among bean common mosaic virus strains and closely related potyviruses

Bean common mosaic virus (BCMV) consists of a large number of strains with complex and controversial relationships among them and with other potyviruses that infect legumes. In order to elucidate the BCMV taxonomic pattern and its evolutionary implications, a phylogenetic analysis has been carried out. The analysis of the coat protein gene and 3' non-coding region (NCR) sequences recently obtained by us and other currently available potyviral sequences confirms the clustering of viruses comprised in BCMV strains with other closely related potyviruses and reveals the great informative content of 3' NCR, suggesting a more relevant role for this region in phylogenetic analysis.

Molecular analysis of the capsid protein gene of a german isolate of barley mild mosaic virus

Barley mild mosaic virus (BaMMV) is one of the agents causing the barley yellow mosaic disease. The sequence corresponding to the 3′end of the BaMMV RNA1 of a German isolate was sequenced and the coding sequence for the 251 amino acid containing capsid protein was determined. Comparison of this sequence to other potyviral sequences and to the corresponding sequence of two Japanese isolates of BaMMV was done. The three different isolates of BaMMV show a high degree of similarity.

Molecular analysis of the capsid protein gene of a german isolate of barley mild mosaic virus

Barley mild mosaic virus (BaMMV) is one of the agents causing the barley yellow mosaic disease. The sequence corresponding to the 3'end of the BaMMV RNA1 of a German isolate was sequenced and the coding sequence for the 251 amino acid containing capsid protein was determined. Comparison of this sequence to other potyviral sequences and to the corresponding sequence of two Japanese isolates of BaMMV was done. The three different isolates of BaMMV show a high degree of similarity.

The use of PCR for cloning of large cDNA fragments of turnip mosaic potyvirus

A method is described whereby turnip mosaic virus RNA (TuMV RNA) was reverse transcribed and the resulting cDNA amplified enzymatically using the Taq DNA polymerase and degenerate oligonucleotide primers. Two degenerate oligonucleotide primers based on regions of homology in the amino acid sequence of the cytoplasmic inclusion protein and the nuclear inclusion b protein from five potyviruses were synthesized. Polymerase chain reactions utilizing these degenerate primers] in association with specific primers amplified a 1.2 kb and a 3.3 kb fragment. These amplified fragments were dC-tailed and cloned into pUC9. Their partial sequence, when compared to potyvirus sequences, showed that they were derived from TuMV RNA and approximately 4.4 kb of viral genome was cloned.

CDNA cloning and sequence analysis of the 3'-terminal region of zucchini yellow mosaic virus RNA

The 3' half of the RNA of the cucurbit potyvirus zucchini yellow mosaic virus (ZYMV) was genetically cloned and the cDNA sequence of a portion of the putative RNA polymerase gene, the complete coat protein gene and the 3' untranslated region was determined. The coat protein and putative polymerase genes are both part of a continuous open reading frame as is the case for other potyviruses whose genomes are expressed as polyproteins. The Q/S protease cleavage site for the N terminus of the coat protein was deduced by alignment of the coat protein and polymerase genes with other potyviral sequences. The resulting protein has 279 amino acids and a calculated Mr of 31,214. The predicted amino acid sequence indicates a ZYMV-unique N-terminal region and potyvirus-characteristic central and C-terminal regions. These data also verify that ZYMV is distinct from the cucurbit potyvirus watermelon mosaic virus 2.

Complementary DNA cloning and expression of the papaya ringspot potyvirus sequences encoding capsid protein and a nuclear inclusion-like protein in Escherichia coli

Three cDNA clones that express viral gene products in Escherichia coli JM83 were derived from a watermelon mosaic virus-1 strain of papaya ringspot virus (PRSV-W). DNAs complementary to portions of the viral RNA were inserted into the pUC8 and pUC9 plasmids, and the expressed polypeptides were fusion products with the amino terminus of β-galactosidase. Clones W1-77 and W2-1 expressed fusion products with apparent molecular weights of 40,000 (40K) and 14K, respectively, which were serologically related to PRSV capsid protein. A 52K product serologically related to a 54K nuclear inclusion protein of tobacco etch virus was produced by clone W1-18. The sequences encoding the capsid and 57K nuclear inclusion-like proteins of PRSV were physically mapped to adjacent positions through Southern blot analyses of clones W1-77 and W1-18.