Odontoglossum Ringspot Virus Coat Protein Research Articles

Simultaneous detection of odontoglossum ringspot virus (ORSV) and cymbidium mosaic virus (CymMV) infecting orchids in Meghalaya, India by Multiplex RT-PCR

• ORSV was found to be more prevalent than CymMV with the incidence percentages of 53.5% and 46.4% respectively. • Mixed infection of ORSV and CymMV was found to be 35.7%. • The Nucleotide sequences of ORSV and CymMV CP genes from Meghalaya showed high sequence identities of 96.90–99.56% and 96.91–99.66% respectively with other available sequences in Genbank. Odontoglossum ringspot virus (ORSV) and cymbidium mosaic virus (CymMV) are the most prevalent viruses infecting orchids all around the world. These viruses cause a serious threat to the orchid industry as they can spread from infected plants to healthy plants through vegetative propagation, contaminated tools, and human hands. This result in negative vigour of infected plants, showing stunted growth, producing fewer flowers, and decreasing flower quality. In this study, 84 leaf samples of orchids were collected from nine horti-hubs and orchid nurseries in Meghalaya. The orchid leaf samples were subjected to multiplex RT-PCR amplification using gene-specific primers targeting the coat protein (CP) genes of CymMV and ORSV. The most prevalent virus detected was ORSV with a 53.5% incidence while CymMV showed a 46.4% incidence. Mixed infections of ORSV and CymMV were detected in 30 samples (35.7%). The nucleotide sequences of ORSV and CymMV CP genes from Meghalaya showed high sequence identities of 96.90–99.56% and 96.91–99.66%, respectively when compared to other sequences in GenBank. The phylogenetic relationship among ORSV isolates and CymMV, based on infected host or geographical origin, showed no clear-cut clustering of isolates.

Identification and Detection Odontoglossum ringspot virus on Native Orchids Collection of Nurserys in Java, Indonesia

Nature orchid are one of original floral in Indonesia. Virus infection is one of the limiting factor in the cultivation of orchid. Infection Odontoglossum ringspot virus (ORSV) was reported infets native orchids collection in Indonesia. The purpose of this study was to identification and ORSV that infects native orchid nurserys collection in Java, Indonesia. Symptomatic samples were collected from 5 nurserys collections, i.e. Rumah Bunga Rizal (Bandung), Bali Tanaman Hias (Cianjur), Borobudur Orchids Center (Magelang), Kebun Anggrek Bungarinte (Yogyakarta), and Titi Orchids (Yogyakarta). Detection and identification was conducted by serological test using ORSV specific antisera, RT-PCR and DNA sequencing. The serological test using ORSV antisera showed that 3 of 11 sampels reacted positively against ORSV antiserum i.e Phalaenopsis amabilis (Cianjur.1, Cianjur.2, and Magelang). RT-PCR of the 3 samples using specific primer of ORSV coat protein (CP) gene amplified a DNA with size ± 474 bp. Homology analysis of those 3 Indonesian isolates showed highest index similiarity (IS) was 99.8% with corresponding sequences from 10 other ORSV isolates. Phylogenetic analysis showed that ORSV Cianjur.1 and Cianjur.2 isolates clustered in separated group far from ORSV isolates in other countries.

RNA Silencing-Mediated Control of <i>Odontoglossum ringspot virus</i> (ORSV) Infection

Odontoglossum ringspot virus (ORSV) infects perennial orchids (Phalaenopsis amabilis) and causes a widespread viral disease. RNA-silencing of viral genes is a promising and effective way of controlling viral infection in plants. An inverted repeat (IR) fragment of the ORSV coat protein gene, cp, was inserted into the pXGY1 vector to generate the silencing construct, pXGY1-ORSV, which was introduced into Nicotiana benthamiana via Agrobacterium-mediated infiltration. A total of 15 homozygous pXGY1-ORSV transgenic N. benthamiana T1 plants were obtained from five transgenic lines, and ORSV cp gene multiplication was reduced by at least 75% - 95% in 12 T2 plants, demonstrating their increased resistance to ORSV. An infectious ORSV clone, pCAMBIA2300-ORSV, was generated to facilitate rigorous analyses of plant viral resistance. Semi-quantitative RT-PCR (sqRT-PCR) and northern-blot analyses revealed that levels of ORSV multiplication and ORSV coat protein were significantly reduced in pXGY1-ORSV transgenic N. benthamiana. Western-blot from pXGY1-ORSV inoculated leaves of ORSV infected P. amabilis also revealed the significant decrease and even degradation of ORSV-CP protein. Disease symptoms were not observed in transgenic plants. These results indicate a high level of ORSV-resistance in pXGY1-ORSV transgenic N. benthamiana.

TransgenicNicotiana benthamianaresistance to synergistic infection of two orchid viruses CymMV and ORSV

Cymbidium mosaic virus (CymMV) and Odontoglossum ringspot virus (ORSV) have been reported as co-infection (or synergistic interaction) resulting in more severe symptoms in orchids. In this project, silencing dual RNA-dependent RNA polymerase (RdRp) transcripts of CymMV and ORSV based on RNA interference (RNAi), including amiRNA-CymMV-ORSV or antisense-CymMV-ORSV constructs were adopted to create transgenic plants that can resist to CymMV and ORSV at the same time. A susceptible host model to both viruses, Nicotiana benthamiana, was generated first to test the feasibility of the RNAi strategy and gene stacking also was applied to increase copy number in transgenic N. benthamiana by cross pollination. Successfully of viral resistant analyses were performed until F3 generation using Southern blot, real-time RT-PCR and western blot. Resistant FF3 transgenic plants had a higher transgene expression as compared to non-resistant plants, coupled with the absence of CymMV and ORSV coat protein which suggests negligible CymMV and ORSV concentration in that resistant plant which allowed the plant to exhibit a normal phenotype after mixed inoculation for three weeks. We believe this is the first report on the transgenic N. benthamiana plants that can resist against both CymMV and ORSV concurrently.

Deteksi Odontoglossum ringspot virus pada Anggrek Asli Koleksi Kebun Raya di Indonesia

Native orchid is one of Indonesian natural resources which play important role as parental materials in breeding program. Virus infection is one of the limiting factors in the cultivation of orchid. The purpose of this study was to detect Odontoglossum ringspot virus (ORSV) from native orchid. Symptomatic orchids were collected from 5 botanical gardens, i.e. Bogor, Cibodas, Purwodadi, Balikpapan, and Enrekang Botanical Gardens. Detection and identification was conducted by serological method using ORSV specific antisera, followed by RT-PCR and DNA sequencing. The serological test showed that 5 samples gave positive reaction against ORSV antiserum, i.e. Phalaenopsis amboinensis (KRB2) and Phalaenopsis amabilis (KRB12) from Bogor Botanical Garden, Phalaenopsis amabilis (KRP18) and Dendrobium salacence (KRP20) from Purwodadi Botanical Garden, dan Phalaenopsis modesta J. J. Sm. (KRBp5) from Balikpapan Botanical Garden. RT-PCR of the 5 samples using specific primer of ORSV coat protein gene was successfully amplified fragment DNA with size ± 474 bp. Homology analysis of those 5 ORSV isolates showed the highest index similiarity of 99.8% with corresponding sequences from 14 other ORSV isolates. Phylogenetic analysis indicated that ORSV KRB2 and KRP18 isolates was clustered in a separate group far from ORSV isolates in other countries. This is the first report of ORSV infection on native orchids collection from 5 botanical gardens in Indonesia.

Simultaneous detection of Cymbidium mosaic virus and Odontoglossum ringspot virus in orchids using multiplex RT-PCR.

A system for simultaneous detection of two orchid-infecting viruses was developed and applied to several orchid species. The detection system involved multiplex reverse transcription-polymerase chain reaction (RT-PCR) and could simultaneously identify Cymbidium mosaic virus (CymMV) and Odontoglossum ringspot virus (ORSV) from the orchid species studied. Multiplex RT-PCR was conducted using two virus-specific primer pairs and an internal control pair of primers to amplify the CymMV and ORSV coat protein regions, and orchid 18S rDNA, respectively. For optimization of multiplex RT-PCR conditions, serial dilutions of total RNA and cDNA were performed and the detection limit of the system was evaluated. The optimized multiplex detection system for CymMV and ORSV was applied to various orchid species, including several cultivars of Doritaenopsis, Cymbidium, Dendrobium, and Phalaenopsis to test the efficacy of this method. Our results indicate that the multiplex RT-PCR detection system will be a rapid, simple, and precise diagnosis tool in a range of orchid species.

DETECTION AND MOLECULAR CHARACTERIZATION OF ODONTOGLOSSUM RINGSPOT VIRUS (ORSV) JAVA AND BALI ISOLATES

<p>Orchids are one of the most important ornamental plants and cultivated in tropical countries, including in Indonesia. Virus infections become an important limiting factor in orchids cultivation because it causes significant losses of the plants. Odontoglossum ringspot virus (ORSV) is one of the most reported viruses infecting orchids and spread widely in the world. The common symptoms are mosaic with line pattern and necrotic ringspot on leaf surface, and also color breaking on flowers. The purpose of this research was to find out the occurrence of ORSV infecting orchids in Java and Bali, genetic relationship among ORSV isolates based on similarities of coat protein (CP) gene, and to analyze the pathogenicity test of ORSV Java and Bali isolates. Survey and samples collection were conducted in seven locations cultivating orchids in Java and Bali. Detection with Reverse Transcription-Polymerase Chain Reaction (RT-PCR) showed that ORSV was infected three leaf samples of Phalaenopsis sp., called ORSV BOC, ORSV KRB and ORSV TNBB isolates. The results showed 474 bp-amplified DNA band as the expression of ORSV CP gene. Phylogenetic analysis based on nucleotide sequences of CP gene showed that ORSV BOC have similarity close to ORSV Germany, whereas ORSV KRB and ORSV TNBB leads to speciation that possible to be a new strain. Pathogenicity test using various healthy plants showed that ORSV BOC may infected and cause systemic symptoms on Chenopodium amaranticolor, Nicotiana tabacum, Dendrobium sp., Cymbidium sp., Chattleya sp., Phalaenopsis sp., Liparis sp., Spatthoglotis sp. and Pectelis sussanae (L.) Raf.</p><p><br /><strong>Keywords:</strong> ORSV, orchids, coat protein, RT-PCR</p>

Monoclonal antibody-based triple antibody sandwich-enzyme-linked immunosorbent assay and immunocapture reverse transcription-polymerase chain reaction for Odontoglossum ringspot virus detection

Odontoglossum ringspot virus (ORSV) infects numerous commercially important orchids and causes significant losses worldwide. The coat protein ( CP) gene of ORSV was cloned and expressed in Escherichia coli by using the pET-32a expression vector, and the expression of recombinant protein was confirmed by Western blotting using anti-ORSV antibodies. The recombinant protein was purified using Ni-NTA agarose, and the purified protein was used as an immunogen to produce monoclonal antibodies (MAbs) and polyclonal antibodies (PAbs). Five murine MAbs against ORSV CP were obtained. Among them, two MAbs (6B4 and 1D1) also reacted with TMV CP. The triple antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA) and immunocapture reverse transcription-polymerase chain reaction (IC-RT-PCR) methods using the MAb (8A5) were then developed for sensitive, specific, and rapid detection of ORSV. TAS-ELISA and IC-RT-PCR could detect ORSV in the infected leaf saps with dilutions of 1:10,240 and 1:81,920 (w/v, g mL −1), respectively. TAS-ELISA and IC-RT-PCR detections indicated that ORSV was prevalent in orchids in the Zhejiang Province of China.

STUDIES ON SYNERGISM AND COMPLEMENTATION OF LONG DISTANCE MOVEMENT OF CYMBIDIUM MOSAIC VIRUS RNA BY ODONTOGLOSSUM RINGSPOT VIRUS COAT PROTEIN

There are two common orchid viruses, namely Cymbidium mosaic virus (CymMV) and Odontoglossum ringspot virus (ORSV). Complementation of movement and coat proteins of these two orchid viruses was studied. We produced four transgenic lines, each harboring one of the movement protein (MP) or coat protein (CP) genes of CymMV or ORSV. The MP of CymMV consists of three overlapping open reading frames (ORFs) called the triple gene block (TGB). We also generated CymMV and ORSV mutants each carrying an inactivated MP or CP. Complementation was studied by infecting transgenic plants with in vitro transcripts generated from these mutants. The cell-to-cell movement of a movement-deficient CymMV could be restored in transgenic plants carrying the ORSV MP transgene. Similarly, CymMV TGB123 transgenic plants were able to rescue the cell-to-cell movement of a movement-deficient ORSV mutant. ORSV CP transgenic plants were able to support systemic movement of a CymMV CP-deficient mutant. Trans-encapsidation was not observed. The complementation of MPs and CPs of CymMV and ORSV facilitates movement of the two viruses in plants, with an exception in long distance movement of ORSV RNA by CymMV CP. The synergism between CymMV and ORSV was examined using viral RNA and CP accumulation in single and double infections.

Introduction of Odontoglossum ringspot virus Coat Protein Gene into Cymbidium niveo-marginatum mediated by Agrobacterium tumefaciens to Produce Transgenic Plants

We cloned the Odontoglossum ringspot virus (ORSV) coat protein (CP) gene from ORSV-infected Epidendrum and constructed a pHPUNOJE-1 vector by ligating this CP gene, green fluorescence protein (GFP) gene, and hygromycin resistant (Hygr) gene into the plasmid pGreenII. This chimeric vector was mediated into A. tumefaciens LBA 4404. By infecting and co-culturing the rhizomes of Cym. niveo-marginatum with A. tumefaciens LBA 4404, ORSV CP gene transgenic shoots were obtained. GFP was observed in 64% of the 81 surviving rhizomes and in 39% of 215 regenerated shoots. The presence of ORSV CP, GFP, and Hygr genes was confirmed in the transgenic shoots by using polymerase chain reaction (PCR) analysis, whereas that of ORSV CP was confirmed by enzyme-linked immunosorbent assay (ELISA).

Reciprocal function of movement proteins and complementation of long-distance movement of Cymbidium mosaic virus RNA by Odontoglossum ringspot virus coat protein

Complementation of movement and coat proteins of the orchid-infecting potexvirus Cymbidium mosaic virus (CymMV) and tobamovirus Odontoglossum ringspot virus (ORSV) was investigated. Nicotiana benthamiana, which is susceptible to both CymMV and ORSV, was used as a model system. Four transgenic lines, each harbouring one of the movement protein (MP) or coat protein (CP) genes of CymMV or ORSV, were constructed. The MP of CymMV consists of three overlapping open reading frames, together called the triple-gene block (TGB). CymMV and ORSV mutants, each carrying an inactivated MP or CP, were generated from the respective biologically active full-length cDNA clones. Complementation was studied by infecting transgenic plants with in vitro transcripts generated from these mutants. The cell-to-cell movement of a movement-deficient CymMV was restored in transgenic plants carrying the ORSV MP transgene. Similarly, CymMV TGB1 transgenic plants were able to rescue the cell-to-cell movement of a movement-deficient ORSV mutant. ORSV CP transgenic plants supported systemic movement of a CymMV CP-deficient mutant. However, in these plants, neither encapsidation of CymMV RNA with ORSV CP nor CymMV CP expression was detected. Long-distance movement of an ORSV CP-deficient mutant was not supported by CymMV CP. The complementation of MPs and CPs of CymMV and ORSV facilitates movement of these viruses in plants, except for long-distance movement of ORSV RNA by CymMV CP.

Genetic variability in the coat protein genes of two orchid viruses: Cymbidium mosaic virus and Odontoglossum ringspot virus.

The variability in coat protein gene sequences of Cymbidium mosaic virus (CymMV) and Odontoglossum ringspot virus (ORSV) that naturally infect orchids worldwide was investigated. Samples were collected from Korea, Singapore and Taiwan. The sequence data were compared with available published coat protein gene sequences of CymMV and ORSV, including those from Japan and Thailand. Among CymMV isolates, the homology was 89.1%-99.7% and 93.2%-100% at the nucleotide and amino acid levels, respectively. Among the ORSV isolates, the homology was 95.5%-100% and 93%-100% at the nucleotide and amino acid levels, respectively. No particular region of variability could be defined in either of the viruses. In deduced amino acid sequence, the N-terminal was more conserved than the C-terminal in both CymMV and ORSV. By comparing all sequences determined in this study and those that are published in the GenBank databases, we did not find clustering based on geographical distribution or sequence identity. Such high sequence conservation suggests that both CymMV and ORSV coat protein genes are suitable candidates to provide resistance to orchids cultivated in different geographical locations.

Expression and purification of a neuropeptide nocistatin using two related plant viral vectors

Both odontoglossum ringspot virus (ORSV) and tobacco mosaic virus (TMV) were investigated as expression viral vectors for the expression of a neuropeptide nocistatin. Chimeras of ORSV and TMV were constructed by fusion of 17 amino acids of mouse nocistatin (mNST) to the C-terminal of the coat protein (CP) gene via a Factor Xa cleavage linker to yield ORSV-mNST and TMV-mNST. Expression of the mNST peptide was demonstrated by immuno-transmission electron microscopy, western blot, mass spectrometry and radioimmunoassay. Serial passaging of the chimeric viruses revealed loss of mNST from TMV-mNST by the fifth passage. The mNST was maintained in ORSV-mNST throughout six passages. The mNST peptide could be effectively cleaved and purified from chimeric ORSV CP. To our knowledge, this is the first successful attempt in obtaining a complete peptide with no additional amino acid sequence after expression and purification through the use of either ORSV or TMV as vectors.

Molecular cloning, sequencing and expression in Escherichia coli of the odontoglossum ringspot virus coat protein gene

The sequence of the 3'-terminal 1865 nucleotides of the genome of the tobamovirus odontoglossum ringspot virus (ORSV) was determined. This sequence contained two open reading frames (ORFs), 912 and 477 nucleotides long. The 912 nucleotide ORF has been identified as the cell-to-cell transport protein gene. The 477 nucleotide ORF was expressed in Escherichia coli, and the product was detected by antibodies specific for the coat protein of ORSV. The amino acid sequence of protein encoded by this ORF shares 84% similarity with the tobacco mosaic virus (TMV) (vulgare) coat protein. The 3'-terminal untranslated region of ORSV comprises 414 nucleotides, 210 nucleotides more than that of TMV (vulgare) RNA.

Systemic expression of a bacterial gene by a tobacco mosaic virus-based vector.

Tobacco mosaic virus (TMV) produces large quantities of RNA and protein on infection of plant cells. This and other features, attributable to its autonomous replication, make TMV an attractive candidate for expression of foreign sequences in plants. However, previous attempts to construct expression vectors based on plant RNA viruses, such as TMV, have been unsuccessful in obtaining systemic and stable movement of foreign genes to uninoculated leaves in whole plants. A hybrid viral RNA (TB2) was constructed, containing sequences from two tobamoviruses (TMV-U1 and odontoglossum ringspot virus). Two bacterial sequences inserted independently into TB2 moved systemically in Nicotiana benthamiana, although they differed in their stability on serial passage. Systemic expression of the bacterial protein neomycin phosphotransferase was demonstrated. Hybrid RNAs containing both TMV-U1 and the inserted bacterial gene sequences were encapsidated by the odontoglossum ringspot virus coat protein, facilitating their transmission and amplification on passaging to subsequent plants. The vector TB2 provides a rapid means of expressing genes and gene variants in plants.

Odontoglossum ringspot virus coat protein: sequence and antigenic comparisons with other tobamoviruses

Comparative immunochemical analysis of different tobamoviruses indicated that the previously reported coat protein sequence of Odontoglossum ringspot virus was likely erroneous. This sequence has been determined again by direct sequencing of the genomic RNA and was found to differ from the previously proposed sequence in 31 of the 157 amino acid residues. The extent of antigenic cross-reactivity between ORSV protein and other tobamovirus proteins was measured by ELISA and found to correlate satisfactorily with the degree of sequence homology.