Odontoglossum Ringspot Virus Research Articles

Molecular Characterization of Odontoglossum Ringspot Virus (ORSV) in Java and Bali, Indonesia

Orchids are one of the important ornamental plants that were cultivated in tropical countries, including in Indonesia. Virus infections has been important limiting factor in orchids cultivation because it could decrease the orchids quality. Odontoglossum ringspot virus (ORSV) is one of the most reported viruses infecting orchids, which spread widely in the world. During 2010-2011 surveys of viral diseases were conducted in Jawa and Bali, Indonesia. The orchids were found infected by virus, showed symptoms of mosaic, mottle, chlorotic, necrotic, streak, wilting leaf and ringspot on leaf surface. Detection with Reverse Transcription-Polymerase Chain Reaction (RT-PCR) showed that only 5 from 88 samples were infected by ORSV with total incidence of 5.7%. Three leaf samples of Phalaenopsis sp., were infected, then called ORSV BOC, ORSV KRB and ORSV TNBB isolates, respectively. The results obtained by amplification of DNA band with 474 bp in length as expression of Coat Protein (CP) gene. Phylogenetic analysis based on nucleotide sequences of CP gene showed that ORSV BOC have similarity to ORSV Germany, whereas, ORSV KRB and ORSV TNBB lead to speciation that possibly to be a new strain. This study was proved that ORSV have entered and spread widely by infected orchids in orchids landscape (nursery), semi-natural forests (botanical gardens) and natural forest (national park) in Java and Bali, Indonesia.

First report of Cymbidium mosaic virus and Odontoglossum ring spot virus in Argentina

Oncidium Sharry Baby orchids from nursery greenhouses in Buenos Aires, Argentina with viral symptoms were processed for transmission electron microscope observations. Two types of viral particles, morphologically different, were observed. The aim of this work is identification of viral particles infecting the orchids. Serological analysis and partial genome sequence confirmed the presence of Cymbidium mosaic virus (CymMV) and Odontoglossum ring spot virus (ORSV). To our knowledge this is the first report of ORSV and CymMV in Argentina.

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>

Problemas fitosanitarios que amenazan la conservación de las orquídeas de Costa Rica

Orchids from private collections, nurseries and wild areas, located in different ecological regions of Costa Rica, were studied in order to know the pests and diseases affecting them. 16 insect and two mite genera were identified as pests on different orchids and 28 pathogens were found as causal agents of infec- tious diseases. The most frequent pests were: Tenthecoris orchidearum, Stethobaris sp., Xylosandrus com- pactus, Pseudococcus longispinus, Diaspis boisduvalii and Tenuipalpus pacificus. The main diseases found were caused by: Phytophthora spp., Pythium spp., Fusariun oxysporum, Fusarium spp., Sclerotium rolfsii, Colletotrichum gloeosporioides, Sphenospora spp., Uredo spp., Cymbidium Mosaic virus and Odontoglossum Ring Spot Virus. This results demonstrate the diversity of organisms producing sanitary problems on orchids in Costa Rica, and suggests the inclusion of pest and diseases management in strate- gies for orchid conservation.

Application of an Integrated Omics Approach for Identifying Host Proteins That Interact With Odontoglossum ringspot virus Capsid Protein.

The glutamic acid at position 100 (E(100)) in the capsid protein (CP) of Odontoglossum ringspot virus (ORSV) plays an important role in long-distance viral movement in Nicotiana benthamiana. The ORSV(E100A) mutant, which has a glutamic acid to alanine substitution, shows a loss of systemic infectivity in N. benthamiana. Transmission electron microscopy and size-exclusion chromatography assays showed that E(100) is essential for CP-CP interaction and viral particle assembly. To identify the ORSV triggering or response genes and CP-interacting proteins (CP-IP), an integrated omics approach based on next-generation sequencing and proteomics profiling was used in this study. The whole-transcriptomes of healthy and ORSV-infected leaves of N. benthamiana were analyzed, and the gene information was used to create a N. benthamiana protein database that was used for protein identification following mass spectrometry analysis. The integrated omics approach identified several putative host proteins that interact with ORSV CP(WT) and were categorized as photosystem subunits, defense-associated proteins, and cell division components. The expression pattern and CP interaction of these CP-IP were examined by semiquantitative reverse transcription polymerase chain reaction and an in vitro binding assay, respectively, to verify the in silico data. Among these proteins, a proteinase inhibitor of N. benthamiana (NbPI2) was highly associated with CP(E100A) as compared with CP(WT), and NbPI1 and NbPI2 were highly induced in ORSV-infected plants. NbPI1- and NbPI2-silenced plants (via a Tobacco rattle virus-induced gene-silencing system) did not exhibit a difference in ORSV infection. Thus, whether NbPI1 and NbPI2 play a role in plant immunity requires further investigation. In summary, the integrated omics approach provides massive and valuable information to identify the ORSV CP-IP and these CP-IP will help us to understand the movement of this virus and plant-virus interaction.

Deteksi Odontoglossum ringspot virus Terhadap Anggrek Alam di Hutan Wonosadi, Gunung Kidul

Wonosadi forest is managed forests by indigenous forest communities and is located in the hamlet of Duren and Sidorejo, Beji village, subdistrict Ngawen, Gunung Kidul regency, Yogyakarta. One of the biological richness contained in Wonosadi forest is very valuable natural orchids and need to be preserved. Management to the preservation of nature in the woods Wonosadi orchids can be done through natural orchid protection against infectious pathogens, including viruses. Odontoglossum ringspot virus (ORSV) is one type of orchid viruses infect most widely reported and has spread widely in the world, including in Indonesia. ORSV can cause severe damage to the orchid. Therefore, it is necessary to study to detect ORSV that infect wild orchids in the forest Wonosadi molecular basis for detecting the presence and pattern of infection ORSV as well as possible to reduce the severity of disease caused by infection with the virus. The purpose of this study is to assess the natural orchid ORSV infection in molecular Wonosadi forest that can be used as a database in an effort to protect against natural orchid Wonosadi forest. In the long term, the results of this study are very useful to support conservation efforts and determine appropriate conservation efforts for wild orchids in the forest Wonosadi. The initial phase of the study conducted a survey and collection of samples of wild orchids in the forest leaves that show symptoms of infection Wonosadi ORSV. Furthermore, molecular analysis performed with gene amplification Coat Protein ORSV using Reverse engineering Trancriptase Polymerase Chain Reaction (RT-PCR).Keywords: orchid soil, ORSV, RT-PCR, Wonosadi forest

Elimination of mixed ‘Odontoglossum ringspot’ and ‘Cymbidium mosaic’ viruses from Phalaenopsis hybrid ‘V3’ through shoot-tip culture and protocorm-like body selection

Phalaenopsis is one of the most popular orchid plants in the global market. Several viruses have been reported to negatively impact its growth, yield and quality. A mixed infection of Odontoglossum ringspot virus and Cymbidium mosaic virus was detected in Phalaenopsis hybrid “V3” (Phal. Yukimai × Phal. Taisuco Kochdian) plants in Taiwan. In the present communication, we report a relatively simple protocol for elimination of these two viruses using shoot-tip culture, and early isolation and selection of virus free protocorm-like body (PLB) through subcultures. The indexing of viruses in the field grown plants, PLBs and tissue culture plants was carried out by the Indirect enzyme-linked immunosorbent assay (ELISA), and one-step multiplex reverse transcription polymerase chain reaction (RT-PCR). The induction of PLBs in shoot-tips was achieved on 1/2× Murashige and Skoog's basal medium supplemented with 1% sucrose and 0.9% Bacto-agar. PLBs could be maintained, proliferated and converted to plantlets on 1 g/L Hyponex medium supplemented with 1% sucrose, 6% potato pulp, 0.5 g/L tryptone, 0.25% activated charcoal and 0.6% Bacto-agar. The regenerated plantlets from virus-free PLBs acclimatized easily in a greenhouse and showed 100% survival rate. All the tissue culture-raised Phalaenopsis plants in the greenhouse tested negative for the two viruses. Our study demonstrates that some PLB lines selected at the first subculture as virus-free were found to be infected with virus at second subculture, however, re-occurrence of virus was never found in PLB lines at third subculture onwards. Hence, at least 3 subcultures are necessary to authenticate that the cultures are free of viruses. Simple culture media without plant growth regulators used in the present study minimizes the chances of somaclonal variations and ensures genetic uniformity of cultured plantlets, a highly desirable trait in the orchid industry. The method developed in the present study has potential of virus elimination, mass propagation of genetically uniform and virus-free Phalaenopsis plants.

Genetic Diversities of Cymbidium mosaic virus and Odontoglossum ringspot virus Isolates Based on the Coat Protein Genes from Orchids in Guangdong Province, China

AbstractOrchids are some of the most important ornamental flowers. Cymbidium mosaic virus (CymMV) and Odontoglossum ringspot virus (ORSV) are the most prevalent and economically important viruses affecting orchids in China. In this study, 20 CymMV and 28 ORSV isolates were selected for genetic diversity analysis. The CymMV isolates shared 84.6–100% and 89.5–100% identities of coat protein (CP) at the nucleotide (nt) and amino acid (aa) levels, respectively. The identities of ORSV isolates were 96.4–100% (nt) and 92.5–99.4% (aa). The CP genes of CymMV were found to have genetic diversity, and the CP genes of ORSV were genetically conservative. These results can aid in designing effective disease‐control strategies.

Incidence of Cymbidium mosaic virus and Odontoglossum ringspot virus affecting Oncidium orchids in Hainan Island, China

Oncidium is the most popular cut flower for the orchid industry in Hainan Island and several viruses have been reported to negatively impact their growth and yield. A survey of Cymbidium mosaic virus (CymMV) and Odontoglossum ringspot virus (ORSV) in Oncidium orchids cultivated in Hainan Island was conducted during 2009–2012 with indirect enzyme-linked immunosorbent assay (ELISA). A total of 171,585 samples from adult plant, tissue-culture flask, seedling, and medium seedling were tested for CymMV and ORSV. Of the 24,125 adult plant samples from eighteen commercial orchid farms, 77.58% were infected with CymMV, 16.04% with ORSV, and 10.19% with both CymMV and ORSV. Of the 67,080 samples cultivated in tissue-culture flasks from eight tissue culture laboratories, 41.18% were infected with CymMV, 6.11% with ORSV, and 4.46% with both CymMV and ORSV. Of the 54,450 seedling samples from eight seedling nurseries, 53.20% were infected with CymMV, 9.00% with ORSV, and 5.23% with both CymMV and ORSV. And of the other 25,930 medium seedling samples from twelve medium seedling nurseries, 44.96% were infected with CymMV, 7.90% with ORSV, and 5.15% with both CymMV and ORSV. Plant material must be tested for the presence of viruses before being used for mass production by tissue culture, and sanitation during propagation of orchid plants and harvesting of flowers is essential in order to prevent the virus from spreading. These findings emphasize the need to implement a virus-free certification programme that would greatly enhance the management of these two prevalent viruses in the Hainan orchid industry.

Direct detection of orchid viruses using nanorod-based fiber optic particle plasmon resonance immunosensor

A fiber optic particle plasmon resonance (FOPPR) immunosensor is developed for label-free detection of orchid viruses that use gold nanorods (AuNRs) as the sensing material. The AuNRs are employed to create a near-infrared sensing window to solve the color interference problem of sample matrix for direct sensing of target analyte. This work cannot be achieved using gold nanospheres (AuNSs) because the signal of sample color absorption largely overlaps the signal of molecular recognition events in the visible spectrum, making the signal interpretation much more difficult. The AuNRs are immobilized on the unclad fiber core surface, and functionalized by antibodies which can specifically recognize the corresponding Cymbidium mosaic virus (CymMV) or Odontoglossum ringspot virus (ORSV) for rapid viral infection diagnosis. The refractive index resolution of the AuNR-FOPPR sensor is estimated to be 8×10−6 RIU. The limits of detection (LODs) for CymMV and ORSV in leaf saps are 48 and 42pg/mL, respectively, which are better than the LODs of 1200pg/mL for both viruses obtained by enzyme-linked immunosorbent assay (ELISA). Exploiting the AuNR-FOPPR sensing strategy not only solves the color interference problem encountered by using AuNSs, but provides faster analysis, better reproducibility, and lower detection limit than ELISA. The sensor can distinguish between healthy and infected orchids in 10min, and can further provide the quantitative analysis of infection level. It is potentially applicable to the quality control of orchid cultivation industry, but not limited to this, especially for creating special spectral sensing window for particular samples.

NIR-assisted orchid virus therapy using urchin bimetallic nanomaterials in phalaenopsis

The use of nanoparticles has drawn special attention, particularly in the treatment of plant diseases. Cymbidium mosaic virus (CymMV) and Odontoglossum ring spot virus (ORSV) are the most prevalent and serious diseases that affect the development of the orchid industry. In this study we treated nanoparticles as a strategy for enhancing the resistance of orchids against CymMV and ORSV. After chitosan-modified gold nanoparticles (Au NPs) were injected into Phalaenopsis leaves, the injected leaves were exposed to 980 nm laser for light–heat conversion. To evaluate virus elimination in the treated Phalaenopsis leaves, the transcripts of coat protein genes and the production of viral proteins were assessed by reverse transcription-Polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. The expression of coat protein genes for both CymMV and ORSV was significantly lower in the chitosan-modified Au NP-treated Phalaenopsis leaves than in the control. Similarly, the amount of coat proteins for both viruses in the Phalaenopsis leaves was lower than that in the control (without nanoparticle injection). We propose that the temperature increase in the chitosan-modified Au NP-treated Phalaenopsis tissues after laser exposure reduces the viral population, consequently conferring resistance against CymMV and ORSV. Our findings suggest that the application of chitosan-modified Au NPs is a promising new strategy for orchid virus therapy.

Proteomics-Based Analysis of <i>Phalaenopsis amabilis</i> in Response toward <i>Cymbidium</i> Mosaic Virus and/or <i>Odontoglossum</i> Ringspot Virus Infection

Stress response at the protein level to viral infection in orchid plants has not been extensively investigated to date. To understand the proteomic basis of Phalaenopsis amabilis’s responses to Cymbidium Mosaic virus (CymMV), and/or Odontoglossum ring spot virus (ORSV), the total proteins were extracted from Phalaenopsis amabilis leaves infected with CymMV, ORSV, or both respectively. Differentially expressed proteins were identified by two-dimensional electrophoresis, and 27 of these proteins that had significant changes were further examined by mass spectrometry. Comparing CymMV-infected leaves with mock-inoculated ones, 2 proteins were significantly up-regulated, 9 were significantly down-regulated and 1 previously undetected protein was identified. 10 proteins were significantly up-regulated, 3 significantly down-regulated and 1 previously undetected protein was identified in ORSV-infected leaves. 6 proteins were significantly up-regulated and 9 significantly down-regulated proteins were found in co-infected leaves. These identified proteins are involved in disease resistance, stress response, transcriptional regulation, energy metabolism, protein modification and the previously unknown proteins were not involved with known protein pathways. Proteins significantly up-regulated were ATP sulfurylase, down-regulated proteins included glutamate decarboxylase isozyme 2, RNA polymerase alpha subunit and chloroplastic peptide deformylase 1A were proteins with similar alteration trend after all infection treatments. Significantly up-regulated were Thioredoxin H-type and down-regulated Cytosolic phosphoglycerate kinase I which were proteins that have been shown to be specifically regulated by the infection with CymMV. Significantly up-regulated were proteins like Rubisco large subunit, Triosephosphate isomerase, NADP-specific isocitrate dehydrogenase and Cinnamoyl CoA reductase CCR2 by the infection of ORSV. Protein regulation in coinfected leaves followed a pattern similar to that of any of the single virus infection results. The<span

First Report of Cymbidium mosaic virus and Odontoglossum ringspot virus in Orchids in Mexico

In 2010, a survey for viral diseases in commercial, orchid-producing greenhouses was carried out in Morelos, Mexico. Many symptomatic plants were observed. The most common leaf symptoms were yellow mottle, yellow streaks, and chlorotic and necrotic ringspots. Leaf samples were collected from eight symptomatic plants from the following genera: Encyclia, Oncidium, Shomburghia, Brassia, Guarianthe, Cattleya, Epidendrum, Vanilla, Xilobium, Laelia, and Brassocattleya. Samples were tested using double-antibody sandwich (DAS)-ELISA (Agdia, Elkhart, IN) with antiserum for Cymbidium mosaic virus (CymMV), Odontoglossum ringspot virus (ORSV), Cymbidium ringspot mosaic virus, and Tobacco mosaic virus (TMV) and a general antiserum for potyviruses. At least one plant from each genus was positive to CymMV and ORSV as individual or mixed infections. Encyclia and Laelia plants were the most frequently found with mixed infections by both viruses. All genera were negative for TMV and potyviruses. Total RNA extracts were obtained from all ELISA-positive samples by a modified silica capture protocol (2). Reverse transcription (RT)-PCR was carried out with general polymerase (RdRp) gene primers corresponding to the Potexvirus group (3) and specific primers for the coat protein gene (CP) of CymMV and ORSV (1). The PCR amplification from a positive sample of each genus was resolved in agarose gels. Amplification products of the expected size were obtained for CymMV and ORSV. Five CymMV RdRp gene clones from five different plants of Laelia (GenBank Accession Nos. HQ393958, HQ393959, HQ393960, HQ393961, and HQ393962), two CP gene clones of CP gene of CymMV from two different plants of Oncidium (GenBank Accession Nos. HQ393956 and HQ393957), and three CP clones of CP of ORSV from three different plants of Encyclia (GenBank Accession Nos. HQ393953, HQ393954, and HQ393955) were sequenced. The nucleotide sequences of the Mexican orchid CymMV isolates were 96 to 97% identical to CymMV sequences in the GenBank, while those of ORSV were 99 to 100% identical to deposited ORSV sequences. To our knowledge, this is the first report of CymMV and ORSV in orchids in Mexico, which are two of the most important quarantine virus in orchids in Mexico.

Development of a sensitive diagnostic assay to detect Cymbidium mosaic virus and Odontoglossum ringspot virus in members of the Orchidaceae

SummaryThe Orchidaceae is a family of flowering plants native to China, members of which possess high ornamental and pharmacological value. Many members of the family Orchidaceae are susceptible to infection by both Odontoglossum ringspot tobamovirus (ORSV) and Cymbidium mosaic potexvirus (CymMV). Symptoms of viral infection include ringspots, mosaics, mottles, chlorotic streaks, and stripes on the leaves, and viral infections cause significant losses in the commercial value of these plants. However, there are no effective measures to control ORSV and CymMV. Therefore, it is vital to identify viral epidemics through the rapid and efficient detection of these two viruses, in order to limit their economic damage. We have developed an assay for detecting co-infection of members of the Orchidaceae by ORSV and CymMV. The coat protein (CP) genes from ORSV and CymMV were cloned and the CPs were expressed in vitro and used to generate two virus-specific polyclonal antisera that were validated by western blotting. Polyclonal anti-ORSV-CP and anti-CymMV-CP antisera were used simultaneously to develop a bi-immunocapture reverse transcription-polymerase chain reaction (BIC RT-PCR) assay. We demonstrated that the BIC RT-PCR assay was sensitive and specific, and provided a valuable tool for the early detection of ORSV and CymMV in leaf sap extracts from plants of the Orchidaceae.

High sequence conservation among Odotoglossum ringspot virus isolates from orchids

The variability in the nucleotide (nt) and amino acid (aa) sequences of the coat protein (CP) of Odontoglossum ringspot virus (ORSV), which naturally infects orchids worldwide, was investigated. The CP genes of 48 ORSV isolates originating from different locations in Korea were amplified using RT-PCR and sequenced. The encoded CP consists of 158 aa. The CP sequences of the Korean isolates were compared at the nt and aa levels with those of the previously published ORSV isolates originating from different countries. The Korean isolates share 94.8-100% and 92.4-100% CP identity to ORSV isolates from other countries at the nt and aa levels, respectively. No particular region of variability could be found in either sequence of the viruses. In the deduced aa sequence, the N-terminal region was more conserved than the C-terminal region in ORSV. The phylogenetic tree analysis and recombination analysis revealed that there was no distinct grouping between geographic locations and sequence identity, and nor distinct intra-specific recombination events among ORSV isolates.

Ribgrass Mosaic Tobamovirus Occurred on Chinese Cabbage in Korea

A tobamovirus, Ribgrass mosaic virus (RMV), was identified newly from chinese cabbage (Brassica campestris L. pekinensis) in Korea. Virus disease incidence of RMV on chinese cabbage was 37.9% in alpine area on August in 1993. RMV induced the symptoms of necrotic ring spots, necrotic streak on midrib and malformation. RMV, Ca1 and Ca3 isolate, could infect 35 species out of 45 plants including Chenopodium amaranticolor. Physical properties of RMV Ca1 isolate were very stable as 10.8 over for dilution end point, <TEX>$95^{\circ}C$</TEX> for temperature inactivation point and 18 weeks for longevity in vitro. RMV had the soil transmission rate of 75.0% for the chinese cabbages, 'Chunhawang' and 'Seoul' cultivars. The purified virions of RMV had the typical ultraviolet absorption spectrum of maximum at 260 nm and minimum at 247 nm. RMV of Ca1 isolate was related serologically with antisera of Tobacco mosaic virus (TMV)-Cym, TMV-O and Pepper mottle virus, but not related with antiserum of Odontoglossum ring spot virus. coat protein gene of RMV-Ca1, sized 473 nucleotides, encoded 158 amino acid residues. Nucleotide identity of RMV-Ca1 CP gene was 96.4% with RMV-Shanghai (GenBank accession No. of AF185272) from China and 96.0% with RMV-Impatiens (GenBank accession No. of AM040974) from Germany. Identity of amino acids between RMV-Ca1 and the two RMV isolates was 96.8%. Specific three primers were selected for rapid and easy genetic detection of RMV using Virion Captured (VC)/RT-PCR method.

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.

Serological Detection of Cymbidium mosaic and Odontoglossum ringspot viruses in Orchids with Polyclonal Antibodies Produced Against Their Recombinant Coat Proteins

AbstractCymbidium mosaic and Odontoglossum ringspot viruses infecting orchids were identified by coat protein (CP) properties. The Cymbidium mosaic virus (CymMV) CP gene is 672 nt long, potentially encoding 223 amino acids (aa). The Odontoglossum ringspot virus (ORSV) CP gene is 477 nt long, potentially encoding 158 aa. The CP gene of CymMV and ORSV isolates originating from different locations was highly conserved both at the nucleotide and amino acid levels (94–100%). Polyclonal antibodies against CymMV and ORSV were separately produced using bacterially expressed recombinant CP as immunogens. Antisera to CymMV (titre 1 : 2000) and ORSV (titre 1 : 250) detected the viruses by direct antigen‐coated enzyme‐linked immunosorbent assay (DAC‐ELISA) in orchid samples collected from Sikkim, India. Survey results indicated the prevalence of mixed infection of CymMV and ORSV in Cymbidium spp. The immunoreagents we developed will be useful for virus indexing in orchid certification programmes.