Cucumber Mosaic Virus Coat Protein Gene Research Articles

Vasconcellea cauliflora – a new natural host of cucumber mosaic virus

Vasconcellea cauliflora plants exhibiting chlorotic spots, mottling and mosaic with yellowing on leaves were observed in Pune, India in October 2020. The disease incidence was 16%. The samples were screened for cucumber mosaic virus (CMV), papaya ringspot virus, tobacco mosaic virus and zucchini yellow mosaic virus using ELISA. Sap inoculation on healthy V. cauliflora plants using CMV positive DAS-ELISA samples reproduced field symptoms. The transmission upon mechanical inoculation was 100%. For further confirmation, RT-PCR was carried out using CMV coat protein gene specific primers. Amplicons of expected size (c. 650 bp) were obtained in all symptomatic and sap inoculated samples. No amplification was observed in the asymptomatic samples. BLASTn analysis revealed that the amplicon sequence shares more than 99% identity with CMV isolates and in phylogenetic analysis it clustered with CMV IB isolates. This is the first record of V. cauliflora as a natural host of CMV worldwide.

Cucumber mosaic virus and turnip mosaic virus occurrence in garlic mustard in Ukraine

Garlic mustard (Alliaria petiolata) is an herbaceous biennial plant native to Europe. In Ukraine, in addition to becoming a serious invader, garlic mustard can serve as a host to several viruses, which may affect agricultural crops. In view of this, the purpose of the study was to identify the virome of garlic mustard growing in Ukraine. Plant samples collected in Kyiv regions were tested for the presence of cucumber mosaic virus (CMV), turnip mosaic virus (TuMV), turnip yellow mosaic virus (TYMV), watermelon mosaic virus II (WMV), and turnip crinkle virus (TCV) by serological and/or molecular methods. According to the results found in the present study, symptomatic A. petiolata obtained in 2021 were infected with CMV (60%), TuMV (20%), or co-infected with CMV + TuMV (20%). TYMV, WMV II, and TCV were not detected in any of the collected samples. The cDNA fragments encoded the coat protein (CP) gene of CMV and TuMV were sequenced and named as CMV-Ap and TuMV-Ap, respectively. In phylogenetic analysis, the CMV-Ap closely resembled the German isolate MW582807 (Sarracenia sp.), with 99.8% nucleotide identity and belongs to subgroup II of CMV. In the phylogenetic tree, TuMV-Ap clustered with isolates AP017803, AP017764, AP017791, and JQ073722, and represented the highest identity (98.6%) to Iranian isolate IRNTRa9 (AP017803) from Rapistrum rugosum and Turkish isolate TUR49 (AP017872) from Raphanus raphanistrum. The sequences of CMV-Ap and TuMV-Ap were deposited in the GenBank under Accession Numbers MZ540213 and OM799323, respectively. The results obtained in the study indicate the important role of infected garlic mustard as alternative host and natural reservoir of CMV and TuMV from which these economically important viruses can spread to other wild and cultivated plants. This is the first molecular evidence of TuMV infection in A. petiolata from Ukraine.

Development of transgenic cucumber mosaic virus (CMV) resistant gerbera plants expressing CMV coat protein gene

Gerbera (Gerbera jamesonii L.) has its immense importance to the floriculture industry worldwide. The gerbera flower production has been hampered by various viruses, among them cucumber mosaic virus (CMV) has shown considerable damage.As natural resistance to CMV is absent in gerbera, here, we have made an attempt to develop transgenic gerbera plants expressing coat protein (CP) gene of CMV via Agrobacterium mediated transformation of base petiole explants for genetic resistance to CMV infection. Among the 44 putative transgenic gerbera plant acclimatized, 39 were found positive for integration of CP gene by polymerase chain reaction and southern hybridization assay using their specific primer and probe respectively. Northern hybridization assay using CP gene specific probe confirmed the transcription of transgene in all 39 transgenic plants. These plants showed translation of CP during DAS-ELISA when tested with antiserum specific to CP of CMV. These 39 plants when challenged by mechanical inoculations with CMV gerbera isolate showed virus resistance in 53% (21 out of 39) plants, virus tolerance (delayed mild symptom) in 33% (13/39) plants, while rest 12.8% (5/39) plants showed severe disease symptoms. The CP mediated resistance of CMV in transgenic gerbera is being reported for the first time from India.

Genetic Diversity of Cucumber Mosaic Virus from Catharanthus roseus, Jasminum sambac, Patchouly, Cubeb and Java-tea

<p><em>Cucumber mosaic virus (CMV) symptoms are found in </em>Catharanthus roseus, patchouly (Pogostemon cablin), cubeb (Piper chaba), Jasminum sambac and Java-tea (Orthosiphon aristatus); however, their genetic characterization has not been studied.<em> The study aimed to molecularly characterize the CMV isolates from </em>Catharanthus roseus, patchouly, cubeb, Jasminum sambac and Java-tea.<em> </em><em>Disease plant samples showing mosaic and yellow mosaic symptom</em>s<em> were collected from Petak Pamer Garden, ISMCRI, Bogor. Molecular characterization was carried out by reverse transcription</em>-<em>polymerase chain reaction (RT-PCR) assay using a specific primer of CMV coat protein gene and DNA sequenced. Sequence analysis was performed using the BLAST, Bioedit, Genedoc, Mega 5 programs. The RT-PCR technique succeeded in amplifying a DNA band measuring 650 bp, according to the prediction of the primary design. BLAST analyses revealed that all of these CMV isolates belonged to subgroup IB. Nucleotide sequence homology of CMV from </em>C. roseus,<em> </em>patchouly, P. chaba, <em>and</em> J. sambac, <em>were more than 95.00%. Based on </em>the <em>phylogenetic tree, these four isolates were closely related to CMV isolate from Japan (AB070622). Homology of </em>the <em>nucleotide sequence of CMV from </em>Java-tea<em> </em><em>with </em>the <em>other four isolates w</em>as<em> below 95.00%. This isolate clustered with CMV isolate from Indonesia </em>(<em>AB042294</em>) <em>and</em> <em>was</em> <em>separated with another four isolates according to </em>the <em>phylogeny tree. In the amino acid sequence alignment, </em>Java-tea <em>isolates had five different amino acids compared to the other four isolates. This result indicates the poss</em>i<em>bility of CMV transmission between patchouly</em>, Java-tea, C. roseus <em>and</em><em> </em>J. sambac, <em>so it must be anticipated to prevent its spread. </em></p>

Opuntin B, the antiviral protein isolated from prickly pear (Opuntia ficus-indica (L.) Miller) cladode exhibits ribonuclease activity

An antiviral protein, designated Opuntin B, was purified from Prickly Pear (Opuntia ficus-indica (L.) Miller) Cladode by heat treatment of the extract, protein precipitation by ammonium sulfate treatment followed by ion-exchange chromatography. Assessment of enzymatic activity of the purified protein showed that it degrades total plant genomic RNA, while causing electrophoretic mobility shifting of Cucumber mosaic virus (CMV) RNAs. However, heat-denatured viral RNA became sensitive to degradation upon treatment with antiviral protein. Opuntin B had no DNase activity on native and heat-denatured apricot genomic DNA, and on PCR-amplified coat protein gene of CMV. Using CMV as prey protein and Opuntin B as bait protein, no interaction was found between the antiviral protein and viral coat protein in far western dot blot analysis.

“Rapid diagnosis of Cucumber mosaic virus in banana plants using a fluorescence-based real-time isothermal reverse transcription-recombinase polymerase amplification assay”

Cucumber mosaic virus (CMV) is a widespread plant virus infecting important vegetables, plantation and flower crops. Currently, CMV is detected by enzyme-linked immunosorbent assays (ELISA) and reverse transcription-polymerase chain reaction (RT-PCR) assays. ELISA requires polyclonal antibodies and is time-consuming. PCR requires skilled manpower and complex procedures of RNA isolation as well as a thermal cycler. To overcome these difficulties, a portable rapid, simple and visual fluorescence-based reverse transcription-recombinase polymerase amplification (portable RT-exo-RPA) assay for the detection of CMV was developed. A specific primer pair of 30–33 bp targeting a conserved region of the coat protein (CP) gene of CMV and a probe to function in the RT-exo-RPA assays were designed and synthesized. A total of 62 symptomatic as well as 58 asymptomatic banana plant samples, collected from banana orchards located in Jalgaon, Maharashtra, India, were evaluated for CMV infections using crude leaf extracts as templates by a reverse transcription-recombinase polymerase amplification (RT-RPA) assay as well as a real-time RT-exo-RPA assay and the results were compared with those of a reverse transcription-polymerase chain reaction (RT-PCR) assay using purified total plant RNAs as templates. CMV was as efficiently detected using the crude leaf extract template in the RT-RPA and real-time RT-exo-RPA assays as using the purified RNA template in the RT-PCR assay. To dispense with the use of real-time PCR, a portable RT-exo-RPA assay was developed and the alternative methods for the visualization of CMV detection using either a fluorometer or direct viewing with a UV transilluminator were evaluated. To our knowledge, this is the first report of the rapid and reliable diagnosis of CMV infections by a real-time RT-exo-RPA assay using a crude leaf extract as template.

Large-Scale Synonymous Substitutions in Cucumber Mosaic Virus RNA 3 Facilitate Amino Acid Mutations in the Coat Protein.

The effect of large-scale synonymous substitutions in a small icosahedral, single-stranded RNA viral genome on virulence, viral titer, and protein evolution were analyzed. The coat protein (CP) gene of the Fny stain of cucumber mosaic virus (CMV) was modified. We created four CP mutants in which all the codons of nine amino acids in the 5' or 3' half of the CP gene were replaced by either the most frequently or the least frequently used synonymous codons in monocot plants. When the dicot host (Nicotiana benthamiana) was inoculated with these four CP mutants, viral RNA titers in uninoculated symptomatic leaves decreased, while all mutants eventually showed mosaic symptoms similar to those for the wild type. The codon adaptation index of these four CP mutants against dicot genes was similar to those of the wild-type CP gene, indicating that the reduction of viral RNA titer was due to deleterious changes of the secondary structure of RNAs 3 and 4. When two 5' mutants were serially passaged in N. benthamiana, viral RNA titers were rapidly restored but competitive fitness remained decreased. Although no nucleic acid changes were observed in the passaged wild-type CMV, one to three amino acid changes were observed in the synonymously mutated CP of each passaged virus, which were involved in recovery of viral RNA titer of 5' mutants. Thus, we demonstrated that deleterious effects of the large-scale synonymous substitutions in the RNA viral genome facilitated the rapid amino acid mutation(s) in the CP to restore the viral RNA titer.IMPORTANCE Recently, it has been known that synonymous substitutions in RNA virus genes affect viral pathogenicity and competitive fitness by alteration of global or local RNA secondary structure of the viral genome. We confirmed that large-scale synonymous substitutions in the CP gene of CMV resulted in decreased viral RNA titer. Importantly, when viral evolution was stimulated by serial-passage inoculation, viral RNA titer was rapidly restored, concurrent with a few amino acid changes in the CP. This novel finding indicates that the deleterious effects of large-scale nucleic acid mutations on viral RNA secondary structure are readily tolerated by structural changes in the CP, demonstrating a novel part of the adaptive evolution of an RNA viral genome. In addition, our experimental system for serial inoculation of large-scale synonymous mutants could uncover a role for new amino acid residues in the viral protein that have not been observed in the wild-type virus strains.

First Report of Mosaic Disease caused by Cucumber mosaic virus Infecting Passion Fruit in Ecuador

HomePlant DiseaseVol. 102, No. 2First Report of Mosaic Disease caused by Cucumber mosaic virus Infecting Passion Fruit in Ecuador PreviousNext DISEASE NOTES OPENOpen Access licenseFirst Report of Mosaic Disease caused by Cucumber mosaic virus Infecting Passion Fruit in EcuadorS. Yeturu, L. Paz, D. Intriago, J. Castro, H. Álvarez, and W. VieraS. YeturuSearch for more papers by this author, L. PazSearch for more papers by this author, D. IntriagoSearch for more papers by this author, J. CastroSearch for more papers by this author, H. ÁlvarezSearch for more papers by this author, and W. Viera†Corresponding author: W. Viera; E-mail: E-mail Address: william.viera@iniap.gob.ecSearch for more papers by this authorAffiliationsAuthors and Affiliations S. Yeturu , National Institute of Agricultural Research, Fruit Program, Quito, Ecuador L. Paz D. Intriago , National Institute of Agricultura Research, Plant Protection Department, Yaguachi, Ecuador J. Castro , National Institute of Agricultural Research, Fruit Program, Mocache, Ecuador H. Álvarez , National Institute of Agricultural Research, Fruit Program, Portoviejo, Ecuador W. Viera † , National Institute of Agricultural Research, Fruit Program, Quito, Ecuador. Published Online:7 Dec 2017https://doi.org/10.1094/PDIS-06-17-0814-PDNAboutSectionsSupplemental ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat Passion fruit (Passiflora edulis) is a native of tropical America and is cultivated throughout tropical and subtropical regions around the world. It belongs to the family Passifloraceae and is commonly known as yellow passion fruit, maracuyá, yellow granadilia, and pommelianejaume (Sunitha and Devaki 2009). Ecuador is the main exporter of maracuyá pulp in the world. It is grown in an area of 13,389 ha, with a production of 58,522 t (MAGAP 2016). During September 2015, mosaic, blistering, curling, and necrosis symptoms were seen on passion fruit plants in Quevedo, Los Ríos, Ecuador. Ten seedlings of Cucurbita pepo (squash) were inoculated with sap from symptomatic passion fruit leaves using 0.01 M phosphate buffer (pH 7.0). Vein clearing and blistering symptoms were observed 11 days post inoculation (dpi). Field-infected and sap-inoculated passion fruit and C. pepo samples showed positive reactions against Cucumber mosaic virus (CMV) polyclonal antibodies using TAS-ELISA (Agdia, U.S.A.). The sap-inoculated passion fruit leaves were also positive for CMV in TAS-ELISA. Total RNA was isolated from a field-infected passion fruit leaf sample using Trizol (Sigma). In reverse transcription (RT)-PCR, a DNA fragment of ∼500 bp was amplified using specific primers for the coat protein (CP) gene of CMV (Liu et al. 2012). The amplicon was sequenced and deposited in NCBI GenBank (accession no. KX470408). Sequence comparison (BioEdit v. 7.05) of the CP gene of the Ecuadorian isolate with other CMV isolates (KT302171-China, DQ302719-China, AJ585521-Australia, FJ403473-China, JN559384-China, EU250361-Taiwan, EF213025-China, AJ871492-Taiwan, AF368192-China, KC527745-South Korea, GU111229-India, AJ829779-Spain, KR535607-South Korea, AF418577-Brazil, KU144674-Mexico, GU453918-U.S.A., AY429432-China, and FN257306-Italy) showed maximum identity of 99.4% at the nucleotide level and 99.1% identity at the amino acid level with KT302171-China. CMV belongs to the genus Cucumovirus and family Bromoviridae. It is transmitted by aphids in a nonpersistent manner and has a wide host range that includes over 1,200 species belonging to 100 plant families. CMV causes significant economic losses in vegetable crops, horticulture crops, and weed plants (Zitter and Murphy 2009). To our knowledge, this is the first report of CMV on passion fruit in Ecuador.

The effect of salicylic and jasmonic acids on tomato physiology and tolerance to Cucumber mosaic virus (CMV)

Cucumber mosaic virus (CMV) is one of the most important plant viruses responsible for sharp reductions in the production of many cultivated plants. Activities of antioxidant enzymes, photosynthetic capacity, proline and total soluble carbohydrates (TSC) content were measured in the leaves of tomato (Solanum lycopercicum cv. Falat) plants treated with phytohormones (salicylic and jasmonic acids and their combination) and inoculated with CMV at 0, 1, 2, 4, 6, 8, and 15 days after the treatments. Based on the results, catalase (CAT) activity decreased in the healthy and infected plants, but peroxidase (POD) activity increased in the CMV-infected plants signifying that POD is more active in H2O2 scavenging in tomato. Because the hormone treatments inhibited the reduction in the enzyme activity, it may be considered as a controlling method against CMV. Superoxide dismutase (SOD) activity was lower in the control until 6 days post inoculation (dpi), but increased after 8 dpi. The infected plants and the hormone-treated plants showed an increased SOD activity from 0 to 15 dpi. Phenylalanine ammonia lyase (PAL) activity also increased in all the treatments over the time period (0-15 dpi). Net photosynthesis (NP) rate and chlorophyll content decreased under the virus infection and hormone treatment, whereas control plants had the highest NP and chlorophyll content. Proline accumulation occurred in the infected and hormone- treated plants, but TSC content decreased in comparison to the control. Reduction of TSC content was not significant in the hormone and virus- treated plants. Expression of CMV coat protein gene (CMV-CP) was decreased by approximately 34% in SA+JA+CMV treatment in comparison to the CMV-infected plants. In conclusion, CMV had harmful effect on physiological traits of tomato plants, but hormone application induced resistance. This resistance may be accomplished through the combination of both hormone-related signaling pathways which likely established a strong resistance network together.

Positive role of phytochromes in Nicotiana tabacum against Cucumber mosaic virus via a salicylic acid‐dependent pathway

Previous literature indicates that both light and photoreceptors may play key roles in host susceptibility to viruses. Studies have already suggested that phytochrome B is essential for the defence strategies of Nicotiana tabacum against Cucumber mosaic virus (CMV). Here, further investigations are made into the roles of phytochromes in N. tabacum against CMV. The recombinant vector pRNAi‐LIC‐PHYA/PHYB was used to produce tobacco phyA, phyB and phyAphyB mutants via the Agrobacterium‐mediated transformation method. The results suggest that both phyA and phyB deficiency increase the susceptibility of N. tabacum to CMV infection. Mutant plants showed more obvious CMV‐induced symptoms and higher transcriptional levels of the CMV coat protein gene than wild type plants, and the effect was clearly synergistic in the phyAphyB double mutant. Furthermore, the data show that mutations in phyA/phyB up‐regulate the sensitivity of N. tabacum to CMV by repressing the salicylic acid (SA)‐mediated defence pathway in tobacco plants. Additionally, the results indicate that phytochrome signalling strongly modulates the response of N. tabacum to endogenous SA but not to endogenous jasmonic acid. Taken together, these results reinforce the idea that intact phytochrome signalling plays a positive role in the ability of N. tabacum to resist CMV and is required for the induction and development of SA‐mediated defence responses in CMV‐inoculated N. tabacum.

OCCURRENCE OF CUCUMBER MOSAIC VIRUS IN OLEANDER (NERIUM OLEANDER) IN HUNGARY

In June 2016 mosaic symptoms characteristic of viral in- fection were observed in a ten year-old potted oleander ( Ner- ium oleander ) maintained in Velence (Hungary). Inoculation of test plants ( Chenopodium , Cucumis and Nicotiana spp.) with extracts of diseased oleander leaves demonstrated the presence of a mechanically transmitted plant virus marked Nol. The Nol isolate caused local lesions on Chenopodium quinoa , mosaic on Cucumis sativus and local etched ring pat- tern followed by vein clearing and recovery on the top leaves of Nicotiana tabacum cv. Xanthi-nc. These symptoms sug- gested the presence of B pathotype (subgroup II strain) of Cucumber mosaic virus (CMV). CMV was identified in the oleander leaves using CMV specific lateral flow immunoas- says (Bioreba AgriStrip). To confirm the presence of CMV, t otal RNA was extract- ed from N . clevelandii infected with CMV-Nol and used in RT-PCR with subgroup I and II CMV coat protein (CP) gene specific primers, respectively (Salanki et al ., 1997). The DNA fragment of the expected size (1460 bp) was amplified from tobacco leaves using subgroup II specific primers solely. The PCR product was cloned into pGEM-T Easy Vector (Pro- mega, USA), sequenced (Biomi, Hungary) and the complete CP gene nucleotide sequence was deposited in GenBank (accession No. KX951465). The complete CP gene of CMV- Nol showed 99% nucleotide and 100% amino acid sequence identity with a Serbian CMV isolate (232Mrg, accession No. KP034963.1). This is the first record on the natural occur- rence of CMV in oleander in Hungary.

Serological Methods to Confirm Expression of Coat Protein Gene From an Iranian Isolate of Cucumber Mosaic Virus in Escherichia coli

Background: Cucumber mosaic virus (CMV) has isometric particles with a diameter of about 28 - 29 nm. Detection and prevention are the critical steps in the control of plant viruses. Detection in a large number of samples is still done by serological methods due to their robustness and perhaps low cost. Objectives: To this end, our aim was to express the CMV CP gene in E. coli to be used as the antigen for antibody production in the future. Materials and Methods: Coat Protein (CP) gene cDNA from an isolate (B13) of Cucumber Mosaic Virus (CMV) was subcloned from pTZ57RCMVCP to pET21a expression vector and transformed to E. coli strain Rosetta. Expression of CMV CP was successful and confirmed by Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE), wherein a ~30- kDa protein band was revealed. Induction by Isopropyl-Thiogalactoside (IPTG) at final concentrations of 0.5 to 2 mM appeared to produce similar results as to the amount of the expressed protein, which was judged by intensity of the band on SDS-PAGE. Results: The identity of the expressed protein was confirmed by immunoassays such as western blot, Dot-Immunobinding Assay (DIBA) and Enzyme-Linked Immunosorbent Assay (ELISA) by the use of anti-CMV antibody. Conclusions: This is the first report of expression of CMV CP gene in Iran, which is important for the preparation of anti-CMV antibody and paving the way for the use of the virus coat protein as a nanomaterial.

Nitrate reductase-dependent nitric oxide production is required for regulation alternative oxidase pathway involved in the resistance to Cucumber mosaic virus infection in Arabidopsis

Nitrate reductase (NR), a key enzyme of nitrogen metabolism, catalyzes the reduction of nitrite to produce nitric oxide (NO). In this study, we investigated the role of NR-dependent NO in alleviating the cucumber mosaic virus (CMV)-caused damage in Arabidopsis thaliana (Arabidopsis). Quantitative real-time polymerase chain reaction analysis indicated that NR genes NIA1 and NIA2 transcripts were significantly increased by CMV infection in Arabidopsis. Further evidence showed that the nia1nia2 mutant and cPTIO-pretreated plants exhibited more serious symptoms (more reactive oxygen species accumulation, higher H2O2 content, malonaldehyde content, electrolyte leakage and transcription of CMV coat protein gene) compared with the wild-type (WT) after CMV infection. Analysis of putative molecular mechanism revealed that the expression of genes encoding pathogenesis-related 1 protein, pathogenesis-related 2 protein and alternative oxidase1a protein in nia1nia2 mutant were significantly decreased compared to the WT, and this phenomenon could be reversed by pretreatment with sodium nitroprusside (SNP). However, pretreatment with SNP could not effectively decrease the damage in aox1a mutant caused by CMV. Taken together, our results indicated that the NR-dependent NO was required for regulation the salicylic acid-mediated defense response and cyanide-resistant respiration pathway involved in the resistance to CMV infection, and alternative oxidase pathway played an important role in the defense response mediated by NO.

First Report of Cucumber mosaic virus in Catharanthus roseus in Korea.

Catharanthus roseus, commonly known as Madagascar rosy periwinkle (also called vinca), is a tropical perennial herb of the family Apocyanaceae. Periwinkle is a bedding plant widely used in Korea because of its drought tolerance, low maintenance, and varied flower colors. In May 2013, virus-like foliar symptoms, including a mosaic with malformation of leaves, were observed on a periwinkle plant in a greenhouse located in Chonbuk Province, Korea. Cucumber mosaic virus (CMV) was identified in the symptomatic plant by serological testing for the presence of CMV coat protein (CP) with an immune-strip kit developed by our laboratory. The presence of CMV was confirmed by serological detection with a commercially available double-antibody sandwich (DAS)-ELISA kit (Agdia, Elkhart, IN). Sap from the serologically positive sample was mechanically inoculated to test plants using 10 mM phosphate buffer (pH 7.0). The virus (named CMV-Vin) caused necrotic local lesions on Chenopodium amaranticolor at 5 days-post-inoculation (dpi), while mild to severe mosaic was observed in Capsicum annuum, Cucumis sativus, Cucurbita pepo 'Cheonggobong,' Nicotiana glutinosa, N. tabacum'Samsun NN,' Physalis angulate, and Solanum lycopersicum 'Pink-Top' 10 to 14 dpi. Examination of the inoculated plant leaves by DAS-ELISA and electron microscopy (leaf dips) showed positive reactions to CMV and the presence of spherical virions ~28 nm in diameter, respectively. To verify whether CMV was the causal agent for the disease symptoms observed in naturally infected periwinkle, virus-free periwinkle (10 plants) was mechanically inoculated by sap from local lesions on C. amaranticolor inoculated with CMV-Vin. At 6 weeks after inoculation, all plants produced systemic mosaic and distortion of leaves, resulting in strong DAS-ELISA reactions for CMV, whereas mock-inoculated periwinkle plants remained symptomless and virus-free. The presence of CMV-Vin in all naturally infected and mechanically inoculated plants was further verified by reverse transcription (RT)-PCR. Total RNAs were extracted with a RNeasy Plant Mini Kit (Qiagen, Valencia, CA) and RT-PCR was carried out with the One-Step RT-PCR Kit (Invitrogen, Carlsbad, CA) using a pair of primers, CMVCPFor and CMVCPRev (1), which amplified the entire CP gene. RT-PCR products (657 bp) were obtained from all naturally infected and mechanically inoculated plants as well as from a positive control (viral RNAs from virions), but not from healthy tissues. The amplified RT-PCR products were directly sequenced using BigDye Termination kit (Applied Biosystems, Foster City, CA). Multiple alignment of the CMV-Vin CP sequence (Accession No. AB910598) with CP sequences of other CMV isolates using MEGA5 software revealed that 91.8 to 99.0% and 71.0 to 73.0% identities to those of CMV subgroup I and subgroup II, respectively. These results provide additional confirmation of CMV-Vin infection. Being perennial, periwinkle plants could serve as a reservoir for CMV to infect other ornamentals and cultivated crops (2). To our knowledge, this is the first report of CMV infection on periwinkle in Korea.

First Report of Cucumber mosaic virus in Saintpaulia ionantha in Korea.

African violet (Saintpaulia ionantha) is an ornamental species of the family Gesneriaceae and is characterized by fleshy leaves and colorful flowers. This popular, exotic ornamental, originally from Kenya and Tanzania, is vegetatively produced from cutting and tissue culture (1). In May 2013, virus-like foliar symptoms, including a mosaic with dark green islands and chlorosis surrounding the veins, were observed on an African violet plant in a greenhouse located in Icheon, Korea. Cucumber mosaic virus (CMV) was identified in the symptomatic plant by serological testing for the presence of CMV coat protein (CP) with a commercial immunostrip kit (Agdia, Elkhart, IN). The presence of CMV was confirmed by serological detection with a commercially available double-antibody sandwich (DAS)-ELISA kit (Agdia). Sap from the serologically positive sample was mechanically inoculated to test plants using 10 mM phosphate buffer (pH 7.0). The virus (named CMV-AV1) caused necrotic local lesions on Chenopodium amaranticolor at 5 days post-inoculation (dpi), while mild to severe mosaic was observed in Nicotiana glutinosa, N. tabacum 'Samsun NN,' Cucurbita pepo 'Super-Top,' Physalis angulate, and Solanum lycopersicum 'Unicorn' 10 to 14 dpi. Examination of the inoculated plant leaves by DAS-ELISA and electron microscopy (leaf dips) showed positive reactions to CMV and the presence of spherical virions ∼28 nm in diameter, respectively. To verify whether CMV-AV1 is the cause of disease symptoms observed in African violet, virus-free African violet (10 plants) was mechanically inoculated by sap from local lesions on C. amaranticolor inoculated with CMV-AV1. At 8 weeks after inoculation, all plants produced systemic mosaic and chlorosis surrounding veins, resulting in strong DAS-ELISA reactions for CMV, whereas mock-inoculated African violet plants remained symptomless and virus-free. The presence of CMV-AV1 in all naturally infected and mechanically inoculated plants was further verified by reverse transcription (RT)-PCR. Total RNAs were extracted with the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany), according to the manufacturer's instructions. RT-PCR was carried out with the One-Step RT-PCR Kit (Invitrogen, Carlsbad, CA) using a pair of primers, CPTALL3 and CPTALL5 (2), amplifying the entire CP gene and part of an intergenic region and 3'-noncoding region of CMV RNA3. RT-PCR products (960 bp) were obtained from all naturally infected and mechanically inoculated plants as well as from positive control (viral RNAs from virions), but not from healthy tissues. The amplified RT-PCR products were purified with QIAquick PCR Purification Kit (Qiagen) and sequenced using BigDye Termination kit (Applied Biosystems, Foster City, CA). Multiple alignment of the CMV-AV1 CP sequence (Accession No. AB842275) with CP sequences of other CMV isolates using MEGA5 software revealed that 91.8 to 99.0% and 71.0 to 73.0% identities to those of CMV subgroup I and subgroup II, respectively. These results provide additional confirmation of CMV-AV1 infection. CMV may pose a major threat for production of African violet since the farming of African violet plants is performed using the vegetative propagation of the African violet leaves in Korea. In particular, mosaic and chlorosis symptoms in African violet cause damage to ornamental quality of African violet. To our knowledge, this is the first report of CMV infection of African violet in the world.

Immunodiagnostics of cucumber mosaic virus using antisera developed against recombinant coat protein

Cucumber mosaic virus (CMV), causing mosaic disease in banana cultivars, is the most widespread virus after Banana bunchy top virus. The entire coat protein (CP) gene of CMV was amplified by polymerase chain reaction (PCR) and engineered to be expressed in bacterial expression vector pQE30, under the control of isopropyl-thio-β-d galactopyranoside (IPTG)-inducible T7lac promoter. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) revealed expressed CP as ∼25 kDa protein. Expression conditions for maximum recovery of soluble recombinant protein were standardised. The recombinant CP (r-CP) fused with His-tag was purified from Escherichia coli using nickel-nitrilotriacetic acid (Ni-NTA) resin and used as an antigen for the production of polyclonal antisera. This antiserum was used to develop enzyme-linked immunosorbent assay (ELISA) and immunocapture reverse transcription-polymerase chain reaction (IC-RT-PCR) based assay for the detection of CMV isolates. The antiserum had a titre of 1:8000 when tested with r-CP. The reaction of polyclonal antisera was much better than that of commercial antibody. On comparing IC-RT-PCR with direct antigen coating-enzyme-linked immunosorbent assay (DAC-ELISA) using polyclonal antisera, IC-RT-PCR was found to be more sensitive, hence can be used to detect the presence of virus in tissue culture based banana plant as well as in various host plants.

Genetic transformation and development of Cucumber mosaic virus resistant transgenic plants of Chrysanthemum morifolium cv. Kundan

Chrysanthemum morifolium Ramat. cv. Kundan has been found susceptible to infections of Cucumber mosaic virus (CMV) which drastically affects the quality and quantity of blooms and poses significant constraints in commercial cultivation of chrysanthemum in India. Therefore, development of inbuilt resistance in C. morifolium against CMV seems to be essential. Agrobacterium-mediated transformation of petiole explants of C. morifolium was attempted using p RoK2 binary vector harbouring coat protein (CP) gene of CMV under the control of CaMV 35S promoter. A total of 257 explants were transformed and 73 putative transgenic plants from seven independent co-cultivation events were obtained with ∼6% transformation efficiency. Molecular analysis of these plants confirmed the successful integration of CP transgene in 63% plants, of which 12.3% plants were able to transcript and translate the transgene. Expression of coat protein did not evoke any abnormal phenotype. Transgenic plants showed delayed resistance when challenged by CMV-chrysanthemum strain which produced good quality blooms as compared to the susceptible ones.

Cucumber mosaic virus as the expression system for a potential vaccine against Alzheimer's disease

A primary therapeutic goal in Alzheimer's disease (AD) is to reduce the quantity of amyloid β protein (Aβ) present in the brain. To develop an effective, safe system for vaccination against Alzheimer's disease, the plant virus Cucumber mosaic virus (CMV) was engineered genetically to express Aβ-derived fragments that stimulate mainly humoral immune responses. Six chimeric constructs, bearing the Aβ1–15 or the Aβ4–15 sequence in positions 248, 392 or 529 of the CMV coat protein (CP) gene, were created. Viral products proved to be able to replicate in their natural host. However, only chimeric Aβ1–15-CMVs were detected by Aβ1–42 antiserum in Western blot analysis. Experimental evidence of Immunoelectron microscopy revealed a complete decoration of Aβ1–15-CMV 248 and Aβ1–15-CMV 392 following incubation with either anti-Aβ1–15 or anti-Aβ1–42 polyclonal antibodies. These two chimeric CMVs appear to be endowed with features making them possible candidates for vaccination against Alzheimer's disease.

Allergenicity Assessment of Genetically Modified Cucumber Mosaic Virus (CMV) Resistant Tomato (Solanum lycopersicon)

Cucumber mosaic virus (CMV) has been identified as the causal agent of several disease epidemics in most countries of the world. Insect-mediated virus diseases, such as those caused by CMV, caused remarkable loss of tomato (Solanum lycopersicon) production in Taiwan. With expression of the CMV coat protein gene (Cmvcp) in a local popular tomato cultivar L4783, transgenic tomato line R8 has showed consistent CMV resistance through T(0) to T(8). In this report, the allergenicity of the CMV coat protein (CMV cp) expressed in transgenic tomato R8 was assessed by investigation of the expression of the transgene source of protein, sequence similarity with known allergens, and resistance to pepsin hydrolysis. There is no known account for either the CMV or its coat protein being an allergen. The result of a bioinformatic search also showed no significant homology between CMV cp and any known allergen. The pepsin-susceptible property of recombinant CMV cp was revealed by a simulated gastric fluid (SGF) assay. Following the most recent FAO/WHO decision tree, all results have indicated that CMV cp was a protein with low possibility to be an allergen and the transgenic tomato R8 should be considered as safe as its host.

Characterization, detection and identification of transgenic chili pepper harboring coat protein gene that enhances resistance to cucumber mosaic virus

Previously, two events (H15 and B20) of transgenic pepper (Capsicum annuum L.) that enhanced resistance to Cucumber mosaic virus (CMV) by the introduction of CMV coat protein (CP) gene were constructed. Presently, a single copy number of the CP gene was revealed in H15 and B20 by Southern blot. To predict possible unintended effects due to transgene insertion in an endogenous gene, we carried out sequencing of the 5'-flanking region of the CP gene and a Blastbased search. The results revealed that insertion of the transgene into genes encoding putative proteins may occur in the H15 and B20 transgenic event. Mutiplex polymerase chain reaction (PCR) for simultaneous detection and identification of transgenic pepper was conducted with a set of nine primers. Both transgenic event were differentiated from non-transgenic event by the presence of 267 bp and 430 bp PCR products indicative of CP gene specific primer pairs and primer pairs targeting the CP gene and 35S promoter. H15 and B20 uniquely possessed a 390 bp and 596 bp PCR product, respectively. The presence of a 1115 bp product corresponding to intrinsic pepper actin gene confirmed the use of pepper DNA as the PCR template. The primer set and PCR conditions used presently may allow the accurate and simple identification of CMV resistant transgenic pepper.