Coat Protein Open Reading Frame Research Articles

Regulation of Positive-Strand Accumulation by Capsid Protein During Brome mosaic virus Infection In Planta.

A hallmark feature of (+)-strand RNA viruses of eukaryotic cells is that progeny (+)-strands are accumulated 100-fold over (-)-strands. Previous experimental evidence suggests that, in Brome mosaic virus (BMV), a plant-infecting member of the alphavirus-like superfamily, the addition of RNA3 and, specifically, translation of the wild-type (WT) coat protein (CP) gene contributes to increased accumulation of (+)-strands. It is unclear whether this stimulation of (+)-strand accumulation by CP is due to direct regulation of viral RNA replication or RNA stabilization via encapsidation. Analysis of BMV progeny RNA in Nicotiana benthamiana plants revealed that expression of RNA3 variants that did not express WT CP led to a severe defect in BMV (+)-strand accumulation. The (+)-strand accumulation could be rescued when CP was complemented in trans. To verify whether stimulation of (+)-strand accumulation is coupled with encapsidation, two independent mutations were engineered into CP open reading frames. An N-terminal deletion that prevented CP binding to the viral RNAs resulted in a severe reduction of BMV (+)-strand accumulation but stimulated (-)-strand accumulation over the WT. On the other hand, a C-terminal mutation affecting CP dimerization caused a significant decrease in (+)-strand accumulation but had no detectable effect on (-)-strand accumulation. Nucleotide sequences in the movement protein-coding region were also found to contribute to (+)-strand accumulation, in part by providing packaging signals for efficient RNA3 encapsidation. Overall, these results show that RNA encapsidation is a significant determinant of BMV RNA intracellular accumulation.

A Stem-Loop Structure in Potato Leafroll Virus Open Reading Frame 5 (ORF5) Is Essential for Readthrough Translation of the Coat Protein ORF Stop Codon 700 Bases Upstream.

Translational readthrough of the stop codon of the capsid protein (CP) open reading frame (ORF) is used by members of the Luteoviridae to produce their minor capsid protein as a readthrough protein (RTP). The elements regulating RTP expression are not well understood, but they involve long-distance interactions between RNA domains. Using high-resolution mass spectrometry, glutamine and tyrosine were identified as the primary amino acids inserted at the stop codon of Potato leafroll virus (PLRV) CP ORF. We characterized the contributions of a cytidine-rich domain immediately downstream and a branched stem-loop structure 600 to 700 nucleotides downstream of the CP stop codon. Mutations predicted to disrupt and restore the base of the distal stem-loop structure prevented and restored stop codon readthrough. Motifs in the downstream readthrough element (DRTE) are predicted to base pair to a site within 27 nucleotides (nt) of the CP ORF stop codon. Consistent with a requirement for this base pairing, the DRTE of Cereal yellow dwarf virus was not compatible with the stop codon-proximal element of PLRV in facilitating readthrough. Moreover, deletion of the complementary tract of bases from the stop codon-proximal region or the DRTE of PLRV prevented readthrough. In contrast, the distance and sequence composition between the two domains was flexible. Mutants deficient in RTP translation moved long distances in plants, but fewer infection foci developed in systemically infected leaves. Selective 2'-hydroxyl acylation and primer extension (SHAPE) probing to determine the secondary structure of the mutant DRTEs revealed that the functional mutants were more likely to have bases accessible for long-distance base pairing than the nonfunctional mutants. This study reveals a heretofore unknown combination of RNA structure and sequence that reduces stop codon efficiency, allowing translation of a key viral protein.IMPORTANCE Programmed stop codon readthrough is used by many animal and plant viruses to produce key viral proteins. Moreover, such "leaky" stop codons are used in host mRNAs or can arise from mutations that cause genetic disease. Thus, it is important to understand the mechanism(s) of stop codon readthrough. Here, we shed light on the mechanism of readthrough of the stop codon of the coat protein ORFs of viruses in the Luteoviridae by identifying the amino acids inserted at the stop codon and RNA structures that facilitate this "leakiness" of the stop codon. Members of the Luteoviridae encode a C-terminal extension to the capsid protein known as the readthrough protein (RTP). We characterized two RNA domains in Potato leafroll virus (PLRV), located 600 to 700 nucleotides apart, that are essential for efficient RTP translation. We further determined that the PLRV readthrough process involves both local structures and long-range RNA-RNA interactions. Genetic manipulation of the RNA structure altered the ability of PLRV to translate RTP and systemically infect the plant. This demonstrates that plant virus RNA contains multiple layers of information beyond the primary sequence and extends our understanding of stop codon readthrough. Strategic targets that can be exploited to disrupt the virus life cycle and reduce its ability to move within and between plant hosts were revealed.

Probing of RNA structures in a positive sense RNA virus reveals selection pressures for structural elements.

In single stranded (+)-sense RNA viruses, RNA structural elements (SEs) play essential roles in the infection process from replication to encapsidation. Using selective 2′-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-Seq) and covariation analysis, we explore the structural features of the third genome segment of cucumber mosaic virus (CMV), RNA3 (2216 nt), both in vitro and in plant cell lysates. Comparing SHAPE-Seq and covariation analysis results revealed multiple SEs in the coat protein open reading frame and 3′ untranslated region. Four of these SEs were mutated and serially passaged in Nicotiana tabacum plants to identify biologically selected changes to the original mutated sequences. After passaging, loop mutants showed partial reversion to their wild-type sequence and SEs that were structurally disrupted by mutations were restored to wild-type-like structures via synonymous mutations in planta. These results support the existence and selection of virus open reading frame SEs in the host organism and provide a framework for further studies on the role of RNA structure in viral infection. Additionally, this work demonstrates the applicability of high-throughput chemical probing in plant cell lysates and presents a new method for calculating SHAPE reactivities from overlapping reverse transcriptase priming sites.

A versatile papaya mosaic virus (PapMV) vaccine platform based on sortase-mediated antigen coupling

BackgroundFlexuous rod-shaped nanoparticles made of the coat protein (CP) of papaya mosaic virus (PapMV) have been shown to trigger innate immunity through engagement of toll-like receptor 7 (TLR7). PapMV nanoparticles can also serve as a vaccine platform as they can increase the immune response to fused peptide antigens. Although this approach shows great potential, fusion of antigens directly to the CP open reading frame (ORF) is challenging because the fused peptides can alter the structure of the CP and its capacity to self assemble into nanoparticles—a property essential for triggering an efficient immune response to the peptide. This represents a serious limitation to the utility of this approach as fusion of small peptides only is tolerated.ResultsWe have developed a novel approach in which peptides are fused directly to pre-formed PapMV nanoparticles. This approach is based on the use of a bacterial transpeptidase (sortase A; SrtA) that can attach the peptide directly to the nanoparticle. An engineered PapMV CP harbouring the SrtA recognition motif allows efficient coupling. To refine our engineering, and to predict the efficacy of coupling with SrtA, we modeled the PapMV structure based on the known structure of PapMV CP and on recent reports revealing the structure of two closely related potexviruses: pepino mosaic virus (PepMV) and bamboo mosaic virus (BaMV). We show that SrtA can allow the attachment of long peptides [Influenza M2e peptide (26 amino acids) and the HIV-1 T20 peptide (39 amino acids)] to PapMV nanoparticles. Consistent with our PapMV structural model, we show that around 30% of PapMV CP subunits in each nanoparticle can be fused to the peptide antigen. As predicted, engineered nanoparticles were capable of inducing a strong antibody response to the fused antigen. Finally, in a challenge study with influenza virus, we show that mice vaccinated with PapMV-M2e are protected from infection.ConclusionsThis technology will allow the development of vaccines harbouring long peptides containing several B and/or T cell epitopes that can contribute to a broad and robust protection from infection. The design can be fast, versatile and can be adapted to the development of vaccines for many infectious diseases as well as cancer vaccines.

An Analysis of the Complete Genome Sequence and Subgenomic RNAs Reveals Unique Features of the Ampelovirus, Grapevine leafroll-associated virus 1

Despite being the first closterovirus documented in grapevines (Vitis sp.), the molecular biology of Grapevine leafroll-associated virus 1 (GLRaV-1, genus Ampelovirus, family Closteroviridae) is still in its infancy. In this study, the complete genome sequence of two GLRaV-1 isolates was determined to be 18,731 (isolate WA-CH) and 18,946 (isolate WA-PN) nucleotides (nt). The genome of WA-CH and WA-PN isolates encodes nine putative open reading frames (ORFs) and the arrangement of these ORFs in both isolates was similar to that of Australian and Canadian isolates. In addition to two divergent copies of the coat protein (CP), the genome of GLRaV-1 isolates contain CP-homologous domain in four genes, making the virus unique among Closteroviridae members. The 5' and 3' nontranslated regions (NTRs) of WA-CH and WA-PN isolates showed differences in size and sequence composition, with 5' NTR having variable number of ∼65-nt-long repeats. Using the 5' NTR sequences, a reverse transcription-polymerase chain reaction and restriction fragment length polymorphism method was developed to distinguish GLRaV-1 variants in vineyards. Northern analysis of total RNA from GLRaV-1-infected grapevine samples revealed three subgenomic RNAs (sgRNAs), corresponding tentatively to CP, p21, and p24 ORFs, present at higher levels, with p24 sgRNA observed at relatively higher abundance than the other two sgRNAs. The 5' terminus of sgRNAs corresponding to CP, CPd1, CPd2, p21, and p24 were mapped to the virus genome and the leader sequence for these five sgRNAs determined to be 68, 27, 15, 49, and 18 nt, respectively. Taken together, this study provided a foundation for further elucidation of the comparative molecular biology of closteroviruses infecting grapevines.

A Sequence-Independent, Unstructured Internal Ribosome Entry Site Is Responsible for Internal Expression of the Coat Protein of Turnip Crinkle Virus.

To maximize the coding potential of viral genomes, internal ribosome entry sites (IRES) can be used to bypass the traditional requirement of a 5' cap and some/all of the associated translation initiation factors. Although viral IRES typically contain higher-order RNA structure, an unstructured sequence of about 84 nucleotides (nt) immediately upstream of the Turnip crinkle virus (TCV) coat protein (CP) open reading frame (ORF) has been found to promote internal expression of the CP from the genomic RNA (gRNA) both in vitro and in vivo An absence of extensive RNA structure was predicted using RNA folding algorithms and confirmed by selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) RNA structure probing. Analysis of the IRES region in vitro by use of both the TCV gRNA and reporter constructs did not reveal any sequence-specific elements but rather suggested that an overall lack of structure was an important feature for IRES activity. The CP IRES is A-rich, independent of orientation, and strongly conserved among viruses in the same genus. The IRES was dependent on eIF4G, but not eIF4E, for activity. Low levels of CP accumulated in vivo in the absence of detectable TCV subgenomic RNAs, strongly suggesting that the IRES was active in the gRNA invivo Since the TCV CP also serves as the viral silencing suppressor, early translation of the CP from the viral gRNA is likely important for countering host defenses. Cellular mRNA IRES also lack extensive RNA structures or sequence conservation, suggesting that this viral IRES and cellular IRES may have similar strategies for internal translation initiation.IMPORTANCE Cap-independent translation is a common strategy among positive-sense, single-stranded RNA viruses for bypassing the host cell requirement of a 5' cap structure. Viral IRES, in general, contain extensive secondary structure that is critical for activity. In contrast, we demonstrate that a region of viral RNA devoid of extensive secondary structure has IRES activity and produces low levels of viral coat protein in vitro and in vivo Our findings may be applicable to cellular mRNA IRES that also have little or no sequences/structures in common.

A CGMMV genome-replicon vector with partial sequences of coat protein gene efficiently expresses GFP in Nicotiana benthamiana

A highly infectious clone of Cucumber green mottle mosaic virus (CGMMV), a cucurbit-infecting tobamovirus was utilized for designing of gene expression vectors. Two versions of vector were examined for their efficacy in expressing the green fluorescent protein (GFP) in Nicotiana benthamiana. When the GFP gene was inserted at the stop codon of coat protein (CP) gene of the CGMMV genome without any read-through codon, systemic expression of GFP, as well as virion formation and systemic symptoms expression were obtained in N. benthamiana. The qRT-PCR analysis showed 23 fold increase of GFP over actin at 10days post inoculation (dpi), which increased to 45 fold at 14dpi and thereafter the GFP expression was significantly declined. Further, we show that when the most of the CP sequence is deleted retaining only the first 105 nucleotides, the shortened vector containing GFP in frame of original CP open reading frame (ORF) resulted in 234 fold increase of GFP expression over actin at 5dpi in N. benthamiana without the formation of virions and disease symptoms. Our study demonstrated that a simple manipulation of CP gene in the CGMMV genome while preserving the translational frame of CP resulted in developing a virus-free, rapid and efficient foreign protein expression system in the plant. The CGMMV based vectors developed in this study may be potentially useful for the production of edible vaccines in cucurbits.

Requirement for Host RNA-Silencing Components and the Virus-Silencing Suppressor when Second-Site Mutations Compensate for Structural Defects in the 3' Untranslated Region.

Turnip crinkle virus (TCV) contains a structured 3' region with hairpins and pseudoknots that form a complex network of noncanonical RNA:RNA interactions supporting higher-order structure critical for translation and replication. We investigated several second-site mutations in the p38 coat protein open reading frame (ORF) that arose in response to a mutation in the asymmetric loop of a critical 3' untranslated region (UTR) hairpin that disrupts local higher-order structure. All tested second-site mutations improved accumulation of TCV in conjunction with a partial reversion of the primary mutation (TCV-rev1) but had neutral or a negative effect on wild-type (wt) TCV or TCV with the primary mutation. SHAPE (selective 2'-hydroxyl acylation analyzed by primer extension) structure probing indicated that these second-site mutations reside in an RNA domain that includes most of p38 (domain 2), and evidence for RNA:RNA interactions between domain 2 and 3'UTR-containing domain 1 was found. However, second-site mutations were not compensatory in the absence of p38, which is also the TCV silencing suppressor, or in dcl-2/dcl4 or ago1/ago2 backgrounds. One second-site mutation reduced silencing suppressor activity of p38 by altering one of two GW motifs that are required for p38 binding to double-stranded RNAs (dsRNAs) and interaction with RNA-induced silencing complex (RISC)-associated AGO1/AGO2. Another second-site mutation substantially reduced accumulation of TCV-rev1 in the absence of p38 or DCL2/DCL4. We suggest that the second-site mutations in the p38 ORF exert positive effects through a similar downstream mechanism, either by enhancing accumulation of beneficial DCL-produced viral small RNAs that positively regulate the accumulation of TCV-rev1 or by affecting the susceptibility of TCV-rev1 to RISC loaded with viral small RNAs. Genomes of positive-strand RNA viruses fold into high-order RNA structures. Viruses with mutations in regions critical for translation and replication often acquire second-site mutations that exert a positive compensatory effect through reestablishment of canonical base pairing with the altered region. In this study, two distal second-site mutations that individually arose in response to a primary mutation in a critical 3' UTR hairpin in the genomic RNA of turnip crinkle virus did not directly interact with the primary mutation. Although different second-site changes had different attributes, compensation was dependent on the production of the viral p38 silencing suppressor and on the presence of silencing-required DCL and AGO proteins. Our results provide an unexpected connection between a 3' UTR primary-site mutation proposed to disrupt higher-order structure and the RNA-silencing machinery.

Encapsidation of Host RNAs by Cucumber Necrosis Virus Coat Protein during both Agroinfiltration and Infection.

Next-generation sequence analysis of virus-like particles (VLPs) produced during agroinfiltration of cucumber necrosis virus (CNV) coat protein (CP) and of authentic CNV virions was conducted to assess if host RNAs can be encapsidated by CNV CP. VLPs containing host RNAs were found to be produced during agroinfiltration, accumulating to approximately 1/60 the level that CNV virions accumulated during infection. VLPs contained a variety of host RNA species, including the major rRNAs as well as cytoplasmic, chloroplast, and mitochondrial mRNAs. The most predominant host RNA species encapsidated in VLPs were chloroplast encoded, consistent with the efficient targeting of CNV CP to chloroplasts during agroinfiltration. Interestingly, droplet digital PCR analysis showed that the CNV CP mRNA expressed during agroinfiltration was the most efficiently encapsidated mRNA, suggesting that the CNV CP open reading frame may contain a high-affinity site or sites for CP binding and thus contribute to the specificity of CNV RNA encapsidation. Approximately 0.09% to 0.7% of the RNA derived from authentic CNV virions contained host RNA, with chloroplast RNA again being the most prominent species. This is consistent with our previous finding that a small proportion of CNV CP enters chloroplasts during the infection process and highlights the possibility that chloroplast targeting is a significant aspect of CNV infection. Remarkably, 6 to 8 of the top 10 most efficiently encapsidated nucleus-encoded RNAs in CNV virions correspond to retrotransposon or retrotransposon-like RNA sequences. Thus, CNV could potentially serve as a vehicle for horizontal transmission of retrotransposons to new hosts and thereby significantly influence genome evolution. Viruses predominantly encapsidate their own virus-related RNA species due to the possession of specific sequences and/or structures on viral RNA which serve as high-affinity binding sites for the coat protein. In this study, we show, using next-generation sequence analysis, that CNV also encapsidates host RNA species, which account for ∼0.1% of the RNA packaged in CNV particles. The encapsidated host RNAs predominantly include chloroplast RNAs, reinforcing previous observations that CNV CP enters chloroplasts during infection. Remarkably, the most abundantly encapsidated cytoplasmic mRNAs consisted of retrotransposon-like RNA sequences, similar to findings recently reported for flock house virus (A. Routh, T. Domitrovic, and J. E. Johnson, Proc Natl Acad Sci U S A 109:1907-1912, 2012). Encapsidation of retrotransposon sequences may contribute to their horizontal transmission should CNV virions carrying retrotransposons infect a new host. Such an event could lead to large-scale genomic changes in a naive plant host, thus facilitating host evolutionary novelty.

Genomic characterization of Ambrosia asymptomatic virus 1 and evidence of other Tymovirales members in the Oklahoma tallgrass prairie revealed by sequence analysis

The Plant Virus Biodiversity and Ecology project was undertaken to better understand the nature of plant-viral interactions and the occurrence of non-pathogenic viruses. Plants from the Tallgrass Prairie Preserve (TPP), Osage County, Oklahoma, were surveyed from 2005 to 2008 for the presence of viruses, resulting in the detection, using a virus-like particle enrichment method, of the genome a novel virus, Ambrosia asymptomatic virus 1 (AAV1), from Ambrosia psilostachya DC (western ragweed). Here, we present the genomic organization and genetic variability of AAV1. The virus has a single-stranded RNA genome of about 7408 nt, which has six open reading frames (ORFs). Phylogenetic analysis of the replicase and coat protein ORFs of the virus indicates strongly that the virus should be placed in the genus Mandarivirus. No evidence of recombination was detected. We also report the detection in the TPP of two known viruses and seven other putative viruses, members of the order Tymovirales.

DISTRIBUTION AND GENETIC DIVERSITY OF TOMATO MOSAIC VIRUS ISOLATES IN IRAN

The host range and genetic variability of Tomato mosaic virus (ToMV) from Iran were investigated using isolates from different vegetables and chenopodiaceous weeds collected from 10 Iranian provinces. Of 849 symptomatic vegetables and 45 asymptomatic chenopodiaceous weeds sampled, 85 different vegetables and three nettle-leaf goosefoot (Chenopodium murale L.) samples were infected with ToMV, as indicated by DAS-ELISA. Analysis of the biological properties of selected ToMV isolates revealed three pathological groups, indicating the presence of different ToMV populations in Iran. Iranian isolates NL93 and NO1, belonging to group II, did not infect solanaceous plants whereas four other Iranian ToMV isolates along with other global isolates in group I mostly infected the solanaceous host species tested. Phylogenetic analyses of the complete ToMV coat protein open reading frame sequences indicated two clades on the basis of nucleotide and deduced amino acid sequences, and suggested the genetic relationship between isolates from various geographic origin due to the exchange of infected plant materials between Asian countries. The broad diversity of Iranian ToMV isolates showed that the ToMV populations in Iran consist of several strains that have high nucleotide diversity.

First Report of Alfalfa mosaic virus Infecting Lavandula × intermedia in Croatia.

Lavandin (Lavandula × intermedia Emeric ex Loiseleur) is cultivated on a large scale in some South European countries for the extraction of essential oils or as an ornamental plant for gardens and landscapes. In May of 2012, virus-like symptoms including bright yellow calico mosaic, leaf distortion, and growth reduction were observed on 15% of lavandin plants in a commercial nursery in Banovo Brdo locality, Baranja County, Republic of Croatia. Leaves from 15 symptomatic lavandin plants were collected and examined by double-antibody sandwich (DAS)-ELISA using commercial antisera (Bioreba AG, Reinach, Switzerland) against two viruses known to infect Lavandula spp.: Alfalfa mosaic virus (AMV) and Cucumber mosaic virus (CMV) (2,3). Commercial positive and negative controls and extracts from healthy lavandin leaves were included in each ELISA. Only AMV was detected serologically in all 15 tested samples. Five plants each of Chenopodium quinoa, C. amaranticolor, and Nicotiana benthamiana were mechanically inoculated with sap from an ELISA-positive sample (70-12) using 0.01 M phosphate buffer (pH 7). Local chlorotic spots accompanied by systemic mosaic on both Chenopodium species and bright yellow mosaic on N. benthamiana were observed 6 and 12 days post-inoculation, respectively. Test plants were assayed by DAS-ELISA and all inoculated plants of each species tested positive for AMV. The presence of AMV in all symptomatic lavandin plants was further confirmed by reverse transcription (RT)-PCR assay. Total nucleic acid was extracted using RNeasy Plant Mini Kit (Qiagen, Hilden, Germany). RT-PCR was performed with the One-Step RT-PCR Kit (Qiagen) using AMV specific primer pair CP AMV1 (5'-TCCATCATGAGTTCTTCAC-3') and CP AMV2 (5'-AGGACTTCATACCTTGACC-3') (1). Total RNAs obtained from the Serbian AMV isolate from alfalfa (GenBank Accession No. FJ527748) and healthy L. × intermedia plant served as the positive and negative control, respectively. The 751-bp amplicons, covering the partial coat protein (CP) gene and 3'-UTR, were obtained from all 15 samples that were serologically positive to AMV as well as from positive control. No amplification product was observed when extract from healthy L. × intermedia plant was used as template in the RT-PCR assay. The RT-PCR product derived from isolate 70-12 was directly sequenced in both directions using the same primer pair as in RT-PCR and deposited in GenBank (JX996119). Multiple sequence alignment of the CP open reading frame was performed by MEGA5 software (4) and revealed that the isolate 70-12 showed the highest nucleotide identity of 99.4% (99.5% amino acid identity) with Serbian AMV isolate from tobacco (FJ527749). To our knowledge, this is the first report of AMV on L. × intermedia in Croatia. Because lavandin is an aromatic plant traditionally and widely grown in Croatia, the presence of AMV could be a limiting factor for its successful production.

Lamium maculatum is a Natural Host for Cucumber mosaic virus.

Lamium maculatum L. (spotted dead-nettle) is a flowering perennial ornamental that is commonly grown as a landscape plant for an effective ground cover. In June 2010, severe mosaic accompanied by reddish brown necrosis and leaf deformation was noticed on 80% of L. maculatum growing in shade under trees and shrubs in Sarajevo (Bosnia and Herzegovina). Leaves from 10 symptomatic L. maculatum plants were sampled and analyzed by double-antibody sandwich (DAS)-ELISA using commercial diagnostic kits (Bioreba AG, Reinach, Switzerland) against Cucumber mosaic virus (CMV), Tomato spotted wilt virus (TSWV), and Impatiens necrotic spot virus (INSV), the most important viral pathogens of ornamental plants (1,2). Commercial positive and negative controls and extracts from healthy L. maculatum leaves were included in each assay. All samples tested negative for TSWV and INSV and positive for CMV. The virus was mechanically transmitted to test plants and young virus-free plants of L. maculatum using 0.01 M phosphate buffer (pH 7). The virus caused chlorotic local lesions on Chenopodium quinoa, while systemic mosaic was observed on Capsicum annuum 'Rotund,' Nicotiana rustica, N. glutinosa, N. tabacum 'White Burley,' and Phaseolus vulgaris 'Top Crop.' The virus was transmitted mechanically to L. maculatum and induced symptoms resembling those observed on the source plants. Inoculated plants were assayed by DAS-ELISA and all five inoculated plants of each species tested positive for CMV. The presence of CMV in L. maculatum as well as mechanically infected N. glutinosa plants was further confirmed by RT-PCR. Total RNA from symptomatic leaves was isolated using RNeasy Plant Mini Kit (Qiagen, Hilden, Germany) and RT-PCR was performed with the One-Step RT-PCR Kit (Qiagen) following the manufacturer's instructions. The primer pair, CMVAu1u/CMVAu2d, that amplifies the entire coat protein (CP) gene and part of 3'- and 5'-UTRs was used for both amplification and sequencing (4). Total RNA obtained from the Serbian CMV isolate from pumpkin (GenBank Accession No. HM065510) and a healthy L. maculatum plant were used as positive and negative controls, respectively. All naturally and mechanically infected plants as well as the positive control yielded an amplicon of the expected size (850 bp). No amplicon was observed in the healthy control. The amplified product derived from isolate 3-Lam was purified (QIAquick PCR Purification Kit, Qiagen), directly sequenced in both directions and deposited in GenBank (JX436358). Sequence analysis of the CP open reading frame (657 nt), conducted with MEGA5 software, revealed that the isolate 3-Lam showed the highest nucleotide identity of 99.4% (99.1% amino acid identity) with CMV isolates from Serbia, Australia, and the USA (GQ340670, U22821, and U20668, respectively). To our knowledge, this is the first report of the natural occurrence of CMV on L. maculatum worldwide and it adds a new host to over 1,241 species (101 plant families) infected by this virus (3). This is also an important discovery for the ornamental industry since L. maculatum is commonly grown together with other ornamental hosts of CMV in nurseries and the urban environment as well as in natural ecosystems.

First Report of Cucumber mosaic virus in Desert Rose in Taiwan.

Desert rose (Adenium obesum (Forssk.) Roem. & Schult, family Apocynaceae) is native to southeastern Africa, and is a perennial potted ornamental with colorful flowers that are popular in Taiwan. Symptoms of mosaic and chlorotic ringspots and line patterns on leaves were observed in July 2010, on all eight plants in a private garden in Potzu, Chiayi, Taiwan. Spherical virus particles with a diameter of approximately 28 nm were observed in crude sap prepared from symptomatic leaves. Virus culture was established by successive local lesion isolation in Chenopodium quinoa and was maintained in the systemic host Nicotiana tabacum van Hicks. The virus was mechanically transmissible to indicator plants and induced symptoms similar to those incited by Cucumber mosaic virus (CMV). Observed symptoms included local lesions on inoculated leaves of C. amaranticolor and systemic mosaic in Cucumis sativus, Lycopersicon esculentum, N. benthamiana, N. glutinosa, and N. rustica. On N. tabacum, necrotic ringspots developed on inoculated leaves followed by systemic mosaic. Serological tests using ELISA assays and western blotting indicated that the virus reacted positively to a rabbit antiserum prepared to CMV (4). Amplicons of an expected size (1.1 kb) were obtained in reverse transcription-PCR with primers specific to the 3'-half of CMV RNA 3 (3) using total RNA extracted from infected desert rose and N. tabacum. The amplified cDNA fragment was cloned and sequenced (GenBank Accession No. AB667971). Nucleotide sequences of the coat protein open reading frame (CP ORF) (657 nt) had 92 to 96% and 76 to 77% sequence identity to those of CMV in subgroups I (GenBank Accession Nos. NC_001440, D00385, M57602, D28780, and AB008777) and II (GenBank Accession Nos. L15336, AF127976, AF198103, and M21464), respectively. Desert roses infected by Tomato spotted wilt virus (TSWV) (1) and CMV (2) have been reported previously. In spite of the plants showing mosaic symptoms similar to that caused by CMV (2) and chlorotic ringspots and line patterns caused by TSWV (1), only CMV was detected in and isolated from these infected desert roses. However, the possibility of mixed infection of CMV and other viruses were not excluded in this research. To our knowledge, this is the first report of CMV infection in desert rose plants occurring in Taiwan.

A Sequence in Coat Protein Open Reading Frame Is Required for Turnip Yellow Mosaic Virus Replication

Turnip yellow mosaic virus (TYMV) is a spherical plant virus that has a single 6.3 kb positive strand RNA genome. Information for TYMV replication is limited, except that the 3’-terminal sequence and 5’-untranslated region are required for genome replication. When a foreign sequence was inserted at the position upstream of the coat protein (CP) open reading frame (ORF), replication of the recombinant TYMV was comparable to wild type, as long as an RNAi suppressor was provided. In contrast, when the foreign sequence was inserted between the CP ORF and the 3’-terminal tRNA-like structure, replication of the recombinant virus was not detected. This result suggests that the CP ORF contains an essential replication element which should be appropriately spaced with respect to the 3’-end. Analysis of TYMV constructs containing a part or a full additional CP ORF indicates that the 3’ quarter of the CP ORF is required for TYMV replication.

DEVELOPMENT OF A DIAGNOSTIC PROTOCOL FOR CUCUMBER MOSAIC VIRUS FOR SCREENING BANANA (MUSA SPP.) PLANTING MATERIAL IN IVORY COAST

Cucumber mosaic virus (CMV) is the primary virus affecting commercial banana (Musa spp.) plantations in Ivory Coast, with the disease also present within nursery systems. However, currently no virus indexing of planting material is performed for banana in the country. This current study attempts to quantify levels of detection of CMV within banana material using flinders technology associates (FTA) cards in order to identify a reliable and reproducible method suitable for local conditions. A suite of PCR primers were developed against CMV coat protein open reading frame (CP ORF) sequence available on GenBank. TaqMan assay generated comparable CT values with either FTA or cetyltrimethyl ammonium bromide (CTAB) derived templates and was able to detect CMV RNA in leaf samples. The conventional real time polymerase chain reaction (RT-PCR) assay, RNeasy and FTA derived RNA templates reliably amplified CMV, whilst CTAB-derived RNA gave variable results. The TaqMan ® reaction was much more sensitive than the conventional RT-PCR for CMV detection in banana leaf samples. The suitability of FTA cards as a matrix for virus nucleic acid capture provides scope for its use in the transportation of banana virus samples to laboratories for diagnosis and characterisation, circumventing constraints of time-related degeneration of sample integrity. Sequence data of the CMV CP ORF region of 16 isolates from Cote d'Ivoire clustered the isolates to CMV Sub-Group IA. This is the first successful application of Real-time PCR and FTA technology for a banana virus in Africa.

A Mutation in the Lettuce Infectious Yellows Virus Minor Coat Protein Disrupts Whitefly Transmission but Not In Planta Systemic Movement

The Lettuce infectious yellows virus (LIYV) RNA 2 mutant p1-5b was previously isolated from Bemisia tabaci-transmitted virus maintained in Chenopodium murale plants. p1-5b RNA 2 contains a single-nucleotide deletion in the minor coat protein (CPm) open reading frame (ORF) that is predicted to result in a frameshift and premature termination of the protein. Using the recently developed agroinoculation system for LIYV, we tested RNA 2 containing the p1-5b CPm mutant genotype (agro-pR6-5b) in Nicotiana benthamiana plants. We showed that plant infection triggered by agro-pR6-5b spread systemically and resulted in the formation of virions similar to those produced in p1-5b-inoculated protoplasts. However, virions derived from these mutant CPm genotypes were not transmitted by whiteflies, even though virion concentrations were above the typical transmission thresholds. In contrast, and as demonstrated for the first time, an engineered restoration mutant (agro-pR6-5bM1) was capable of both systemic movement in plants and whitefly transmission. These results provide strong molecular evidence that the full-length LIYV-encoded CPm is dispensable for systemic plant movement but is required for whitefly transmission.

Oral administration of plant-based rotavirus VP6 induces antigen-specific IgAs, IgGs and passive protection in mice

Plant viruses show significant potential as expression vectors for producing foreign proteins in plants. In this study, codon-optimized VP6 gene (sVP6) of human rotavirus was engineered as a replacement to the coat protein (CP) open reading frame of Beet black scorch virus (BBSV). In vitro-generated RNA transcripts corresponding to the engineered virus were infectious when inoculated onto the leaves of Chenopodium amaranticolor. Molecular analysis revealed that sVP6 was efficiently expressed and accounted for 0.25% of the total soluble protein (TSP) in plant leaves on 7 dpi. On average, a high level 1.54 μg of sVP6 was expressed in each gram of infected leaves. Oral immunization of female BALB/c mice with the plant-based sVP6 protein induced high titers of anti-VP6 mucosal IgA and serum IgG. 60% of suckling pups born from immunized dams were protected against the virulent rotavirus challenge and those infected pups developed less severe diarrhea. These results suggested that it is feasible to induce lactogenic immunity against an enteric pathogen through oral vaccination, by using the antigen produced in a new BBSV-based plant protein expression system.

Development ofTobacco necrosis virus Aas a vector for efficient and stable expression of FMDV VP1 peptides

Plant virus-based expression systems provide attractive alternatives for production of animal virus-originated antigenic peptides. In the present study, an infectious cDNA clone of Tobacco necrosis virus A Chinese isolate (TNV-A(C)) was used for expression of different peptides derived from Foot and mouth disease virus (FMDV) serotype O VP1 fused downstream of the coat protein (CP) open reading frame (ORF). Chenopodium amaranticolor inoculated with in vitro transcripts of the chimaeras developed symptoms similar to those caused by wild-type TNV-A(C). Western blot and RT-PCR detection of the infected leaves demonstrated that the chimaeras were infective, and a large number of self-assembled virions could be purified and observed under electron microscopy. Immunogold labelling revealed that highly expressed FMDV VP1 peptides could be displayed on the surfaces of virus particles. Additional immunoblotting and DNA sequence analyses showed that most of the chimaeras contained unmodified foreign peptides even after six successive passages in C. amaranticolor and three passages in Nicotiana benthamiana. Our results also suggest that the amino acid sequence and peptide length have a substantial influence on viral morphogenesis and systemic infections. Finally, animal experiments showed that purified chimaeric virus particles (CVPs) could induce a strong immune response against FMDV structural protein VP1 via an intramuscular route. And when inoculated nasally, CVPs could induce systemic and mucosal immune responses in mice.

First Report of Cucumber mosaic virus Infecting Pinewood Coneflower (Rudbeckia bicolor) in Korea

Department of Plant Medicine, Chungbuk National University, Cheongju 361-763, Korea(Received on December 21, 2009; Accepted on February 15, 2010)A virus isolate causing symptoms of yellow mosaic, fernleaves, malformation and plant necrosis on Rudbeckiabicolor was prevalent around Pyeongchang area inKorea. The causal virus was identified as Cucumbermosaic virus (CMV) using characteristics from bio-logical, serological and molecular analyses and namedas CMV-Rb. CMV-Rb caused mosaic on Nicotianabenthamiana, N. tabacum, Capsicum annuum, and Lyco-persicon esculentum. However, typical local lesions didnot develop on inoculated Pisum sativum, Cucurbitamoschata, Datura stramonium and Tetragonia expansaplants. Full-length genome sequences of CMV-RbRNAs 1, 2 and 3 were obtained using 12 primer pairs byRT-PCR analysis. The genome of CMV-Rb RNA seg-ments 1, 2, and 3 consists of 3363nt, 3049nt, and 2214ntin length, respectively. In order to ascertain their taxo-nomic identity, nucleotide and the deduced amino acidsequence analyses RNAs 1, 2 and 3 of CMV-Rb isolateswere conducted with previously reported sequences ofCMV strains and/or isolates. CMV-Rb RNAs showedabout 90 to 99% sequence identity to those of subgroupI strains suggesting that CMV-Rb is more closely relat-ed to CMV isolates belong to subgroup I. To our know-ledge, this is the first report of CMV on Rudbeckiabicolor in Korea.Keywords : CMV-Rb, phylogenetic analyses, Rudbeckiabicolor, subgroup ICucumber mosaic virus (CMV), the type member of thegenus Cucumovirus, infects more than 1,000 species ofplants and induces the typical mosaic symptoms in manyhost plants. Some of the intermediate symptoms, however,include blight, fernleaf, ringspot, and stunt. In Korea, atleast 109 natural host plant species has been reported, i.e.,20 in upland crops, 20 in vegetable crops, 28 in floral crops,acacia in wood, 40 in wild plants (List of plant disease inKorea, 2004, The Korean Society of Plant Pathology).Recently, CMV infections were also reported on Hydrangeamacrophylla (Lee et al., 2001), water chickweed (Choi etal., 2004), Isodon inflexus Kudo, Jeffersonia dubia Benth,Phryma leptostachya var. asiatica Hara (Lee et al., 2008).However, no natural outbreaks by CMV have been reportedon Rudbeckia bicolor, but Bidens mottle virus (BiMoV)was only identified (Brunt et al., 1996; Choi, 2001; Jin,2003; Lee, 1981; Lee et al., 2003; Logan et al., 1984).The genome of CMV consists of three genomic RNAs(RNA 1, 2 and 3; Roossinck, 2001). In infected host plants,it also produces two subgenomic RNAs (4 and 4A; Palukaitiset al., 1992). Based upon biological, serological, and, mole-cular biological characteristics of CMV isolates, they wereclassified into two subgroups, i.e., subgroup I and II (Owenand Palukaitis, 1988). The nucleotide sequence identitybetween subgroup I and II isolates range from 69 to 77%,while showing above 90% nucleotide sequence identitywithin each subgroup isolates. More detailed nucleotidesequence analyses, especially with the coat protein (CP)and 5' nontranslated region (NTR) of RNA 3 further sug-gested subdivision of subgroup I into subgroups IA and IB(Roossinck et al., 1999). This subgrouping was more clear-ly supported from the analysis of nucleotide sequences of1a and 3a open reading frames (ORFs) than those of the 2aand CP ORFs (Roossinck, 2002). In this paper, we reportthe biological and serological characteristics as well ascomplete nucleotide sequence analyses of CMV-Rb isolate.We believe this is the first report of natural CMV infectionon Rudbeckia bicolor in Korea.Pinewood coneflower (R. bicolor), family Asteraceae, iswidely grown as an ornamental crop and a wildflower. In2005, we could easily observe yellow mosaic, sometimes