First complete genome sequence of an isolate of cowpea severe mosaic virus from South America.

In this study, the complete nucleotide sequence of a Brazilian isolate of cowpea severe mosaic virus (CPSMV) is presented for the first time. To date, the CPSMV-DG isolate, from the USA, is the only one with the complete known genome. High-throughput sequencing (Illumina HiSeq) and Sanger sequencing of the total RNA extract from a cowpea plant collected in Teresina city, Brazil, revealed the genome sequence of the CPSMV-Ter1 isolate. RNA-1 and RNA-2 are, respectively, 5921 and 3465 nucleotides (nt) long without the poly(A) tail, and show 77.91% and 76.08% nt sequence identity with CPSMV-DG, considered the type isolate of the species. The open reading frames (ORFs) were determined and the cleavage sites of the polyproteins were predicted. Although the two isolates show a similar genomic organization, there was a low percentage of sequence identity between Ter1 and DG. Furthermore, pairwise comparisons of a partial RNA-1 fragment between CPSMV-Ter1 and 11 CPSMV isolates from Brazil indicated 94.6 to 94.8% nt and 98.9% to 99.4% aa sequence identities.

Complete genome sequence of a novel comovirus infecting common bean.

The complete genome sequence of a novel comovirus identified in Guanajuato, Mexico, in a common bean plant (Phaseolus vulgaris L.) coinfected with Phaseolus vulgaris alphaendornavirus 1 (PvEV-1) and Phaseolus vulgaris alphaendornavirus 2 (PvEV-2) is presented. According to the current ICTV taxonomic criteria, this comovirus corresponds to a new species, and the name "Phaseolus vulgaris severe mosaic virus" (PvSMV) is proposed for this virus based on the observed symptoms of "severe mosaic" syndrome caused by comoviruses in common bean. PvSMV is closely related to bean pod mosaic virus (BPMV), and its genome consists of two polyadenylated RNAs. RNA-1 (GenBank accession number MN837498) is 5969 nucleotides (nt) long and encodes a single polyprotein of 1856 amino acids (aa), with an estimated molecular weight (MW) of 210 kDa, that contains putative proteins responsible for viral replication and proteolytic processing. RNA-2 (GenBank accession number MN837499) is 3762 nt long and encodes a single polyprotein of 1024 aa, with an estimated MW of 114 kDa, that contains putative movement and coat proteins. Cleavage sites were predicted based on similarities in size and homology to aa sequences of other comoviruses available in the GenBank database. Symptoms associated with PvSMV include mosaic, local necrotic lesions, and apical necrosis. This is the first report of a comovirus infecting common bean in Mexico.

Complete genome sequence of bean rugose mosaic virus, genus Comovirus.

Since the first report in Costa Rica in 1971, bean rugose mosaic virus (BRMV) has been found in Colombia, El Salvador, Guatemala and Brazil. In this study, the complete genome sequence of a soybean isolate of BRMV from Paraná State, Brazil, was determined. The BRMV genome consists of two polyadenylated RNAs. RNA1 is 5909 nucleotides long and encodes a single polypeptide of 1856 amino acids (aa), with an estimated molecular weight of 210 kDa. The RNA1 polyprotein contains the polypeptides for viral replication and proteolytic processing. RNA2 is 3644 nucleotides long and codes for a single polypeptide of 1097 aa, containing the movement and coat proteins. This is the first complete genome sequence of BRMV. When compared with available aa sequences of comoviruses, the highest identities of BRMV coat proteins and proteinase polymerase were 57.5 and 58 %, respectively. These were below the 75 and 80 % identity limits, respectively, established for species demarcation in the genus. This confirms that BRMV is a member of a distinct species in the genus Comovirus.

Geospatial and temporal analyses of Bean pod mottle virus epidemics in soybean at three spatial scales.

A statewide survey was carried out from 2005 through 2007 to quantify, map, and analyze the spatial dynamics and seasonal patterns of Bean pod mottle virus (BPMV) prevalence and incidence within Iowa. In all, 8 to 16 soybean fields were arbitrarily sampled from 96 counties in 2005 and all 99 counties in 2006 and 2007. Field- and county-scale BPMV prevalence and incidence data were mapped using geographic information systems software. BPMV prevalence was highest in the 2006 soybean growing season, when BPMV was detected in 38.7% of all soybean fields, 91.9% of all counties, and 100% of the agricultural climate districts. BPMV incidence at the field scale was highest in 2006, when mean statewide end-of-season incidence was 24.4%. Spatial analyses indicated that BPMV incidence was spatially clustered at the county scale in all three growing seasons. Prevalence at the county scale was clustered in 2005 and 2007 but not in 2006. Semivariogram analyses at the field scale indicated the presence of significant (P ≤ 0.05) spatial dependence (clustering) at distances ≤23.4 km in 2005, 297.7 km in 2006, and 45.2 km in 2007. Data for county-scale incidence displayed a north (low incidence) to south (high incidence) BPMV gradient in each year of the survey. High county-scale BPMV prevalence and incidence levels in 2006 were significantly associated with BPMV prevalence and incidence in 2007 (P ≤ 0.05). Soybean fields with narrow row spacings (≤38 cm) were associated with higher levels of BPMV incidence. Soybean fields infected with BPMV had a higher probability of infection by Phomopsis pod and stem blight than did non-BPMV-infected fields. This study provides new quantitative tools and information to better understand the seasonal, temporal, and geographical distribution of BPMV disease risk at several spatial scales.

Advancing broad bean true mosaic virus detection using conventional RT-PCR and real-time RT-PCR with novel primer set design.

Broad bean true mosaic virus (BBTMV) infects broad beans and peas, reducing yield. As BBTMV is transmitted through broad beans, many countries have implemented regulations to prevent the distribution of infected seeds. Currently, enzyme-linked immunosorbent assay (ELISA) is commonly used to detect BBTMV. While the PCR-based method is preferred for seed virus detection due to its sensitivity and speed. A BBTMV-specific PCR detection method has not yet been reported. A universal detection method currently exists that utilizes reverse transcription PCR (RT-PCR) for the Comovirus genus, to which BBTMV belongs. However, sequence analysis is required for species identification. To address this limitation, we developed and verified RT-PCR detection methods using newly designed BBTMV-specific primers. RT-PCR and real-time RT-PCR with these primers were approximately 5 × 105-106 times more sensitive than ELISA and 100-1000 times more sensitive than previously reported RT-PCR methods. Using RT-PCR and real-time RT-PCR employing these primers, we could detect BBTMV with same sensitivity when more than 3.0 × 105 copies were present per gram of broad bean seeds. Our newly developed detection methods can test for BBTMV with high sensitivity and speed.

A stem-loop structure in the 5' untranslated region of bean pod mottle virus RNA2 is specifically required for RNA2 accumulation.

Bean pod mottle virus (BPMV) is a bipartite, positive-sense (+) RNA plant virus of the family Secoviridae. Its RNA1 encodes all proteins needed for genome replication and is capable of autonomous replication. By contrast, BPMV RNA2 must utilize RNA1-encoded proteins for replication. Here, we sought to identify RNA elements in RNA2 required for its replication. The exchange of 5' untranslated regions (UTRs) between genome segments revealed an RNA2-specific element in its 5' UTR. Further mapping localized a 66 nucleotide region that was predicted to fold into an RNA stem-loop structure, designated SLC. Additional functional analysis indicated the importance of the middle portion of the stem and an adjacent two-base mismatch. This is the first report of a cis-acting RNA element in RNA2 of a bipartite secovirus.

Construction of an infectious cDNA clone of radish mosaic virus, a crucifer-infecting comovirus.

Radish mosaic virus (RaMV) is a crucifer-infecting comovirus that has been detected worldwide. Here, we report the successful construction of a full-length infectious cDNA clone of RaMV. The full-length cDNA clones corresponding to RNA1 and RNA2 of a Japanese isolate of RaMV were cloned into the pBlueScript plasmid or the binary vector pCAMBIA1301 downstream of the cauliflower mosaic virus 35S promoter. Mechanical inoculation or agroinoculation of Nicotiana benthamiana with these vectors resulted in systemic RaMV infections causing symptoms similar to those caused by the wild-type parental virus. The presence of progeny virus was verified by western blot analysis and electron microscopy.

Viral nanoparticles as tools for intravital vascular imaging.

A significant impediment to the widespread use of noninvasive in vivo vascular imaging techniques is the current lack of suitable intravital imaging probes. We describe here a new strategy to use viral nanoparticles as a platform for the multivalent display of fluorescent dyes to image tissues deep inside living organisms. The bioavailable cowpea mosaic virus (CPMV) can be fluorescently labeled to high densities with no measurable quenching, resulting in exceptionally bright particles with in vivo dispersion properties that allow high-resolution intravital imaging of vascular endothelium for periods of at least 72 h. We show that CPMV nanoparticles can be used to visualize the vasculature and blood flow in living mouse and chick embryos to a depth of up to 500 microm. Furthermore, we show that the intravital visualization of human fibrosarcoma-mediated tumor angiogenesis using fluorescent CPMV provides a means to identify arterial and venous vessels and to monitor the neovascularization of the tumor microenvironment.

One-step detection of Bean pod mottle virus in soybean seeds by the reverse-transcription loop-mediated isothermal amplification.

BACKGROUND: Bean pod mottle virus (BPMV) is a wide-spread and destructive virus that causes huge economic losses in many countries every year. A sensitive, reliable and specific method for rapid surveillance is urgently needed to prevent further spread of BPMV. METHODS: A degenerate reverse-transcription loop-mediated isothermal amplification (RT-LAMP) primer set was designed on the conserved region of BPMV CP gene. The reaction conditions of RT-LAMP were optimized and the feasibility, specificity and sensitivity of this method to detect BPMV were evaluated using the crude RNA rapidly extracted from soybean seeds. RESULTS: The optimized RT-LAMP parameters including 6 mM MgCl2, 0.8 M betaine and temperature at 62.5-65°C could successfully amplify the ladder-like bands from BPMV infected soybean seeds. The amplification was very specific to BPMV that no cross-reaction was observed with other soybean viruses. Inclusion of a fluorescent dye makes it easily be detected in-tube by naked eye. The sensitivity of RT-LAMP assay is higher than the conventional RT-PCR under the conditions tested, and the conventional RT-PCR couldn't be used for detection of BPMV using crude RNA extract from soybean seeds. CONCLUSION: A highly efficient and practical method was developed for the detection of BPMV in soybean seeds by the combination of rapid RNA extraction and RT-LAMP. This RT-LAMP method has great potential for rapid BPMV surveillance and will assist in preventing further spread of this devastating virus.

Phylogenetic and serological analysis of turnip ringspot virus and radish mosaic virus isolates.

Turnip ringspot virus (TuRSV) has been proposed to be a member of a new species in the genus Comovirus. Its remarkable host-range similarity to radish mosaic virus (RaMV) may have led to its misrecognition in the past. Findings from both sequence analysis and serological tests support the assignment of TuRSV to a new comovirus species. In addition, phylogenetic analysis suggests that the two genome segments of some TuRSV isolates have a heterogeneous origin.

Diversity among isolates of cowpea severe mosaic virus infecting cowpeas in northeastern Brazil.

Eleven isolates of cowpea severe mosaic virus (CPSMV), a member of the genus Comovirus, were selected from 50 samples collected of nine cowpea fields in Northeastern Brazil (Piauí, Ceará, Rio Grande do Norte, Paraíba, Pernambuco, Alagoas, Sergipe, Bahia, and Distrito Federal) and partially sequenced. The RNA1 partial sequence, corresponding to the helicase, viral genome-linked protein, picornain 3C-like protease, and the RNA-directed RNA polymerase genes from CPSMV, had high identity among isolates, varying from 98 to 100%. No evidence was found for intermolecular or intramolecular recombination. Phylogenetic analysis confirmed that the Brazilian CPSMV isolates are substantially different from the CPSMV strain USA. Despite the low variability found among Brazilian CPSMV isolates, there were notable differences in the symptomatology of infected cowpea plants, ranging from mild to moderate. Previous reports have demonstrated an association between CPSMV symptom determinants and helicase. However, we found no correlation between the helicase mutations and symptoms caused by CPSMV. Nevertheless, all isolates with mutation R to K in the protease provoked severe symptoms. This type of information can provide a foundation for the development of strategies to produce durable resistant cowpea lines. It is crucial for strategies based on DNA sequence-dependent technologies, such as inhibition with RNAi.

A reverse transcription-cross-priming amplification method with lateral flow dipstick assay for the rapid detection of Bean pod mottle virus.

Bean pod mottle virus (BPMV) is a destructive virus that causes serious economic losses in many countries every year, highlighting the importance of its effective detection. In this study, we developed a fast reverse transcription-cross-priming amplification (RT-CPA) coupled with lateral flow dipstick (LFD) diagnostic method for BPMV detection. The RT-CPA-LFD assay that targets the coat protein gene of BPMV was highly specific against diagnosing four other common viruses transmitted by soybean seeds, i.e., Southern bean mosaic virus (SBMV), Tomato ringspot virus (ToRSV), Arabis mosaic virus (ArMV), and Tobacco ringspot virus (TRSV). The sensitivities of the real-time fluorescent RT-CPA and the RT-CPA-LFD assay were at least 50 pg/µl and 500 pg/µl, respectively. Despite a compromise in the limit of detection of the RT-CPA method compared with TaqMan-MGB real-time RT-PCR, our results demonstrated a notably better performance in the detection of field samples of BPMV-infested soybean seeds. With the advantages of efficiency and convenience by visual determination, the RT-CPA-LFD assay presents a potential application for the rapid and accurate detection of BPMV in routine tests.

Landscape epidemiology of bean pod mottle comovirus: molecular evidence of heterogeneous sources.

Bean pod mottle virus (BPMV) RNAs are grouped into subgroups (sgI and sgII). A BPMV partial diploid reassortant (IA-Di1) from the perennial Desmodium illinoense contained both RNA1 subgroups and an RNA1 recombinant. The RNA2 of IA-Di1 was characteristic of sgII. Additionally, ten BPMV isolates from a soybean field adjacent to the locality of IA-Di1 shared >98.5% nucleotide identity with RNA1 sgII of IA-Di1. The data demonstrate the co-existence of two differing consensus BPMV RNA1 subgroups in adjacent habitats and illustrate variation in virus genetic structure that can occur in a contiguous plant community.

Comparative analysis of the genomes of two isolates of cowpea aphid-borne mosaic virus (CABMV) obtained from different hosts.

The complete genomic sequences of two cowpea aphid-borne mosaic virus (CABMV) isolates from Brazil, MG-Avr from passion fruit (which also infects cowpea), and BR1 from peanut (which also infects cowpea, but not passion fruit), were determined. Their nucleotide sequences are 89% identical and display 85% identity to that of CABMV-Z. Both isolates have the typical potyvirus genome features. P3 and VPg are the most conserved proteins, with 99% amino acid sequence identity between the two isolates, and P1 is the most variable, with 50% identity. A significant variation exists at the 5'-end of the genome between the Brazilian isolates and CABMV-Z. However, this variation does not correlate with the biological properties of these three isolates.

Robust RNAi-based resistance to mixed infection of three viruses in soybean plants expressing separate short hairpins from a single transgene.

Transgenic plants expressing double-stranded RNA (dsRNA) of virus origin have been previously shown to confer resistance to virus infections through the highly conserved RNA-targeting process termed RNA silencing or RNA interference (RNAi). In this study we applied this strategy to soybean plants and achieved robust resistance to multiple viruses with a single dsRNA-expressing transgene. Unlike previous reports that relied on the expression of one long inverted repeat (IR) combining sequences of several viruses, our improved strategy utilized a transgene designed to express several shorter IRs. Each of these short IRs contains highly conserved sequences of one virus, forming dsRNA of less than 150 bp. These short dsRNA stems were interspersed with single-stranded sequences to prevent homologous recombination during the transgene assembly process. Three such short IRs with sequences of unrelated soybean-infecting viruses (Alfalfa mosaic virus, Bean pod mottle virus, and Soybean mosaic virus) were assembled into a single transgene under control of the 35S promoter and terminator of Cauliflower mosaic virus. Three independent transgenic lines were obtained and all of them exhibited strong systemic resistance to the simultaneous infection of the three viruses. These results demonstrate the effectiveness of this very straight forward strategy for engineering RNAi-based virus resistance in a major crop plant. More importantly, our strategy of construct assembly makes it easy to incorporate additional short IRs in the transgene, thus expanding the spectrum of virus resistance. Finally, this strategy could be easily adapted to control virus problems of other crop plants.

Complete nucleotide sequence of the Toledo isolate of turnip ringspot virus.

The complete genomic sequence of the Toledo isolate of the comovirus, turnip ringspot virus (TuRSV), was found to consist of 2 polyadenylated RNAs. RNA 1 is 6082 nucleotides long and encodes a single predicted polypeptide of 1860 amino acids. The predicted RNA 1 polyprotein contains the polypeptides for viral replication and proteolytic processing. RNA 2, that is 3985 nucleotides long, codes for a single predicted 1095 amino acid polypeptide containing the movement and coat proteins. Phylogenetic analysis indicates that TuRSV is most closely related to radish mosaic virus, and these crucifer-infecting pathogens form a distinct clade within the comoviruses.

Construction and biological characterization of an Agrobacterium-mediated infectious cDNA of squash mosaic virus.

Squash mosaic virus (SqMV), a member of the species Squash mosaic virus in the genus Comovirus (family Comoviridae), is an important seed-borne virus that causes serious economic losses in cucurbit crops. Here, we constructed infectious cDNA clones of SqMV genomic RNAs (RNA1 and RNA2) under the control of the cauliflower mosaic virus (CaMV) 35S promoter by Gibson assembly. The infectious cDNA clones of SqMV could infect zucchini squash (Cucurbita pepo) plants systemically by agrobacterium-mediated inoculation. The virus progeny from the infectious clones showed no difference from the wild type in terms of pathogenicity and symptom induction. It could be mechanically transmitted to zucchini squash (Cucurbita pepo), pumpkin (Cucurbita moschata), cucumber (Cucumis sativus), and muskmelon (Cucumis melo) but not watermelon (Citrullus lanatus) or Nicotiana benthamiana. This is the first report of construction of a SqMV infection clone and will facilitate the investigation of viral pathogenesis and host interactions.

Inactivation and purification of cowpea mosaic virus-like particles displaying peptide antigens from Bacillus anthracis.

Chimeric cowpea mosaic virus (CPMV) particles displaying foreign peptide antigens on the particle surface are suitable for development of peptide-based vaccines. However, commonly used PEG precipitation-based purification methods are not sufficient for production of high quality vaccine candidates because they do not allow for separation of chimeric particles from cleaved contaminating species. Moreover, the purified particles remain infectious to plants. To advance the CPMV technology further, it is necessary to develop efficient and scalable purification strategies and preferably eliminate the infectivity of chimeric viruses. CPMV was engineered to display a 25 amino acid peptide derived from the Bacillus anthracis protective antigen on the surface loop of the large coat protein subunit. The engineered virus was propagated in cowpea plants and assembled into chimeric virus particles displaying 60 copies of the peptide on the surface. An effective inactivation method was developed to produce non-infectious chimeric CPMV virus-like particles (VLPs). Uncleaved VLPs were separated from the contaminating cleaved forms by anion exchange chromatography. The yield of purified chimeric VLPs was 0.3 g kg(-1) of leaf tissue. The results demonstrate the ability to generate multi-gram quantities of non-infectious, chimeric CPMV VLPs in plants for use in the development of peptide-based vaccines.

Rapid detection of fifteen known soybean viruses by dot-immunobinding assay.

A dot-immunobinding assay (DIBA) was optimized and used successfully for the rapid detection of 15 known viruses [Alfalfa mosaic virus (AMV), Bean pod mottle virus (BPMV), Bean yellow mosaic virus (BYMV), Cowpea mild mottle virus (CPMMV), Cowpea severe mosaic virus (CPSMV), Cucumber mosaic virus (CMV), Peanut mottle virus (PeMoV), Peanut stunt virus (PSV), Southern bean mosaic virus (SBMV), Soybean dwarf virus (SbDV), Soybean mosaic virus (SMV), Soybean vein necrosis virus (SVNV), Tobacco ringspot virus (TRSV), Tomato ringspot virus (ToRSV), and Tobacco streak virus (TSV)] infecting soybean plants in Oklahoma. More than 1000 leaf samples were collected in approximately 100 commercial soybean fields in 24 counties of Oklahoma, during the 2012-2013 growing seasons. All samples were tested by DIBA using polyclonal antibodies of the above 15 plant viruses. Thirteen viruses were detected, and 8 of them were reported for the first time in soybean crops of Oklahoma. The highest average incidence was recorded for PeMoV (13.5%) followed by SVNV (6.9%), TSV (6.4%), BYMV, (4.5%), and TRSV (3.9%), while the remaining seven viruses were detected in less than 2% of the samples tested. The DIBA was quick, and economical to screen more than 1000 samples against 15 known plant viruses in a very short time.

Oriented immobilisation of engineered single-chain antibodies to develop biosensors for virus detection.

Single chain variable fragment (scFv) molecules were selected from a synthetic phage display library then cloned into a generic vector for expression of the scFv fused to the light chain constant domain of human immunoglobulin with a C-terminal cysteine residue (scFvC(L)cys). A heterobifunctional maleimide linker was synthesised and a strategy for functionalization of gold with the scFvC(L)cys fusion proteins elaborated. Successful covalent attachment of functional scFvC(L)cys was demonstrated using a surface plasmon resonance-based sensor. The results showed that the immobilised scFvC(L)cys molecules were functional and specific binding curves (with response relative to the concentration of virus antigen) were obtained over more than 25 cycles of binding and dissociation. ScFv molecules lacking the C-terminal cysteine performed poorly in similar experiments. The work demonstrates the feasibility of using simple scFv selection and cloning procedures combined with oriented immobilisation of scFvC(L)cys fusion proteins for robust antigen sensing surfaces in immunosensor or other biotechnological applications.