The dinucleotide composition of sugarcane mosaic virus is shaped more by protein coding regions than by host species.

Sugarcane mosaic virus (SCMV), which belongs to the Potyvirus genus of the family Potyviridae, causes mosaic diseases in canna, sugarcane and maize worldwide. Previously, the genetic variations, timescale, codon usage patterns and host adaptions of SCMV were determined. However, the dinucleotide composition and the dinucleotide bias from hosts or the protein coding regions of the virus have yet to be investigated. In this study, comprehensive analyses of the dinucleotide composition and dinucleotide bias from hosts, lineages and protein coding regions of SCMV were performed using 131 complete genomic sequences. We found that UpG and CpA were largely overrepresented while UpA, CpC, and CpG were largely underrepresented in the polyprotein and 11 protein coding region data sets. SCMV dinucleotide composition bias is more strongly dependent on the protein coding regions than on hosts. A weak association between the dinucleotide composition and SCMV lineages was also observed. Our analysis provides a novel perspective on the molecular evolutionary mechanisms of SCMV and may provide a better understanding of future research on the origin and evolutionary patterns of SCMV.

ICTV Virus Taxonomy Profile: Nanoviridae.

Nanoviridae is a family of plant viruses (nanovirids) whose members have small isometric virions and multipartite, circular, single-stranded (css) DNA genomes. Each of the six (genus Babuvirus) or eight (genus Nanovirus) genomic DNAs is 0.9-1.1 kb and is separately encapsidated. Many isolates are associated with satellite-like cssDNAs (alphasatellites) of 1.0-1.1 kb. Hosts are eudicots, predominantly legumes (genus Nanovirus), and monocotyledons, predominantly in the order Zingiberales (genus Babuvirus). Nanovirids require a virus-encoded helper factor for transmission by aphids in a circulative, non-propagative manner. This is a summary of the ICTV Report on the family Nanoviridae, which is available at ictv.global/report/nanoviridae.

Characterization of a highly divergent Sugarcane mosaic virus from Canna indica L. by deep sequencing.

BACKGROUND: Cannas are popular ornamental plants and widely planted for the beautiful foliage and flower. Viral disease is a major threaten to canna horticulture industry. In the city of Beijing, mosaic disease in canna was frequently observed, but the associated causal agent and its biological characterization is still unknown. RESULTS: After small RNA deep sequencing, 36,776 contigs were assembled and 16 of them shared high sequence identities with the different proteins of Sugarcane mosaic virus (SCMV) of the size ranging from 86 to 1911 nt. The complete genome of SCMV isolate (canna) was reconstructed by sequencing all cDNA clones obtained from RT-PCR and 5'\3' RACE amplifications. SCMV-canna isolate showed to have a full RNA genome of 9579 nt in length and to share 78% nt and 85% aa sequence identities with SCMV isolates from other hosts. The phylogenetic tree constructed based on the full genome sequence of SCMV isolates allocated separately the canna-isolate in a distinct clade, indicating a new strain. Recombination analyses demonstrated that SCMV-canna isolate was a recombinant originating from a sugarcane-infecting isolate (major parent, acc. no. AJ310103) and a maize-infecting isolate (minor parent, acc. no. AJ297628). Pathogenicity test showed SCMV-canna could cause typical symptoms of mosaic and necrosis in some tested plants with varying levels of severity but was less virulent than the isolate SCMV-BJ. Field survey showed that the virus was widely distributed. CONCLUSIONS: This study identified SCMV as the major agent causing the prevalent mosaic symptom in canna plants in Beijing and its genomic and biological characterizations were further explored. All these data enriched the knowledge of the viruses infecting canna and would be helpful in effective disease management in canna.

The complete genomic sequence of Sugarcane mosaic virus from Canna spp. in China.

BACKGROUND: Sugarcane mosaic virus (SCMV) is the prevalent virus inducing maize dwarf mosaic and sugarcane mosaic diseases in China. According to the phylogenetic results of the complete genomic and coat protein gene sequences, SCMV was divided into four or five molecular groups, respectively. Previously, we detected SCMV isolates of group SO from Canna spp. in Ji'nan, Shandong province, China. FINDINGS: In this study, we collected two SCMV isolates infecting Canna spp. in Ji'nan (Canna-Ji'nan) and Tai'an (Canna-Tai'an) of Shandong, China. Their complete genome sequences had genome of 9576 nucleotides and contained a large open reading frame encoding a polyprotein of 3063 amino acids. The phylogenetic analysis showed that the both Canna-Ji'nan and Canna-Tai'an were clustered into an independent group based on the complete genome sequence. CONCLUSION: In this study, we report the complete genome sequences of SCMV infecting Canna spp. from Ji'nan and Tai'an. This is the first report on SCMV belonging to SO group.

Canna yellow streak virus: a new potyvirus associated with severe streaking symptoms in canna.

A new potyvirus has been found in canna. A 1700-nucleotide region at the 3' end of the genomic RNA has been sequenced from two isolates. The sequence reveals the virus to be a distinct member of the genus Potyvirus but most closely related to Johnsongrass mosaic virus. A specific primer pair was designed that enabled canna material to be screened specifically for this virus. The virus was consistently found in cannas showing severe virus symptoms. This virus has been found in different canna varieties from the UK, Belgium, Netherlands, France and Israel. The name Canna yellow streak virus (CaYSV) has been proposed for this new virus.

Promoters for pregenomic RNA of banana streak badnavirus are active for transgene expression in monocot and dicot plants.

Two putative promoters from Australian banana streak badnavirus (BSV) isolates were analysed for activity in different plant species. In transient expression systems the My (2105 bp) and Cv (1322 bp) fragments were both shown to have promoter activity in a wide range of plant species including monocots (maize, barley, banana, millet, wheat, sorghum), dicots (tobacco, canola, sunflower, Nicotiana benthamiana, tipu tree), gymnosperm (Pinus radiata) and fern (Nephrolepis cordifolia). Evaluation of the My and Cv promoters in transgenic sugarcane, banana and tobacco plants demonstrated that these promoters could drive high-level expression of either the green fluorescent protein (GFP) or the beta-glucuronidase (GUS) reporter gene (uidA) in vegetative plant cells. In transgenic sugarcane plants harbouring the Cv promoter, GFP expression levels were comparable or higher (up to 1.06% of total soluble leaf protein as GFP) than those of plants containing the maize ubiquitin promoter (up to 0.34% of total soluble leaf protein). GUS activities in transgenic in vitro-grown banana plants containing the My promoter were up to seven-fold stronger in leaf tissue and up to four-fold stronger in root and corm tissue than in plants harbouring the maize ubiquitin promoter. The Cv promoter showed activities that were similar to the maize ubiquitin promoter in in vitro-grown banana plants, but was significantly reduced in larger glasshouse-grown plants. In transgenic in vitro-grown tobacco plants, the My promoter reached activities close to those of the 35S promoter of cauliflower mosaic virus (CaMV), while the Cv promoter was about half as active as the CaMV 35S promoter. The BSV promoters for pregenomic RNA represent useful tools for the high-level expression of foreign genes in transgenic monocots.

Molecular characterisation of Banana mild mosaic virus, a new filamentous virus in Musa spp..

Banana mild mosaic virus (BanMMV) is a previously undescribed filamentous virus infecting Musa spp. The complete genome has been sequenced from PCR clones and consists of 7352 nucleotides, encoding five open reading frames (ORFs) of 205, 25.5, 12.4, 8.0 and 26.8 kDa, respectively. BanMMV was most closely related to fovea- and carlaviruses, based on phylogenetic analysis using a portion of the viral replicase. Analysis of other parts of the genome revealed similarities with fovea-, carla- and potexviruses, but the virus was not clearly aligned to any previously recognised genus.

Additional rep-encoding DNAs associated with banana bunchy top virus.

Banana bunchy top nanovirus (BBTV) has a multicomponent circular single-stranded DNA (cssDNA) genome consisting of at least six components. We have cloned, sequenced and analysed two additional cssDNA components, designated BBTV-S1 and S2, associated with a Taiwanese BBTV isolate. The sequences of BBTV-S1 and S2 comprised 1109 and 1095 nucleotides (nt), respectively, and like BBTV DNA-1, potentially encoded replication initiation proteins (Reps). However, the genome organisation of BBTV-S1 and S2 differed from that of BBTV DNA-1 in that (i) the stem sequence of the CR-SL was not conserved, (ii) the internal gene was absent and (iii) the probable TATA boxes were located 5' of the stem-loop. Further, sequence and phylogenetic analysis of the Rep genes indicated that BBTV DNA-S1 and S2 were distinct from BBTV DNA-1. When different geographical isolates of BBTV were tested for the presence of BBTV-S1/S2, these components were detected in various isolates from Vietnam, Taiwan, the Philippines, Tonga and Samoa but were not detected in isolates from Australia, Egypt, Fiji, and India. Based on these results, BBTV-S1 and S2 do not appear to be integral components of the BBTV genome and represent additional Rep-encoding DNAs associated with BBTV.

Banana bunchy top nanovirus DNA-1 encodes the 'master' replication initiation protein.

Banana bunchy top nanovirus has a multicomponent, circular single-stranded DNA genome comprising at least six integral components, BBTV DNA-1 to -6, which have been consistently associated with bunchy top disease worldwide. At least three other components, BBTV S1, S2 and Y, which have been isolated from Taiwanese BBTV isolates, do not appear to be integral components. We show here that both BBTV DNA-1 and S1, which encode replication initiation (Rep) proteins, were capable of self-replication when bombarded into banana embryogenic cell suspensions. However, only BBTV DNA-1 was capable of directing the replication of two other BBTV genomic components, namely BBTV DNA-3 which encodes the coat protein, and DNA-5 which encodes a retinoblastoma binding-like protein. These results indicate that (i) BBTV DNA-1 is the minimal replicative unit of BBTV and encodes the 'master' viral Rep and (ii) BBTV S1 is possibly a satellite DNA which is unable to replicate integral BBTV components.

Development of a multiplex immunocapture PCR with colourimetric detection for viruses of banana.

A multiplex, immunocapture PCR (M-IC-PCR) was developed for the simultaneous detection of three viruses from crude sap extracts of banana and plantain (Musa spp.). A reverse transcription step was required for Banana bract mosaic virus and Cucumber mosaic virus, which have ssRNA genomes. The detection of Banana bunchy top virus (ssDNA genome) was not adversely affected by inclusion in this step. All the three viruses could be detected simultaneously from a mixed infection. Identification and detection of individual viruses was achieved through the visualisation of discretely sized PCR amplicons by gel electrophoresis. Alternatively, a colourimetric microplate detection system utilising digoxigenin-labelled virus-specific probes was used. The latter assay was up to five times more sensitive than detection by gel electrophoresis and between 25 and 625 times more sensitive than ELISA for the various viruses. Careful selection of PCR primers was necessary to ensure the detection of a wide range of virus isolates and to avoid detrimental interactions between heterologous templates and primers.

Sequence variability in the coat protein gene of two groups of banana bunchy top isolates.

Complete nucleotide sequences of the coat protein gene (DNA-3) of banana bunchy top virus (BBTV) were obtained from five geographical isolates by PCR. Analysis of these sequences revealed two distinct groups of BBTV isolates with those from the Philippines, Taiwan and Vietnam forming the Asian group while the South Pacific/African group consisted of isolates from Australia, Burundi and Fiji. At the nucleotide level, the sequences of DNA-3 were more similar between isolates from the same group (maximum 5.86%) than between members of the two different groups (maximum 13.05%). At the amino acid level, the BBTV coat protein remained highly conserved, with a maximum of < 3% sequence variation between all isolates in this study. There was a significantly higher degree of divergence between the Asian isolates, which may indicate that BBTV has been present in this region for an extended period of time or that there have been multiple introductions of BBTV into bananas. The high level of conservation in the BBTV coat protein suggests that any of the DNA-3 sequences presented in this study would probably be equally effective as transgene in attempts to generate transgenic banana plants with resistance to both groups of BBTV isolates.

Functional analysis of proteins encoded by banana bunchy top virus DNA-4 to -6.

Green fluorescent protein (GFP)-tagging was used to determine the intracellular localization pattern of the proteins encoded by banana bunchy top virus (BBTV) DNA-3, -4 and -6. The protein encoded by BBTV DNA-4, which possesses a hydrophobic N terminus, was found to localize exclusively to the cell periphery while the proteins encoded by BBTV DNA-3 and -6 were found in both the nucleus and the cytoplasm. Co-expression of the DNA-4 protein and the proteins encoded by BBTV DNA-3 and -6 revealed that the DNA-4 protein was able to re-locate the DNA-6 protein, but not the DNA-3 protein, to the cell periphery. The 29 amino acid N-terminal hydrophobic region of the DNA-4 gene product appeared to be essential for specific localization of this protein since deletion of this region abolished its ability to localize to the cell periphery. These results indicate that BBTV may utilize a system analogous to that of the begomoviruses with the BBTV DNA-6 protein acting as a nuclear shuttle protein (NSP) while the DNA-4 protein transports the NSP-DNA complexes to the cell periphery for intercellular transport. The protein encoded by BBTV DNA-5 was found to contain an LXCXE motif and yeast two-hybrid analysis revealed that the DNA-5 protein has retinoblastoma (Rb)-binding activity. This activity was dependent on an intact LXCXE motif since specific mutations to either the C or E residue completely abolished Rb-binding activity. These results indicate that the gene product of BBTV DNA-5 is an Rb-binding-like protein and may play an important role in host-cell cycle manipulation.

Characterization and expression of the coat protein-coding region of banana bract mosaic potyvirus, development of diagnostic assays and detection of the virus in banana plants from five countries in southeast Asia.

We have sequenced the entire coat protein (CP)-coding region and 5' 162 nucleotides of the 3' untranslated region (UTR) of nine different isolates of banana bract mosaic virus (BBrMV) from five different countries. Further, we have sequenced the 3' 621 nucleotides of the NIb-coding region of a Philippines isolate. This is the first report of BBrMV in Thailand, Vietnam and Western Samoa. When the sequences of the CP-coding region and 3' UTR were compared to each other, variability of between 0.3% and 5.6%, and 0.3% and 4. 3%, was observed at the nucleotide and amino acid levels, respectively. Phylogenetic analysis of the BBrMV isolates did not reveal any relationship between the geographic location of the isolates. The BBrMV CP was expressed in Escherichia coli as a fusion protein and the purified recombinant protein was used to produce a high titre BBrMV-specific polyclonal antiserum. This antiserum was used to develop a F(ab')(2) indirect double antibody sandwich ELISA and compared with immuno-capture PCR (IC-PCR) and reverse transcription PCR (RT-PCR) assays for BBrMV detection. RT-PCR was shown to be the most sensitive test followed by ELISA and IC-PCR. http://link.springer. de/link/service/journals/00705/bibs/9144009/91441725.htm

Integration of banana streak badnavirus into the Musa genome: molecular and cytogenetic evidence.

Breeding and tissue culture of certain cultivars of bananas (Musa) have led to high levels of banana streak badnavirus (BSV) infection in progeny from symptomless parents. BSV DNA hybridized to genomic DNA of one such parent, Obino l'Ewai, suggesting integration of viral sequences. Sequencing of clones of Obino l'Ewai genomic DNA revealed an interface between BSV and Musa sequences and a complex BSV integrant. In situ hybridization revealed two different BSV sequence locations in Obino l'Ewai chromosomes and a complex arrangement of BSV and Musa sequences was shown by probing stretched DNA fibers. This is the first report of integrated sequences that possibly lead to a plant pararetrovirus episomal infection by a mechanism differing markedly from animal retroviral systems.

Evidence that badnavirus infection in Musa can originate from integrated pararetroviral sequences.

When some virus- and disease-free Musa spp. (banana and plantain) are propagated by tissue culture, the resulting plants develop infections with banana streak badnavirus (BSV), a pararetrovirus. In sharp contrast to the virion DNA recovered from natural infections, the virion DNA from tissue culture-associated infections of different Musa spp. was highly similar if not identical. Although BSV does not employ integration during the infection cycle, BSV DNA was found to be integrated into the Musa genome. While one integration consisted of a partial BSV genome, a second contained more than one complete genome that was almost identical to BSV recovered from tissue culture-derived plants. The arrangement of this integrated BSV DNA suggests that it can yield an infectious episomal genome via homologous recombination. This report documents the first instance of integrated DNA of a nonintegrating virus giving rise to an episomal viral infection and identifies tissue culture as a possible trigger for the infection, raising the question of whether similar activatable viral sequences exist in the genomes of other plants and animals.

Promoter activity associated with the intergenic regions of banana bunchy top virus DNA-1 to -6 in transgenic tobacco and banana cells.

Promoter regions associated with each of the six ssDNA components of banana bunchy top virus (BBTV) have been characterized. DNA segments incorporating the intergenic regions of BBTV DNA-1 to -6 were isolated and fused to the uidA (beta-glucuronidase) reporter gene to assess promoter activity. In tobacco cell suspensions, the BBTV DNA-2 and -6 promoters generated levels of GUS expression 2-fold greater and similar to the 800 bp CaMV 35S promoter, respectively. Deletion analysis of the BBTV DNA-6 promoter suggested all the necessary promoter elements required for strong expression were located within 239 nucleotides upstream of the translational start codon. In transgenic tobacco plants, the BBTV-derived promoters generally provided a weak, tissue-specific GUS expression pattern restricted to phloem-associated cells. However, in callus derived from tobacco leaf tissue, GUS expression directed by the BBTV DNA-6 promoter was strong and, in some lines, comparable to the CaMV 35S promoter. Detectable promoter activity associated with the BBTV promoters in banana embryogenic cells was only observed using a sensitive green fluorescent protein (GFP) reporter. Promoters derived from BBTV DNA-4 and -5 generated the highest levels of transient activity, which were greater than that of the maize ubi-1 promoter. In transgenic banana plants, the activity of the BBTV DNA-6 promoter was restricted to the phloem of leaves and roots, stomata and root meristems.

Banana bunchy top virus DNA-3 encodes the viral coat protein.

Banana bunchy top virus (BBTV) has a multicomponent genome consisting of at least six circular single-stranded DNAs each with a single large open reading frame (ORF) in the virion sense. A protein of approximately 20 kDa has been associated with purified virions and is assumed to be the viral coat protein. The N-terminus of this protein was sequenced and compared to the predicted amino acid sequence of the large ORF of BBTV DNA-1 to 6. This comparison indicated that the ORF of BBTV DNA-3, which potentially encodes a protein of 19.3 kDa, was the coat protein gene of BBTV. The ORF sequence of BBTV DNA-3 was cloned into a prokaryotic expression vector, pMAL-c2, and the resulting maltose binding-BBTV coat protein fusion product was purified and used for the production of polyclonal antiserum in a rabbit. The resultant antiserum was able to detect the presence of BBTV in infected leaf tissue confirming that the large virion sense ORF of BBTV DNA-3 encodes the viral coat protein.