Complete genome sequence of a putative new caulimovirus which exists as endogenous pararetroviral sequences in Angelica dahurica.

A virus isolate designated Angelica bushy stunt virus (AnBSV), provisionally representing a new species in the genus Caulimovirus, was discovered in the medicinal plant Angelica dahurica. The complete 8,300-nt genomic DNA of AnBSV had seven putative open reading frames containing conserved domains/motifs, which are typical features of caulimoviruses, and showed the greatest nucleotide sequence identity (74% identity and 27% query coverage) to a lamium leaf distortion virus isolate. Interestingly, the new caulimovirus exists as endogenous pararetroviral sequences in the host plant and is considered to have multiple defective plant genome-integrated copies that may lead to the generation of subgenomic DNA species.

Strawberry vein banding virus isolates in eastern Canada are molecularly divergent from other isolates.

The complete sequence of a strawberry vein banding virus (SVBV) isolate collected in Nova Scotia, Canada, and designated NS8, was determined. The 7,856-nucleotide circular double-stranded DNA genome contains seven open-reading frames (ORFs), which is consistent with other SVBV isolates and other members of the genus Caulimovirus. Comparison of NS8 with other whole-genome sequences retrieved from databases revealed that NS8 shares the highest sequence similarity (96.5% identity) with isolate China (accession number HE681085) and the lowest (88.3% identity) with clone pSVBV-E3 (accession number X97304). Despite the overall high sequence similarity between NS8 and China, the coat protein encoding ORF IV of NS8 shares only 90.9% sequence identity with the China isolate. Phylogenetic analysis at the complete-genome level placed NS8 and all Chinese isolates in one clade and clone pSVBV-E3 in a separate clade. Interestingly, phylogenetic analysis of all available ORF IV sequences, including those retrieved from databases and newly sequenced samples in this study from Canada, revealed three distinct clades. All Canadian isolates grouped together as one clade, pSVBV-E3 and several others from Europe, Egypt and the USA grouped as a second clade, and isolates from China formed a third clade. These results demonstrate that SVBV is more divergent than previously reported.

Complete nucleotide sequence of strawberry vein banding virus Chinese isolate and infectivity of its full-length DNA clone.

BACKGROUND: Strawberry vein banding virus (SVBV) is a double-stranded DNA plant virus, which has been found in North America, Australia, Brazil, Japan, Europe and several provinces of China. Infected strawberry plants exhibit mild vein-banding symptoms and chlorosis along the veins. It is one of the most economically important diseases in Asiatic, European and North American strawberry-growing areas. FINDINGS: The complete genome of an SVBV Chinese isolate (SVBV-CN) was isolated and cloned from a naturally infected strawberry (Fragaria × ananassa cv. Sachinoka) sample found in Shenyang city of Liaoning province. Sequence analysis revealed a complete genome of 7864 nucleotides (nts) that indicated SVBV-CN was most closely related to SVBV from the United States (SVBV-US) with a sequence similarity of 85.8 %. Two major clades were identified based on phylogenetic analysis of the complete genome sequences of caulimoviruses. SVBV-CN clustered together with SVBV-US, whereas other caulimoviruses formed a separate branch. Agrobacterium-mediated inoculation of Fragaria vesca with an infectious clone of SVBV-CN results in systemic infection with distinct symptoms of yellowing bands along the main leaf veins. This suggests that the SVBV-CN infectious clone can recapitulate the symptoms observed in naturally infected strawberries, and therefore is likely the causal agent of the original disease observed in strawberries. Furthermore, strawberry plants inoculated with the infectious clone using vacuum infiltration developed symptoms with a very high infection rate of 86-100 % in 4-5 weeks post-inoculation. This compares to an infection rate of 20-40 % in 8-9 weeks post-inoculation using syringe-inoculation. CONCLUSIONS: The complete nucleotide sequence of SVBV from a naturally infected strawberry was determined. Agroinfiltration of strawberry plants using an infectious clone of SVBV-CN resulted in symptoms typically found in infected strawberries from Shenyang city of Liaoning province in China. This is the first report describing an infectious clone of SVBV-CN, and that vacuum infiltration can be potentially used as a new and highly efficient means for inoculation of strawberry plants.

Complete genome sequence of a tentative new caulimovirus from the medicinal plant Atractylodes macrocephala.

A total of nine contigs related to caulimovirus-like sequences were detected using high-throughput paired-end RNA sequencing. An attempt to find the plant sample infected with this type of virus identified the medicinal plant Atractylodes macrocephala Koidzumi showing mild mottle symptoms. Subsequently, the complete DNA genome sequence of the Atractylodes virus was determined. The 8,105-nt genome of the virus was composed of six open reading frames and displayed the highest nucleotide sequence identity (70%) with soybean Putnam virus. Based upon the symptoms observed on the source plant, we propose to refer to this new member of the genus Caulimovirus as atractylodes mild mottle virus.

Platinum anniversary: virus and lichen alga together more than 70 years.

Trebouxia aggregata (Archibald) Gärtner (phylum Chlorophyta, family Trebouxiaceae), a lichen symbiotic alga, has been identified as host of the well-known herbaceous plant virus Cauliflower mosaic virus (CaMV, family Caulimoviridae). The alga had been isolated from Xanthoria parietina more than 70 years ago and has been maintained in a collection since that time. The CaMV detected in this collection entry has now been completely sequenced. The virus from T. aggregata is mechanically transmissible to a herbaceous host and induces disease symptoms there. Its genome differs by 173 nt from the closest European CaMV-D/H isolate from cauliflower. No site under positive selection was found on the CaMV genome from T. aggregata. We therefore assume that the virus's presence in this alga was not sufficiently long to fix any specific changes in its genome. Apart from this symbiotic alga, CaMV capsid protein sequences were amplified from many other non-symbiotic algae species maintained in a collection (e.g., Oonephris obesa, Elliptochloris sp., Microthamnion kuetzingianum, Chlorella vulgaris, Pseudococcomyxa sp.). CaMV-free Chlorella vulgaris was treated with CaMV to establish virus infection. The virus was still detected there after five passages. The virus infection is morphologically symptomless on Chlorella algae and the photosynthesis activity is slightly decreased in comparison to CaMV-free alga culture. This is the first proof as to the natural presence of CaMV in algae and the first demonstration of algae being artificially infected with this virus.

The temporal evolution and global spread of Cauliflower mosaic virus, a plant pararetrovirus.

Cauliflower mosaic virus (CaMV) is a plant pararetrovirus with a double-stranded DNA genome. It is the type member of the genus Caulimovirus in the family Caulimoviridae. CaMV is transmitted by sap inoculation and in nature by aphids in a semi-persistent manner. To investigate the patterns and timescale of CaMV migration and evolution, we sequenced and analyzed the genomes of 67 isolates of CaMV collected mostly in Greece, Iran, Turkey, and Japan together with nine published sequences. We identified the open-reading frames (ORFs) in the genomes and inferred their phylogeny. After removing recombinant sequences, we estimated the substitution rates, divergence times, and phylogeographic patterns of the virus populations. We found that recombination has been a common feature of CaMV evolution, and that ORFs I-V have a different evolutionary history from ORF VI. The ORFs have evolved at rates between 1.71 and 5.81×10(-4) substitutions/site/year, similar to those of viruses with RNA or ssDNA genomes. We found four geographically confined lineages. CaMV probably spread from a single population to other parts of the world around 400-500 years ago, and is now widely distributed among Eurasian countries. Our results revealed evidence of frequent gene flow between populations in Turkey and those of its neighboring countries, with similar patterns observed for Japan and the USA. Our study represents the first report on the spatial and temporal spread of a plant pararetrovirus.

A phylogeographical study of the cauliflower mosaic virus population in mid-Eurasia Iran using complete genome analysis.

The full-length sequences of 34 Iranian cauliflower mosaic virus (CaMV) isolates were compared with others from public nucleotide sequence databases to provide a comprehensive overview of the genetic variability and patterns of genetic exchange in CaMV isolates from Iran. Based on the severity of symptoms and their ability to infect Brassica oleracea var. capitata, Iranian CaMV isolates were grouped into two distinct biotypes: latent/mild mottle (LI/MMo) and severe (S) infection. Recombination breakpoints were detected between the large intergenic region (LIR) and open reading frame (ORF) V (event 2); between ORF VII and ORF II (event 3), between ORF I and ORF III (event 4), and within ORF VI (event 1). Phylogenetic analysis indicated that Iranian CaMV isolates clustered into two subgroups belonging to group I (GI) that were distinct from North American and European isolates from group II (GII). Northeast Iranian isolates (subgroup B) and CaMV isolates from subgroup A closely corresponded to the S and LI/MMo biological groups, respectively. Genome-wide pairwise identity analysis of the CaMV isolates revealed three regions of pairwise identity representation: 92-94 % for GII and 94-96 % and 98-100 % for subgroups A and B. The within-population diversity was lower than the between-population diversity, suggesting the contribution of a founder effect on diversification of CaMV isolates. Amino acid sequences were conserved, with ω values ranging from 0.074 to 0.717 in different proteins. Thirteen amino acids in the deduced proteins of ORFs I, II, III, VI and VII were under positive selection (ω > 1), whereas purifying selection applied to the proteins encoded by ORFs IV and V. This study suggests that variation in the CaMV population can be explained by host-range differentiation and selection pressure. Moreover, recombination analysis revealed that a genomic exchange is responsible for the emergence of CaMV strains, providing valuable new information for understanding the diversity and evolution of caulimoviruses.

Comparative analysis of endogenous plant pararetroviruses in cultivated and wild Dahlia spp.

Two distinct caulimoviruses, Dahlia mosaic virus (DMV) and Dahlia common mosaic virus, and an endogenous plant pararetroviral sequence (DvEPRS) were reported in Dahlia spp. DvEPRS, previously referred to as DMV-D10, was originally identified in the US from the cultivated Dahlia variabilis, and has also been found in New Zealand, Lithuania and Egypt, as well as in wild dahlia species growing in their natural habitats in Mexico. Sequence analysis of three new EPRSs from cultivated dahlias from Lithuania [D10-LT; 7,159 nucleotide level (nt)], New Zealand (D10-NZ, 7,156 nt), and the wild species, Dahlia rupicola, from Mexico (D10-DR, 7,133 nt) is reported in this study. The three EPRSs have the structure and organization typical of a caulimovirus species and showed identities among various open reading frames (ORFs) ranging between 71 and 97 % at the nt when compared to those or the known DvEPRS from the US. Examination of a dataset of seven full-length EPRSs obtained to date from cultivated and wild Dahlia spp. provided clues into genetic diversity of these EPRSs from diverse sources of dahlia. Phylogenetic analyses, mutation frequencies, potential recombination events, selection, and fitness were evaluated as evolutionary evidences for genetic variation. Assessment of all ORFs using phylogenomic and population genetics approaches suggests a wide genetic diversity of EPRSs occurring in dahlias. Phylogenetic analyses show that the EPRSs from various sources form one clade indicating a lack of clustering by geographical origin. Grouping of various EPRSs into two host taxa (cultivated vs. wild) shows little divergence with respect to their origin. Population genetic parameters demonstrate negative selection for all ORFs, with the reverse transcriptase region more variable than other ORFs. Recombination events were found which provide evolutionary evidence for genetic diversity among dahlia-associated EPRSs. This study contributes to an increased understanding of molecular population genetics and evolutionary pathways of these reverse transcribing viral elements.

Biological and molecular variation of Iranian Cauliflower mosaic virus (CaMV) isolates.

Seventeen provinces of Iran were surveyed during 2003-2012 to find Brassicaceae hosts of Cauliflower mosaic virus (CaMV). A total 397 samples were collected from plants with virus-like symptoms. Among those tested by ELISA, 255 samples (67.2 %) were found to be infected with CaMV. Mechanical transmission tests showed that the Iranian isolates have similar biological properties on a number of Brassica and Raphanus plant species and cultivars tested. However, the isolates varied in the severity of symptoms they induced and in the capacity to infect B. oleracea var. capitata, on the basis of which they were grouped into two distinct biotypes L/MMo (latent/mild mottle) and severe (S) infection. The molecular diversity of natural population of CaMV were investigated based on the complete sequences of OFR 6 of 36 Iranian isolates collected from different geographically distant regions in Iran alongside the sequences of 14 previously reported isolates. Phylogenetic analyses indicated that the Iranian CaMV isolates belong to two groups (GI and GII). Most of the Iranian isolates fell into GI with other exotic isolates; however, the isolates from North-East Iran with Xinjiang from China fell into GII. The phylogenetic group GII (the North-East Iranian isolates) closely corresponded to the S biological group however other Iranian isolates corresponded to the L/MMo biological group. The within-population diversity was lower than the between population diversity suggesting the contribution of a founder effect on diversification of CaMV isolates. The Iranian isolates were differentiated from other exotic CaMV isolates and clustered into two RFLP groups using Hpy99I which closely corresponded to the biological and phylogenetic groups. This study showed the evolutionary process in CaMV isolates is shaped by a combination of host range differentiation and nucleotide substitution using the approach of population genetics.

Complete genome sequence of a novel pararetrovirus isolated from soybean.

We report the complete genome sequence of soybean Putnam virus (SPuV), a new pararetrovirus isolated from a soybean field in Putnam County, OH. Comparison of SPuV with other plant-infecting pararetroviruses places it in the genus Caulimovirus of the family Caulimoviridae.

Genomic characterization of pararetroviral sequences in wild Dahlia spp. in natural habitats.

The genome structure and organization of endogenous caulimovirus sequences from dahlia (Dahlia spp), dahlia mosaic virus (DMV)-D10 from three wild species, D. coccinea (D10-DC), D. sherffii (D10-DS) and D. tenuicaulis (D10-DT), were determined and compared to those from cultivated species of dahlia, D. variabilis (DvEPRS). The complete ca. 7-kb dsDNA genomes of D10-DC, D10-DS, and D10-DT had a structure and organization typical of a caulimovirus and shared 89.3 to 96.6% amino acid sequence identity in various open reading frames (ORF) when compared to DvEPRS. The absence of the aphid transmission factor and the truncated coat protein fused with the reverse transcriptase ORF were common among these DMV-D10 isolates from wild Dahlia species.

Dynamics of the multiplicity of cellular infection in a plant virus.

Recombination, complementation and competition profoundly influence virus evolution and epidemiology. Since viruses are intracellular parasites, the basic parameter determining the potential for such interactions is the multiplicity of cellular infection (cellular MOI), i.e. the number of viral genome units that effectively infect a cell. The cellular MOI values that prevail in host organisms have rarely been investigated, and whether they remain constant or change widely during host invasion is totally unknown. Here, we fill this experimental gap by presenting the first detailed analysis of the dynamics of the cellular MOI during colonization of a host plant by a virus. Our results reveal ample variations between different leaf levels during the course of infection, with values starting close to 2 and increasing up to 13 before decreasing to initial levels in the latest infection stages. By revealing wide dynamic changes throughout a single infection, we here illustrate the existence of complex scenarios where the opportunity for recombination, complementation and competition among viral genomes changes greatly at different infection phases and at different locations within a multi-cellular host.

Nucleotide sequence and genome organization of a member of a new and distinct Caulimovirus species from dahlia.

A distinct caulimovirus, associated with dahlia mosaic, was cloned and sequenced. The caulimovirus, tentatively designated as dahlia common mosaic virus (DCMV), had a double-stranded DNA genome of ca. 8 kb. The genome organization of DCMV was found to be typical of members of the genus Caulimovirus and consisted of six major open reading frames (ORFs), ORFs I-VI, and one minor ORF, ORF VII. Sequence comparisons with the DNA genomes of two known caulimoviruses isolated from dahlia, Dahlia mosaic virus (DMV) and an endogenous caulimovirus, DMV-D10, showed that DCMV is a member of a distinct caulimovirus species, with sequence identities among various ORFs ranging from 25 to 80%.

A comparison of four DNA extraction methods for the detection of Citrus yellow mosaic badna virus from two species of citrus using PCR and dot-blot hybridization.

Nucleic acid preparations extracted using four procedures were assessed to determine the suitability of the procedure for PCR-based and DNA dot-blot-based detection of Citrus yellow mosaic badna virus (CMBV) from two citrus species, acid lime and pummelo. It was found that the success of PCR detection depended upon the procedure of DNA extraction whereas the dot-blot detection was successful with all extraction methods examined. CMBV DNA sequences amplified from two citrus species indicated high nucleotide sequence identity to the sequences reported previously from sweet orange. These results will help in choosing the correct DNA extraction procedure to be followed for efficient virus screening of citrus propagules.

Studies on biology and genomic characterization of a caulimo-like virus associated with a leaf distortion disease of Lamium maculatum.

A virus associated with leaf distortion of Lamium maculatum had features characteristic of caulimoviruses. The viral genome (Genbank accession number: EU554423) is 7,713 bp in size, with six open reading frames similar in size and organization to those of known caulimoviruses. Phylogenetic analyses based on the conserved ORF V polyprotein coding region indicated that the Lamium virus is a possible new member of the genus Caulimovirus. The virus was not transmitted by mechanical or graft inoculation, or by Myzus persicae. Because proof of pathogenicity remains to be demonstrated, the virus was named provisionally Lamuim leaf distortion-associated virus (LLDAV).

Genome structure and organization of a member of a novel and distinct species of the genus Caulimovirus associated with dahlia mosaic.

The genome structure and organization of a new and distinct caulimovirus that is widespread in dahlia (Dahlia variabilis) was determined. The double-stranded DNA genome was ca. 7.0 kb in size and shared many of the features of the members of the genus Caulimovirus, such as the presence of genes potentially coding for the movement protein, the inclusion body protein, and the reverse transcriptase (RT), and an intergenic region consisting of a potential 35S promoter. However, the virus differed from the previously described dahlia mosaic caulimovirus and other known caulimoviruses in that the aphid transmission factor (ATF) was absent and the putative coat protein contained a C-terminal deletion and was fused in-frame with the RT. Sequence identity at the amino acid level with known caulimoviruses including a previously reported caulimovirus from dahlia was low and ranged from 32 to 72%. The absence of an ATF and the highly divergent nature of the genomic sequence are characteristics of this new caulimovirus that is widely associated with dahlia.

Characterization of Cestrum yellow leaf curling virus: a new member of the family Caulimoviridae.

Cestrum yellow leaf curling virus (CmYLCV) has been characterized as the aetiological agent of the Cestrum parqui mosaic disease. The virus genome was cloned and the clone was proven to be infectious to C. parqui. The presence of typical viroplasms in virus-infected plant tissue and the information obtained from the complete genomic sequence confirmed CmYLCV as a member of the Caulimoviridae family. All characteristic domains conserved in plant pararetroviruses were found in CmYLCV. Its genome is 8253 bp long and contains seven open reading frames (ORFs). Phylogenetic analysis of the relationships with other members of the Caulimoviridae revealed that CmYLCV is closely related to the Soybean chlorotic mottle virus (SbCMV)-like genus and particularly to SbCMV. However, in contrast to the other members of this genus, the primer-binding site is located in the intercistronic region following ORF Ib rather than within this ORF, and an ORF corresponding to ORF VII is missing.

Cloning, sequencing, and promoter identification of Blueberry red ringspot virus, a member of the family Caulimoviridae with similarities to the "Soybean chlorotic mottle-like" genus.

The double-stranded DNA genome of Blueberry red ringspot virus (BRRV), a member of the family Caulimoviridae, was cloned and sequenced. The genome organization and relationships of the 8303 nt sequence revealed BRRV to be a tentative member of the genus that has been provisionally named "Soybean chlorotic mottle-like viruses", rather than a member of the genus Caulimovirus, in which it had been placed previously. Insertion of the putative 35S promoter homolog of BRRV into promoterless constructs carrying the UidA (beta-glucuronidase) gene resulted in high-level transient expression from cranberry and stable expression from transgenic tobacco. Sequences of 5'-RACE clones derived from transcripts from transgenic tobacco were consistent with the map position of the promoter.

A short open reading frame terminating in front of a stable hairpin is the conserved feature in pregenomic RNA leaders of plant pararetroviruses.

In plant pararetroviruses, pregenomic RNA (pgRNA) directs synthesis of circular double-stranded viral DNA and serves as a polycistronic mRNA. By computer-aided analysis, the 14 plant pararetroviruses sequenced so far were compared with respect to structural organization of their pgRNA 5'-leader. The results revealed that the pgRNA of all these viruses carries a long leader sequence containing several short ORFs and having the potential to form a large stem-loop structure; both features are known to be inhibitory for downstream translation. Formation of the structure brings the first long ORF into the close spatial vicinity of a 5'-proximal short ORF that terminates 5 to 10 nt upstream of the stable structural element. The first long ORF on the pgRNA is translated by a ribosome shunt mechanism discovered in cauliflower mosaic (CaMV) and rice tungro bacilliform viruses, representing the two major groups of plant pararetroviruses. Both the short ORF and the structure have been implicated in the shunt process for CaMV pgRNA translation. The conservation of these elements among all plant pararetroviruses suggests conservation of the ribosome shunt mechanism. For some of the less well-studied viruses, the localization of the conserved elements also allowed predictions of the pgRNA promoter region and the translation start site of the first long ORF.

Cassava vein mosaic virus (CsVMV), type species for a new genus of plant double stranded DNA viruses?

The complete sequence of 8159 nucleotides of the double stranded DNA genome of cassava vein mosaic virus (CsVMV) was determined (# U59751) and revealed a significant difference in genome organization when compared with a previous report (# U20341). When transferred to cassava plants by microbombardment, the full length CsVMV clone was infectious, confirming the genome organization here described. Sequence comparisons between CsVMV and members of the genera Caulimovirus and Badnavirus revealed high homologies between consensus sequences of several proteins that are indispensable for virus replication, including a potential transactivator factor not reported previously. The presence of a sequence complementary to a plant Met tRNA confirms that CsVMV is a plant pararetrovirus and is most closely related to members of the genus Caulimovirus as previously assessed. However, differences in genome organization, number and size of the ORFs, in addition to sequence comparisons with other plant pararetroviruses, shows that either the genetic variability of caulimoviruses is much greater than previously thought, or that CsVMV is the unique representative of a new genus within the Caulimoviridae family. On the basis of this study, it is proposed to upgrade the floating genus Caulimovirus to the family level and to divide the Caulimoviridae family into at least three genera with CsVMV being the type member of a new genus.