Extrachromosomal viral DNA produced by transcriptionally active endogenous viral elements in non-infected banana hybrids impedes quantitative PCR diagnostics of banana streak virus infections in banana hybrids.

The main edible and cultivated banana varieties are intra- and interspecific hybrids of the two main Musa species, Musa acuminata and Musa balbisiana, having diploid genomes denoted A and B, respectively. The B genome naturally hosts sequences of banana streak virus (BSV) named endogenous BSV (eBSV). Upon stress, eBSVs are identified as the origin of BSV infection for at least three BSV species, causing banana streak disease. For each of the three species, BSV and eBSV share >99.9 % sequence identity, complicating PCR-based diagnosis of viral infection in the B genome-containing bananas. Here, we designed a quantitative PCR-based method to only quantify episomal BSV particles produced, overcoming the limitation of eBSV also being detected by qPCR by using it as a 'calibrator'. However, our results revealed unexpected variation of eBSV amplification in calibrator plants composed of a clonal population of 53 replicating virus-free banana hybrids with the same AAB genotype. Our in-depth molecular analyses suggest that this calibrator variation is due to the variable abundance of non-encapsidated extrachromosomal viral DNA, likely produced via the transcription of eBSVs, followed by occasional reverse transcription. We also present evidence that accumulation of viral transcripts in AAB plants is downregulated both at post-transcriptional and transcriptional levels by an RNA interference mechanism that keeps the plants free of virus infection. Finally, we recommend that such eBSV amplification variation be taken into account to establish a quantitative viral diagnostic for banana plants with the B genome.

Complete genome sequence of aucuba ringspot virus.

A new badnavirus, aucuba ringspot virus (AuRV), was identified in plants of Aucuba japonica showing mild mosaic, vein banding, and yellow ringspot symptoms on the leaves. The complete nucleotide sequence of the AuRV genome was determined and found to be 9,092 nt in length, and the virus was found to have a genome organization typical of members of the genus Badnavirus. ORF3 was predicted to encode a polyprotein containing conserved movement protein, coat protein, aspartic protease, reverse transcriptase (RT), and RNase H domains. Phylogenetic analysis suggested that this virus is most closely related to codonopsis vein clearing virus but belongs to a distinct species, based on only 69.6% nucleotide sequence identity within the part of ORF 3 encoding the RT and RNase H domains. The vector of AuRV is unknown, but based on phylogenetic relationships, it is predicted to be a type of aphid.

The Citrus yellow mosaic badnavirus ORFI functions as a RNA-silencing suppressor.

Citrus yellow mosaic badnavirus (CMBV) causes mosaic disease in all economically important citrus cultivars of India, with losses reaching up to 70%. CMBV belongs to the genus Badnavirus, family Caulimoviridae, possessing a circular double-stranded (ds) DNA genome with six open reading frames (ORFs I to VI), whose functions are yet to be deciphered. The RNA-silencing suppressor (RSS) activity has not been assigned to any CMBV ORF as yet. In the present study, it was found that ORFI exhibited RSS activity among all the six CMBV ORFs tested. Studies were done by employing the well-established Agrobacterium-mediated transient assay based on the transgenic Nicotiana benthamiana 16c plant line expressing the green fluorescent protein (GFP). The RSS activity of ORFI was confirmed by the analysis of the GFP visual expression in the agroinfiltrated leaves, further supported by quantification of GFP expression by RT-PCR. Based on the GFP visual expression, the CMBV ORFI was a weak RSS when compared to the p19 protein of tomato bushy stunt virus. In contrast, the ORFII, ORFIV, ORFV, ORFVI, and CP gene did not exhibit any RSS activity. Hence, ORFI is the first ORF of CMBV to be identified with RNA-silencing suppression activity.

Genomic characterization of cycad leaf necrosis virus, the first badnavirus identified in a gymnosperm.

A previously undescribed badnavirus was isolated from Zamia fischeri showing symptoms of chlorosis, necrosis, and ringspot. The virus has bacilliform virions 30 nm in diameter and averaging 120 nm in length. The viral genome is 9227 bp in length and contains three open reading frames characteristic of members of the genus Badnavirus. The largest open reading frame (ORF3) encodes a putative polyprotein, with predicted domains including zinc finger, aspartic protease, reverse transcriptase (RT) and RNase H. The virus is tentatively named "cycad leaf necrosis virus" (CLNV). Within the genus Badnavirus, CLNV was most closely related to sugarcane bacilliform Guadeloupe D virus (FJ439817), sharing 69% identity at the nucleotide level in the RT + RNase H region. This virus is the first badnavirus reported to infect cycads, and it has the largest genome among the currently characterized badnaviruses.

Genomic characterisation of a newly identified badnavirus infecting ivy (Hedera helix).

High-throughput sequencing (HTS) was used to investigate ringspots on ivy (Hedera helix) leaves. De novo assembly of HTS data generated from a total RNA extract from these leaves yielded a contig with sequence similarity to viruses of the genus Badnavirus, family Caulimoviridae. The complete genome sequence of this virus consists of 8,885 nucleotides and has three open reading frames (ORFs). Genome organisation and phylogenetic analysis identifies this newly identified virus as a new member of the genus Badnavirus for which we propose the name "ivy ringspot-associated virus" (IRSaV).

First identification and molecular characterization of a new badnavirus infecting camellia.

A new badnavirus was identified in an ornamental camellia tree with yellow mottle symptom. The complete circular double-stranded DNA genome of this virus was found to consist of 8,203 bp. Its genome organization is typical of badnaviruses, containing three open reading frames (ORFs). ORFs 1 and 2 encode putative proteins with unknown functions. ORF3 encodes a large polyprotein that contains almost all of the conserved domains of badnaviruses. The virus shares 55-62% nucleotide sequence identities with other badnaviruses in the RT+RNase H region. Phylogenetic analyses placed it in group I of the genus Badnavirus. Therefore, this virus, which is tentatively named "camellia Lemon Glow virus", should represent a new species of the genus Badnavirus. This virus was found to be present in approximately a quarter of camellia trees tested.

Complete genome sequence of a tentative new member of the genus Badnavirus identified in Codonopsis lanceolata.

High-throughput sequencing revealed a tentative new badnavirus infecting Codonopsis lanceolata, provisionally named Codonopsis vein clearing virus (CoVCV). The complete 8,112-nt CoVCV genomic DNA sequence (GenBank accession: MK044821) comprises three open reading frames (ORFs) encoding conserved domains, with typical features of badnaviruses. Additionally, BLASTn searches indicated the CoVCV genome sequence is most similar to the grapevine vein clearing virus (GVCV) genome (72% identity and 46% query coverage). Moreover, the polyprotein encoded in CoVCV ORF3 is most similar to the corresponding protein of GVCV, with 60% amino acid sequence identity (89% query coverage). These results suggest that CoVCV is a new member of the genus Badnavirus in the family Caulimoviridae.

Complete genome sequence of a previously undescribed badnavirus occurring in Polyscias fruticosa L. (Ming aralia).

A previously undescribed badnavirus was identified in plants of Polyscias fruticosa (Ming aralia) showing symptoms of mild mosaic and leaf senescence. Characteristic bacilliform virions of the Polyscias badnavirus averaging 30 × 120 nm in size were observed by transmission electron microscopy in partially purified leaf tissue extracts from symptomatic but not asymptomatic plants collected in the USA and Nigeria. The isolate from the USA was complete sequenced. The genome is 7592 bp in length and contains three open reading frames with an arrangement similar to that of other members of the genus Badnavirus. The largest open reading frame (ORF3) encodes a putative polyprotein, with predicted domains including zinc finger, aspartic protease, reverse transcriptase (RT) and RNase H, in that order. The USA and Nigeria isolates of the virus had a high level (98%) of nucleotide sequence identity in the RT+RNase H region. Within the genus Badnavirus, these viruses were most closely related to schefflera ringspot virus (SRV), sharing 63% identity at the nucleotide level. Based on the ICTV species demarcation criteria for the genus Badnavirus (more than 20% nucleotide sequence divergence in the RT+RNase H region), the Polyscias virus is proposed to be a new member of the genus, and the name polyscias mosaic virus (PoMV) is proposed. The complete genome sequence was deposited in the NCBI GenBank database under accession no. MH475918.

Molecular characterization of a new badnavirus infecting green Sichuan pepper (Zanthoxylum schinifolium).

A new virus with a circular double-stranded DNA genome was discovered in green Sichuan pepper with vein clearing symptoms. Its complete genome of 8,014 bp contains three open reading frames (ORF) on the plus strand, which is typical of members of the genus Badnavirus in the family Caulimoviridae. Sequence comparisons revealed that the new virus has the highest nucleotide sequence identity with grapevine vein-clearing virus (GVCV). In particular, the identity of the two viruses in the ORF3 RT-RNase H region is 71.9%, which is below the species demarcation cutoff of 80% for badnaviruses. Phylogenetic analysis also placed the new virus with GVCV in a cluster. The virus was tentatively named "green Sichuan pepper vein clearing-associated virus" (GSPVCaV). The geographical distribution and genetic diversity of GSPVCaV were studied. Another isolate was found to be highly divergent.

Characterization of an Australian isolate of taro bacilliform virus and development of an infectious clone.

The badnavirus taro bacilliform virus (TaBV) has been reported to infect taro (Colocasia esculenta L.) and other edible aroids in several South Pacific island countries, but there are no published reports from Australia. Using PCR and RCA, we identified and characterized an Australian TaBV isolate. A terminally redundant cloned copy of the TaBV genome was generated and shown to be infectious in taro following agro-inoculation. This is the first report of TaBV from Australia and also the first report of an infectious clone for this virus.

Complete genome sequence of a new member of the genus Badnavirus, Dioscorea bacilliform RT virus 3, reveals the first evidence of recombination in yam badnaviruses.

Yams (Dioscorea spp.) host a diverse range of badnaviruses (genus Badnavirus, family Caulimoviridae). The first complete genome sequence of Dioscorea bacilliform RT virus 3 (DBRTV3), which belongs to the monophyletic species group K5, is described. This virus is most closely related to Dioscorea bacilliform SN virus (DBSNV, group K4) based on a comparison of genome sequences. Recombination analysis identified a unique recombination event in DBRTV3, with DBSNV likely to be the major parent and Dioscorea bacilliform AL virus (DBALV) the minor parent, providing the first evidence for recombination in yam badnaviruses. This has important implications for yam breeding programmes globally.

Viral Metagenomic-Based Screening of Sugarcane from Florida Reveals Occurrence of Six Sugarcane-Infecting Viruses and High Prevalence of Sugarcane yellow leaf virus.

A viral metagenomics study of the sugarcane virome in Florida was carried out in 2013 to 2014 to analyze occurrence of known and potentially new viruses. In total, 214 sugarcane leaf samples were collected from different commercial sugarcane (Saccharum interspecific hybrids) fields in Florida and from other Saccharum and related species taken from two local germplasm collections. Virion-associated nucleic acids (VANA) metagenomics was used for detection and identification of viruses present within the collected leaf samples. VANA sequence reads were obtained for 204 leaf samples and all four previously reported sugarcane viruses occurring in Florida were detected: Sugarcane yellow leaf virus (SCYLV, 150 infected samples out of 204), Sugarcane mosaic virus (1 of 204), Sugarcane mild mosaic virus (13 of 204), and Sugarcane bacilliform virus (54 of 204). High prevalence of SCYLV in Florida commercial fields and germplasm collections was confirmed by reverse-transcription polymerase chain reaction. Sequence analyses revealed the presence of SCYLV isolates belonging to two different phylogenetic clades (I and II), including a new genotype of this virus. This viral metagenomics approach also resulted in the detection of a new sugarcane-infecting mastrevirus (recently described and named Sugarcane striate virus), and two potential new viruses in the genera Chrysovirus and Umbravirus.

Characterization of a new badnavirus from Wisteria sinensis.

The complete genome sequence of a previously undescribed badnavirus isolated from a wisteria plant exhibiting mosaic and crinkle symptoms in Beijing, China, was determined. The circular double-stranded DNA genome of this virus was 7362 bp in size with four open reading frames (ORFs 1 to 4) on the plus strand. Sequence analysis showed that this virus shared the highest (69%) nucleotide (nt) sequence identity with pagoda yellow mosaic associated virus (PYMAV). In the RT-RNase H region of the ORF-3 encoded polyprotein, this virus shared 74% nt sequence identity with PYMAV. Phylogenetic analysis provided further evidence that the virus identified in this study is a member of a new species in the genus Badnavirus. The name wisteria badnavirus 1 (WBV1) is proposed for this new virus.

Molecular characterization of previously elusive badnaviruses associated with symptomatic cacao in the New World.

Suspected virus-like symptoms were observed in cacao plants in Trinidad during 1943, and the viruses associated with these symptoms were designated as strains A and B of cacao Trinidad virus (CTV). However, viral etiology has not been demonstrated for either phenotype. Total DNA was isolated from symptomatic cacao leaves exhibiting the CTV A and B phenotypes and subjected to Illumina HiSeq and Sanger DNA sequencing. Based on de novo assembly, two apparently full-length badnavirus genomes of 7,533 and 7,454 nucleotides (nt) were associated with CTV strain A and B, respectively. The Trinidad badnaviral genomes contained four open reading frames, three of which are characteristic of other known badnaviruses, and a fourth that is present in only some badnaviruses. Both badnaviral genomes harbored hallmark caulimovirus-like features, including a tRNAMet priming site, a TATA box, and a polyadenylation-like signal. Pairwise comparisons of the RT-RNase H region indicated that the Trinidad isolates share 57-71% nt sequence identity with other known badnaviruses. Based on the system for badnavirus species demarcation in which viruses with less than 80% nt sequence identity in the RT-RNase gene are considered members of separate species, these isolates represent two previously unidentified badnaviruses, herein named cacao mild mosaic virus and cacao yellow vein banding virus, making them the first cacao-infecting badnaviruses identified thus far in the Western Hemisphere.

The genome sequence of Dioscorea bacilliform TR virus, a member of the genus Badnavirus infecting Dioscorea spp., sheds light on the possible function of endogenous Dioscorea bacilliform viruses.

The complete genome sequence of Dioscorea bacilliform TR virus (DBTRV) was determined. The closest relatives of DBTRV are Dioscorea bacilliform AL virus (DBALV) and Dioscorea bacilliform RT virus 1 (DBRTV1). Specific primers were designed and used to determine the prevalence of DBTRV in a yam germplasm collection. It was found that this virus infects Dioscorea alata and D. trifida plants in Guadeloupe and French Guyana. DTRBV was not detected in any of the tested D. cayenensis-rotundata accessions. In silico analysis provided evidence for the presence of DBTRV-like endogenous sequences in the genome of D. cayenensis-rotundata, pointing to a possible role of these sequences in antiviral defense.

Characterization of Canna yellow mottle virus in a New Host, Alpinia purpurata, in Hawaii.

Canna yellow mottle virus (CaYMV) is an important badnavirus infecting Canna spp. worldwide. This is the first report of CaYMV in flowering ginger (Alpinia purpurata) in Hawaii, where it is associated with yellow mottling and necrosis of leaves, vein streaking, and stunted plants. We have sequenced CaYMV in A. purpurata (CaYMV-Ap) using a combination of next-generation sequencing and traditional Sanger sequencing techniques. The complete genome of CaYMV-Ap was 7,120 bp with an organization typical of other Badnavirus species. Our results indicated that CaYMV-Ap was present in the episomal form in infected flowering ginger. We determined that this virus disease is prevalent in Hawaii and could potentially have significant economic impact on the marketing of A. purpurata as cut flowers. There is a potential concern that the host range of CaYMV-Ap may expand to include other important tropical plants.

Genetic and Phenotypic Characterization of Grapevine vein clearing virus from Wild Vitis rupestris.

Grapevine vein clearing virus (GVCV), a new member of the genus Badnavirus in the family Caulimoviridae, is associated with a vein clearing and vine decline disease that severely affects grape production and berry quality in commercial vineyards in the Midwest region of the United States. In this paper, the genetic and phenotypic characteristics of GVCV-VRU1 and GVCV-VRU2, two isolates from wild Vitis rupestris grapevines in their native habitat, are described. The GVCV-VRU1 genome is 7,755 bp long while the GVCV-VRU2 genome consists of 7,725 bp, both of which are different from the genome of the GVCV-CHA isolate (7,753 bp), which was originally discovered in the grape cultivar 'Chardonel'. The nucleotide sequence identity among GVCV-VRU1, GVCV-VRU2, and GVCV-CHA ranges from 91.6 to 93.4%, and open reading frame (ORF) II is the most divergent ORF with only 83.3 to 88.5% identity. Sequence analysis of the ORF II indicated that GVCV isolates genetically similar to GVCV-VRU1 and GVCV-VRU2 also are present in commercial vineyards. Symptoms of GVCV-VRU1- or GVCV-VRU2-infected wild V. rupestris grapevine appeared initially as translucent vein clearing on young leaves and progressed to vein necrosis on mature leaves. Inoculation of GVCV-VRU1 or GVCV-VRU2 by grafting onto grape cultivar Chardonel resulted in mild mottle and leaf distortion. The natural range of wild V. rupestris grapevines overlaps with commercial vineyards in the Midwestern United States. Therefore, the discovery of GVCV isolates in wild V. rupestris grapevines has important implications for epidemics and management of the GVCV-associated disease.

Unusual genomic features of a badnavirus infecting mulberry.

Mulberry badnavirus 1 (MBV1) has been characterized as the aetiological agent of a disease observed on a mulberry tree in Lebanon (accession L34). A small RNA next-generation sequencing library was prepared and analysed from L34 extract, and these data together with genome walking experiments have been used to obtain the full-length virus sequence. Uniquely among badnaviruses, the MBV1 sequence encodes a single ORF containing all the conserved pararetrovirus motifs. Two genome sizes (6 kb and 7 kb) were found to be encapsidated in infected plants, the shortest of which shares 98.95 % sequence identity with the full L34 genome. In the less-than-full-length deleted genome, the translational frame for the replication domains was conserved, but the particle morphology, observed under electron microscopy, was somehow altered. Southern blot hybridization confirmed the coexistence of the two genomic forms in the original L34 accession, as well as the absence of cointegration in the plant genome. Both long and deleted genomes were cloned and proved to be infectious in mulberry. Differently from other similar nuclear-replicating viruses or viroids, the characterization of the MBV1-derived small RNAs showed a reduced amount of the 24-mer class size.

Detection and molecular characterization of a Grapevine Roditis leaf discoloration-associated virus (GRLDaV) variant in an autochthonous grape from Apulia (Italy).

The complete nucleotide sequence and genome organization of a new Badnavirus isolated from the autochthonous grapevine variety "Bombino nero" from Apulia (Italy) was determined. The genome of this virus consists of 7097 nt and has four open reading frames (ORFs). Analysis of putative proteins encoded by each ORF revealed greatest sequence similarity to Grapevine Roditis leaf discoloration-associated virus w4 (GRLDaV; NC_027131). In a pairwise alignment with GLRDaV w4 genome sequence, the "Bombino Nero" sequence was 109 nt longer with a major 57 nt insertion between positions 2405 and 2413. Furthermore, its putative ORF4 is located after the ORF3, while in the GLRDaV w4 sequence, the putative ORF4 completely overlapped ORF3. Nucleotide analysis classifies this new Badnavirus as a GLRDaV strain, which was named GRLDaV-BN. Multi-year field observations showed that the GLRDaV-BN-infected vine was symptomless.

Complete genome sequence of yacon necrotic mottle virus, a novel putative member of the genus Badnavirus.

The complete genome sequence of a previously undescribed virus isolated from a yacon plant exhibiting necrotic mottle, chlorosis, stunting, and leaf malformation symptoms in Gyeongju, Korea, was determined. The genome of this virus consists of one circular double-stranded DNA of 7661 bp in size. The genome contained four open reading frames (ORFs 1 to 4) on the plus strand that potentially encode proteins of 26, 32, 234, and 25 kDa. Protein BLAST analysis showed that ORF3, which is the largest ORF, has 45 % amino acid sequence identity (with 89 % coverage) to the ORF3 of fig badnavirus 1 (FBV-1), a recently identified badnavirus. Phylogenetic analysis provided further evidence that the virus identified in this study is probably a member of a new species in the genus Badnavirus. The name yacon necrotic mottle virus (YNMoV) is proposed for this new virus.