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Rice tungro, economically the most important viral disease of rice, is a complex disease caused by two morphologically and genomically dissimilar viruses, Rice tungro bacilliform virus (RTBV), a double stranded DNA virus replicating through RNA intermediate and Rice tungro spherical virus (RTSV), a single stranded RNA virus with 3' poly (A) tail. A novel multiplex RT-PCR technique for the simultaneous detection of RTBV and RTSV from the total RNA extracted from tungro-infected plants has been developed. It involves a one-step reaction initiating first strand cDNA synthesis by oligo (dT) primer with poly (A) tailed RTBV transcript and RTSV genomic RNA as template for the PCR amplification. The results indicate that adaptation of this technique will strengthen the screening for tungro resistance among rice varieties and hybrids.

The delivery of the double-stranded DNA viral genome into the nucleus is a critical step for the type member of Caulimoviridae, cauliflower mosaic virus (CaMV). The nucleocapsid (NC) of CaMV is directly involved in this process. A nuclear localization signal located at the N-terminus of the NC was shown to be exposed at the surface of the virion. This nuclear localization signal appears to be important to direct the virus to the nuclear pore complex. The nuclear targeting of the NC needs to be tightly regulated because the process of virus assembly, which also involves the viral NC, occurs in the cytosol. It is now accepted that the N- and C-terminal extensions of the viral NC precursor are efficient regulatory sequences that determine the localization of the viral NC in infected leaves. Proteolytic maturation and phosphorylation of the N- and C-terminal extensions are also important in the regulation of this process. Despite these recent discoveries, the transport of CaMV toward and into the nucleus during early events in the infection cycle remains unclear. In this review, we summarize recent advances that explain the mechanisms of targeting of the CaMV genome to the nucleus and extract from other related animal and plant viruses mechanisms that could hint at the possible strategies used by CaMV to enter the nucleus.

Publication Inra prise en compte dans l'analyse bibliométrique des publications scientifiques mondiales sur les Fruits, les Légumes et la Pomme de terre. Période 2000-2012. http://prodinra.inra.fr/record/256699; International audience; Viruses frequently use insect vectors to effect rapid spread through host populations. In plant viruses, vector transmission is the major mode of transmission, used by nearly 80% of species described to date. Despite the importance of this phenomenon in epidemiology, the specificity of the virus-vector relationship is poorly understood at both the molecular and the evolutionary level, and very limited data are available on the precise viral protein motifs that control specificity. Here, using the aphid-transmitted Cauliflower mosaic virus (CaMV) as a biological model, we confirm that the "noncirculative" mode of transmission dominant in plant viruses (designated "mechanical vector transmission" in animal viruses) involves extremely specific virus-vector recognition, and we identify an amino acid position in the "helper component" (HC) protein of CaMV involved in such recognition. Site-directed mutagenesis revealed that changing the residue at this position can differentially affect transmission rates obtained with various aphid species, thus modifying the spectrum of vector species for CaMV. Most interestingly, in a virus line transmitted by a single vector species, we observed the rapid appearance of a spontaneous mutant specifically losing its transmissibility by another aphid species. Hence, in addition to the first identification of an HC motif directly involved in specific vector recognition, we demonstrate that change of a virus to a different vector species requires only a single mutation and can occur rapidly and spontaneously.

The requirement for helper component(s) (HC) facilitating virus transmission by living organisms, termed vectors, is characteristic of plant viruses in several genera, potyviruses (genus Potyvirus, family Potyviridae) and caulimoviruses (genus Caulimovirus, family Caulimoviridae) being the most intensively studied groups of viruses. The potyviral HC-Pro fulfils several functions, including aphid transmission, long-distance movement and suppression of post-transcriptional gene silencing. The HC of Cauliflower mosaic virus plays an important role in aphid transmission, whereas no direct role in viral infectivity has so far been attributed to this protein. This review shows the current knowledge of major functions of HCs encoded by potyviruses and caulimoviruses.

We analyzed the correlation between the development of symptoms, virus titre and carbohydrate levels in turnip and Arabidopsis during infection with four isolates of Cauliflower mosaic virus (CaMV). Infection did not significantly affect sugar levels in source leaves except at the very late stages (28 d.p.i.), but induced a three-fold but short lived increase in sink leaves. Titres of severe isolates were greater than mild isolates, but we observed no obvious correlation between sugar levels and symptom appearance. In wild-type Arabidopsis, infection did not stimulate increased sugar levels, but did so in a mutant, cnr160, which shows altered growth-response to high carbon and low nitrogen. These results do not support a direct role for sugar-mediated control of symptom development during virus infection.

Transport of the viral genome into the nucleus is an obligatory step in the replication cycle of plant pararetro- and geminiviruses. In both these virus types, the multifunctional coat protein (CP) is thought to be involved in this process. Here, a green fluorescent protein tagging approach was used to demonstrate nuclear import of the CPs ofRice tungro bacilliform virus(RTBV) andMungbean yellow mosaic virus-Vigna(MYMV) inNicotiana plumbaginifoliaprotoplasts. In both cases, at least two nuclear localization signals (NLSs) were identified and characterized. The NLSs of RTBV CP are located within both N- and C-terminal regions (residues 479KRPK/497KRK and 744KRK/758RRK), and those of MYMV CP within the N-terminal part (residues 3KR and 41KRRR). The MYMV and RTBV CP NLSs resemble classic mono- and bipartite NLSs, respectively. However, the N-terminal MYMV CP NLS and both RTBV CP NLSs show peculiarities in the number and position of basic residues.In vitropull-down assays revealed interaction of RTBV and MYMV CPs with the nuclear import factor importinα, suggesting that both CPs are imported into the nucleus via an importinα-dependent pathway. The possibility that this pathway could serve for docking of virions to the nucleus is discussed.

Banana streak disease is caused by several distinct badnavirus species, one of which is Banana streak Obino l'Ewai virus. Banana streak Obino l'Ewai virus has severely hindered international banana (Musa spp.) breeding programmes, as new hybrids are frequently infected with this virus, curtailing any further exploitation. This infection is thought to arise from viral DNA integrated in the nuclear genome of Musa balbisiana (B genome), one of the wild species contributing to many of the banana cultivars currently grown. In order to determine whether the DNA of other badnavirus species is integrated in the Musa genome, PCR-amplified DNA fragments from Musa acuminata, M. balbisiana and Musa schizocarpa, as well as cultivars ‘Obino l'Ewai’ and ‘Klue Tiparot’, were cloned. In total, 103 clones were sequenced and all had similarity to open reading frame III in the badnavirus genome, although there was remarkable variation, with 36 distinct sequences being recognized with less than 85 % nucleotide identity to each other. There was no commonality in the sequences amplified from M. acuminata and M. balbisiana, suggesting that integration occurred following the separation of these species. Analysis of rates of non-synonymous and synonymous substitution suggested that the integrated sequences evolved under a high degree of selective constraint as might be expected for a living badnavirus, and that each distinct sequence resulted from an independent integration event.

A full-length transcript (FLt) promoter was isolated from a genomic clone of Strawberry vein banding virus(SVBV), a double-stranded DNA plant pararetrovirus belonging to the Caulimoviridae family, and its activity was analyzed both in protoplasts and transgenic plants. The 5 � –3 � -boundaries required for maximal promoter activity were determined by 5 � - and 3 � -end deletion analysis of the SVBV promoter fused to a -glucuronidase (GUS) reporter gene. A 371-bp promoter fragment (−352 to +19 from the transcription start site; TSS) was found sufficient for maximal promoter activity in a transient protoplast expression assay, and this was chosen for further analysis. Finer deletion analysis of a 90-bp sequence (coordinates −392 to −302 from TSS) of the SVBV FLt promoter revealed the presence of a negative and a positive regulatory element in this region. In gain-of-function experiment, the fusion of the putative positive regulatory elements with minimal promoter showed very little increment in promoter activity, suggesting that a combinatorial action of various cis-sequences is involved in promoter function. The transcription start site of the full-length transcript promoter was mapped to an A-residue that is located 25-bp downstream of the TATA-box. In protoplast transient expression analysis, the SVBV FLt promoter showed about six-fold higher activity in tobacco compared to maize. A quantitative GUS activity assay showed that in transgenic tobacco plants the average promoter activity was about three-fold higher in roots than in leaves, and this higher activity was due to the accumulation of more GUS specific mRNA in roots. Real-time qRT-PCR analysis and quantitative GUS activity assay showed that the relative strength of the SVBV FLt promoter was greater than the CaMV35S promoter in transgenic tobacco plants. The SVBV FLt promoter is a strong, constitutive promoter and has great application potential in expression of foreign genes in plants. © 2004 Elsevier Ireland Ltd. All rights reserved.

The principal aphid-borne viruses infecting Strawberry (Fragaria spp.) Strawberry crinkle virus (SCV), Strawberry mild yellow edge virus (SMYEV), Strawberry mottle virus (SMoV) and Strawberry vein banding virus (SVBV) can cause serious crop losses. In this paper, a multiplex reverse transcriptase polymerase chain reaction (RT-PCR) method is described for the simultaneous detection of all four viruses in combination with a plant mRNA specific internal control which can be used as an indicator of the effectiveness of the extraction and RT-PCR. In total, 18 strawberry isolates infected naturally were analysed by this method. Every combination of RNA virus was able to be detected and a full complement of all four viruses were found together in three isolates, all taken from wild strawberry (Fragaria chiloensis (L.) Duch.) in Chile. The upper detection limit for the four viruses was at an extract dilution of 1/200. The broad applicability of the RNA specific internal control primers—which produced a PCR fragment of the expected size in 25 of 27 plant species tested—combined with improvements, made in extraction methods described provides potentially a standard method for comparable RT-PCR analyses in a wide variety of plant species.

SUMMARY Taxonomic relationship: Cauliflower mosaic virus (CaMV) is the type member of the Caulimovirus genus in the Caulimoviridae family, which comprises five other genera. CaMV replicates its DNA genome by reverse transcription of a pregenomic RNA and thus belongs to the pararetrovirus supergroup, which includes the Hepadnaviridae family infecting vertebrates. Physical properties: Virions are non-enveloped isometric particles, 53 nm in diameter (Fig. 1). They are constituted by 420 capsid protein subunits organized following T= 7 icosahedral symmetry (Cheng, R.H., Olson, N.H. and Baker, T.S. (1992) Cauliflower mosaic virus: a 420 subunit (T= 7), multilayer structure. Virology, 16, 655-668). The genome consists of a double-stranded circular DNA of approximately 8000 bp that is embedded in the inner surface of the capsid. Viral proteins: The CaMV genome encodes six proteins, a cell-to-cell movement protein (P1), two aphid transmission factors (P2 and P3), the precursor of the capsid proteins (P4), a polyprotein precursor of proteinase, reverse transcriptase and ribonuclease H (P5) and an inclusion body protein/translation transactivator (P6). Hosts: The host range of CaMV is limited to plants of the Cruciferae family, i.e. Brassicae species and Arabidopsis thaliana, but some viral strains can also infect solanaceous plants. In nature, CaMV is transmitted by aphids in a non-circulative manner.

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.

▪ Abstract Sequences of various DNA plant viruses have been found integrated into the host genome. There are two forms of integrant, those that can form episomal viral infections and those that cannot. Integrants of three pararetroviruses, Banana streak virus (BSV), Tobacco vein clearing virus (TVCV), and Petunia vein clearing virus (PVCV), can generate episomal infections in certain hybrid plant hosts in response to stress. In the case of BSV and TVCV, one of the parents contains the integrant but is has not been seen to be activated in that parent; the other parent does not contain the integrant. The number of integrant loci is low for BSV and PVCV and high in TVCV. The structure of the integrants is complex, and it is thought that episomal virus is released by recombination and/or reverse transcription. Geminiviral and pararetroviral sequences are found in plant genomes although not so far associated with a virus disease. It appears that integration of viral sequences is widespread in the plant kingdom and has been occurring for a long period of time.

Plant viruses are most frequently transmitted from one host plant to another by vectors. In noncirculative vector transmission, the virus does not process through a cycle within the vector body. Instead, upon acquisition by the vector, viruses are retained in the mouth parts or the anterior gut; from there, they will be subsequently released in a new host plant. Two molecular strategies have been described for the virus—vector interaction. In the capsid strategy, the virus coat interacts directly with binding sites in the vector mouth parts, whereas an additional nonstructural protein, designated helper component (HC), is required in the helper strategy. The HC and virus particles can be acquired sequentially, and this property introduces the possibility that an HC acquired first by the vector assists the transmission of virus particles located in the same cell, in other cells, or even in other host plants probed by the vector. Such a phenomenon is here called HC-transcomplementation. Surprisingly, the existing definition of HC does not explicitly include the concept of HC-transcomplementation, and it is generally omitted in the literature in any consideration of the virus biology other than the molecular interaction with the vector. Here we propose an extended definition of HC and emphasize the concept of HC-transcomplementation that distinguishes the helper strategy from any other type of vector transmission and may have consequences at the level of the virus population genetics and evolution.

Family Caulimoviridae comprises seven genera of plant viruses whose members replicate by reverse transcription and whose virions contain double-stranded deoxyribonucleic acid. In a recent survey of the international scientific community, Cauliflower mosaic virus (CaMV), the type species of the family, was ranked sixth in the world in a list of the most scientifically or economically important plant viruses. This notoriety is due purely to the major conceptual advances that have been made in plant virology using CaMV as a model pathogen. However, several relatively lesser known viruses in the family, such as Rice tungro bacilliform virus and Cacao swollen shoot virus, are very serious constraints to crop production in tropical regions of the world. In this article, information on the taxonomy, replication cycle, vector transmission, epidemiology and disease management of this important group of plant viruses is summarised. Key Concepts: The Caulimoviridae is the only family of plant viruses with a double-stranded deoxyribonucleic acid (dsDNA) genome, and in common with all viral retroelements, incorporates a reverse transcription step in the replication cycle. Cauliflower mosaic virus is the type species of the family, and has been a very important model plant virus for elucidating fundamental aspects of virus replication, cell-to-cell movement and aphid transmission. Members of the Caulimoviridae are most prominent in tropical regions, where they cause serious diseases such as rice tungro, cacao swollen shoot and banana streak disease. The most important component of a control programme for these viruses is to reduced inoculum levels by using clean planting material, avoiding overlapping crops, and removing diseased plants and alternative hosts of the virus. In some host species, infection can arise as a consequence of activation of viral DNA that is integrated in the nuclear genome of the plant. Keywords: plant viruses; retroelement; pararetrovirus; reverse transcription; cauliflower mosaic virus; rice tungro; cacao swollen shoot; tropical crop

International audience; Virus-infected plants accumulate abundant, 21-24 nucleotide viral siRNAs which are generated by the evolutionary conserved RNA interference (RNAi) machinery that regulates gene expression and defends against invasive nucleic acids. Here we show that, similar to RNA viruses, the entire genome sequences of DNA viruses are densely covered with siRNAs in both sense and antisense orientations. This implies pervasive transcription of both coding and non-coding viral DNA in the nucleus, which generates double-stranded RNA precursors of viral siRNAs. Consistent with our finding and hypothesis, we demonstrate that the complete genomes of DNA viruses from Caulimoviridae and Geminiviridae families can be reconstructed by deep sequencing and de novo assembly of viral siRNAs using bioinformatics tools. Furthermore, we prove that this 'siRNA omics' approach can be used for reliable identification of the consensus master genome and its microvariants in viral quasispecies. Finally, we utilized this approach to reconstruct an emerging DNA virus and two viroids associated with economically-important red blotch disease of grapevine, and to rapidly generate a biologically-active clone representing the wild type master genome of Oilseed rape mosaic virus. Our findings show that deep siRNA sequencing allows for de novo reconstruction of any DNA or RNA virus genome and its microvariants, making it suitable for universal characterization of evolving viral quasispecies as well as for studying the mechanisms of siRNA biogenesis and RNAi-based antiviral defense.

Caulimoviruses have evolved very unusual mechanisms of translation initiation to express the multiple genes in their very compact genomes. Most of the icosahedral Caulimoviridae (caulimo-, soymo-, and probably cavemovirus) produce polycistronic mRNAs that are translated via reinitiation—a mechanism normally prohibited in eukaryotes—enabled by the action of a viral transactivator of translation reinitiation. The mechanism of virus-induced polycistronic translation was first discovered and characterized for Cauliflower mosaic virus (CaMV), which produces transactivator/viroplasmin TAV to allow translation of its 35S pregenomic RNA via recruitment of multiple host factors—mainly components of the host’s translation machinery. The details of this close cooperation between CaMV and the host translation machinery is discussed here and is compared to related processes in other viruses and cells. CaMV is the first plant or mammalian virus shown to interact physically with the target-of-rapamycin (TOR) protein kinase and to activate the TOR signaling pathway. We pay special attention to the physical and functional interactions between CaMV TAV, translation initiation factor 3 (eIF3), reinitiation-supporting factor (RISP), and TOR that are able to overcome cellular barriers to reinitiation.

The extent and importance of endogenous viral elements have been extensively described in animals but are much less well understood in plants. Here we describe a new genus of Caulimoviridae called ‘Florendovirus’, members of which have colonized the genomes of a large diversity of flowering plants, sometimes at very high copy numbers (>0.5% total genome content). The genome invasion of Oryza is dated to over 1.8 million years ago (MYA) but phylogeographic evidence points to an even older age of 20–34 MYA for this virus group. Some appear to have had a bipartite genome organization, a unique characteristic among viral retroelements. In Vitis vinifera, 9% of the endogenous florendovirus loci are located within introns and therefore may influence host gene expression. The frequent colocation of endogenous florendovirus loci with TA simple sequence repeats, which are associated with chromosome fragility, suggests sequence capture during repair of double-stranded DNA breaks., Endogenous viral elements have been extensively described in animals but their significance in plants is less well understood. Here, Geering et al. describe a new group of endogenous pararetroviruses, called florendoviruses, which have colonized the genomes of many important crop species.

Screening of food components using a polymerase chain reaction (PCR) for the presence of genetic elements, such as the widespread cauliflower mosaic virus 35S promoter introduced into genetically modified organisms (GMOs), has become a routine method in modern food analysis. With the aim of developing a high throughput method suitable for automation we established a PCR-enzyme-linked immunosorbent assay (PCR-ELISA). It is based on specific hybridization of an immobilized, biotinylated PCR product with a digoxigenin-labelled internal probe; the label then serves in colorimetric immunodetection. With this fast and convenient method laborious blotting procedures and the use of hazardous ethidium bromide in gel staining are avoided. The optimized protocol for this PCR-ELISA allows the detection of as little as 0.1 ng amplicon in only 2 h. With this new technique we analyzed whole Roundup Ready soybeans as well as soybean flour with GMO contents ranging from 0.1% to 2%.

Cauliflower mosaic virus (CaMV) open reading frame (ORF) III encodes a 15 kDa protein (P3) that is indispensable for viral infectivity. Although P3 has been shown to be a prerequisite for CaMV aphid transmission, its role in viral replication remains unknown. We previously showed that P3 forms a tetramer in planta and that P3 tetramer co-sediments with viral coat protein on sucrose gradient centrifugation, suggesting that a tetramer may be the functional form of P3. We presumed that disulfide bonds were involved in tetramer formation because 1) the tetramer was detected by Western blotting after electrophoresis under non-reducing conditions, and 2) the cysteine-X-cysteine motif is well conserved in CaMV P3 and P3 homologues among Caulimoviruses. Therefore we mutated either or both of the cysteine residues of CaMV P3. The mutant viruses were infectious and accumulated to a similar extent as the wild-type. An analysis of mutant proteins confirmed that the wild-type P3 molecules in the tetramer are covalently bound with one another through disulfide bonds. It was also suggested that mutant proteins are less stable than wild-type protein in planta. Furthermore, sedimentation study suggested that the disulfide bonds are involved in stable association of P3 with CaMV virions or virion-like particles, or both. The mutant viruses could be transmitted by aphids. These results suggested that the covalent bonds in P3 tetramer are dispensable for biological activity of P3 under experimental situations and may have some biological significance in natural infection in the field.

Virion purification from indicator plants and partial sequencing of the replicase region of a 'sweet potato caulimo-like virus' (SPCV) isolate from Madeira, Portugal, are described. Phylogenetic analysis suggests that SPCV is a distinct member of the genus Cavemovirus (family Caulimoviridae). These results explain previous failed attempts to characterize SPCV based on antibodies or primers designed for other members of the Caulimoviridae. Using a quick DNA extraction protocol and PCR primers flanking the RT motif region, we were able to detect SPCV directly in sweet potato, thus saving considerable time during routine virus indexing.

Banana streak virus (BSV) is an important pathogen of bananas and plantains (Musa spp.) throughout the world. We have cloned and sequenced part of the genomes of four isolates of BSV from Australia, designated BSV-RD, BSV-Cav, BSV-Mys, and BSV-GF. These isolates originated from banana cvs. Red Dacca, Williams, Mysore, and Goldfinger, respectively. All clones contained a sequence covering part of open reading frame III and the intergenic region of the badnavirus genome. The sequences were compared with those of other badnaviruses, including BSV-Onne, a previously characterized isolate from Nigeria. The BSV-RD sequence was virtually identical to that of BSV-Onne, differing by only two nucleotides over 1,292 bp. However, BSV-Cav, -Mys, and -GF were divergent in nucleotide sequence. Phylogenetic analyses using conserved sequences in the ribonuclease H domain revealed that all BSV isolates were more closely related to each other than to any other badnavirus. BSV-Cav was most closely related to BSV-Onne, and there was 95.1% identity between the two amino acid sequences. Other relationships between the BSV isolates were less similar, with sequence identities ranging from 66.4 to 78.2%, which is a magnitude comparable to the distance between some of the recognized badnavirus species. Immunocapture-polymerase chain reaction assays have been developed, allowing specific detection and differentiation of the four isolates of BSV.

Rice tungro bacilliform virus (RTBV) is a reverse-transcribing DNA virus which, in association with an RNA virus, Rice tungro spherical virus (RTSV), is responsible for rice tungro disease (22), the most important viral disease of rice in South and Southeast Asia. In rice tungro, RTBV induces most of the symptoms (yellowing and reddening of the leaves, stunting of rice plants) and RTSV is mainly involved in the transmission of both viruses via the green leafhopper Nephotettix virescens (5). RTBV is the type and only known member of the “RTBV-like viruses” genus, which has been classified in the Caulimoviridae family comprising caulimoviruses, badnaviruses, and two other genera (29, 31). The plant viruses which belong to this family have many features in common with retroviruses and are also often referred to, together with the human and animal hepadnaviruses, as pararetroviruses (23, 40, 42). The bacilliform RTBV particles are elongated icosahedrons with a diameter of 30 nm and a length of approximately 130 nm, which varies with the virus isolate (22). The RTBV genome is a circular double-stranded DNA molecule of about 8 kbp, containing two site-specific discontinuities resulting from the replication process by reverse transcription and four large open reading frames (ORFs) (Fig. ​(Fig.1A)1A) (1, 17, 39). The corresponding proteins, P1, P2, P3, and P4, are synthesized by specialized translation mechanisms (10–12) from a pregenomic RNA which is used as the template for viral replication and also serves as a polycistronic mRNA (22). FIG. 1 Schematic representations of the RTBV genome and P3 polyprotein. (A) Genome organization. Viral DNA is represented by a thin double line with the sites of the two discontinuities (Δ1 and Δ2) indicated. The thick arrows outside the DNA ... The roles of P1 (24 kDa) and P4 (46 kDa) are still unknown. P3 is a large polyprotein of 196 kDa (Fig. ​(Fig.1B).1B). Sequence comparisons with retroviral and other pararetroviral proteins suggest that P3 contains domains corresponding to the movement protein (MP), coat protein (CP), aspartic protease (PR), reverse transcriptase (RT), and RNase H (RH), ordered from the N terminus to the C terminus (17, 26, 39, 45). The viral protease is at least partly responsible for the processing of P3. The cleavage sites at the N- and C-terminal extremities of the RT-RH domain have been characterized. It has been demonstrated that the PR-RT-RH polyprotein can be processed to yield two proteins of 55 and 62 kDa (p55 and p62) when expressed in insect cells from the 3′ part of gene III (27). First reports indicated that RTBV particles contain two major CP species of 33 and 37 kDa (p33 and p37) (39). The N terminus of p33 was determined to be at amino acid 502. Considering its size and the position of its N-terminal residue within P3, p33 should contain, in its C-terminal region, the basic domain and the Cys-His motif which are conserved in plant pararetrovirus CPs. This motif is the equivalent of the zinc finger motif of retroviral Gag proteins and consequently is thought to be involved in specific RNA binding during packaging of the pregenomic RNA into virions (40). Recently, Marmey et al. (30) showed that RTBV virions contain only a single coat protein species of 37 kDa, with the second peptide (of 34 kDa) most probably being a degradation product of the 37-kDa protein generated during virus purification. Amino acids 477 and 791 of P3 were deduced, from mass spectral analysis, to correspond to the N- and C-terminal residues, respectively, of the 37-kDa coat protein (p37). ORF II encodes a 12-kDa protein (P2) for which no definite function has been assigned. P2 of RTBV and of the badnavirus commelina yellow mottle virus (CoYMV) were shown to be associated with purified virions (3, 22; A. Druka and R. Hull, personal communication). P2 of RTBV and of the badnavirus cacao swollen shoot virus (CSSV) were also described as sequence-nonspecific nucleic acid binding proteins (24, 25). The C termini of RTBV and CSSV P2, which possess basic, hydrophobic, and proline residues, support the nucleic acid binding activity. Such residues are also present at the C termini of caulimovirus gene III products and of bacterial histone-like proteins (34). Moreover, the C-terminal extremity of cauliflower mosaic virus (CaMV) P3 possesses a nonspecific nucleic acid binding activity (33, 34), suggesting a common role for this protein and the P2 of RTBV or badnaviruses in their respective life cycles. To investigate the role of RTBV P2, we searched for possible interactions between this protein and other RTBV proteins. P2 was shown to interact with the CP domain of P3 both in the yeast two-hybrid system and in vitro. We have characterized this interaction and identified peptide motifs involved in the binding on both proteins. To evaluate the importance of this interaction in the context of viral infection, we introduced point mutations within gene II of the RTBV genome and investigated the infectivity of these mutants by agroinoculation of rice plants. Our results showed that virus viability correlates with the ability of P2 to interact with the CP domain of P3.

The genus Caulimovirus consists of several distinct virus species with a double-stranded DNA genome that infect diverse plant species. A comparative analysis of the sequences of known Caulimovirus species revealed two regions that are conserved in all Caulimovirus species with the exception of Strawberry vein banding virus. Degenerate primers based on these two regions were designed and tested in a polymerase chain reaction-based assay for broad spectrum detection of members of this genus. Cauliflower mosaic virus, Figwort mosaic virus and three distinct caulimoviruses associated with dahlia (Dahlia variabilis) were used to show the utility of this test in detecting diverse caulimoviruses. The primer pair gave an amplicon of expected size (840 bp). Amplicons from each virus were cloned and sequenced to verify their identity. The primer pair and the PCR assay provide approach for the broad spectrum detection of several members of the genus Caulimovirus.

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 andMusasequences and a complex BSV integrant.In situhybridization revealed two different BSV sequence locations in Obino l'Ewai chromosomes and a complex arrangement of BSV andMusasequences 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.

Banana streak virus (BSV), a member of the Badnavirus group of plant viruses, causes severe problems in banana cultivation, reducing fruit yield and restricting plant breeding and the movement of germplasm. Current detection methods are relatively insensitive. In order to develop a PCR-based diagnostic method that is both reliable and sensitive, the genome of a Nigerian isolate of BSV has been sequenced and shown to comprise 7389 bp and to be organized in a manner characteristic of badnaviruses. Comparison of this sequence with those of other badnaviruses showed that BSV is a distinct virus. PCR with primers based on sequence data indicated that BSV sequences are present in the banana genome.

Petunia vein-clearing virus (PVCV) is a plant pararetrovirus that has some features of retrotransposons. It encapsidates dsDNA and has isometric particles and inclusion bodies similar to those of caulimoviruses. The PVCV genome of 7205 bp has two large ORFs in the transcribed strand and a methionine tRNA primer-binding site in its 663-bp intergenic region. The N-terminal position of the large protein (126 kDa) encoded by ORF I has similarity to the movement protein of caulimoviruses. Toward the C-terminus of this same polyprotein are the two distinctive sequence elements [HHCC and DD(35)E] of the integrase function of retroviruses and retrotransposons. ORF II of PVCV encodes a protein of 125 kDa with domains for an RNA-binding element, common to thegaggene of retroelements, followed by consensus sequences for an acid protease, reverse transcriptase, and ribonuclease H. Hence, thegagequivalent (capsid protein) andpolgene of PVCV are part of the same polyprotein. Phylogenetic comparison of the reverse transcriptase of PVCV with that of various other retroelements grouped PVCV between caulimoviruses and the Ty3/gypsyretrotransposons, suggesting that PVCV is a divergent member of the caulimoviruses.

True retroviruses are not known in plants; however, plant pararetroviruses (caulimoviridae) share many retroviral properties, replicating by transcription in the nucleus followed by reverse transcription in the cytoplasm. Pararetroviruses have circular DNA genomes that do not integrate into the host genome, and display several unique expression strategies. Typical of plant pararetroviral pregenomic RNA is a highly structured leader of about 600nt long that is bypassed by scanning ribosomes. Caulimoviruses and Soymoviruses have a further interesting translation mechanism: at least six of the seven open reading frames are translated via polycistronic translation mediated by a specific transactivator (TAV), which modifies the translation complex. TAV also forms large intracellular inclusion bodies, which are the site of translation and virus assembly.

Virus diseases are serious constraints to the productivity and profitability of a wide range of tropical crops. Identification of the causal viruses and understanding their epidemiology is the key to estimating the incidence and economic impact of the diseases they cause, and to devising virus management strategies. Epidemics result from interactions between virus, host plant, vector and environmental factors, and every epidemic can be considered to be a unique pathosystem in which each of the components contributes to the epidemic, and in which none are limiting. Pathogen diagnosis is the key to managing diseases, and we list 18 crops, their main virus/viroid diseases and pathosystem descriptors for each. Six types of pathosystem are described on the basis of the mode of spread, and the different management strategies applicable to each are discussed. The Ninth Report of the International Committee on Taxonomy of Viruses (2012) is an essential reference for virologists working with tropical crops. Key Concepts: Identification of viruses or viroids is the key to managing the diseases they cause. Koch's rules are applied to establish causal associations between viruses/viroids and disease. Viruses/viroids are identified by their biological, morphological and genetic properties using classical and molecular methods. The universal database for viruses/viroids published by the International Committee on Taxonomy of Viruses is the resource for classification. Rapid diagnostic methods rely on nucleotide sequence comparisons with publicly available data bases. Epidemics are driven by virus, vector, host, environment, time and human activity. Vector–virus interaction is the main determinant of the rate, range, timing and pattern of disease spread. Viruses/viroids of tropical plants have unique pathosystems. A full description of a pathosystem is required to sustainably manage virus epidemics. Management of epidemics can be achieved by destabilising or down-regulating one or more components of the relevant pathosystem. Keywords: Geminiviridae; Potyviridae; Tospovirus; Caulimoviridae; Nanoviridae; Pospiviroidae; Sobemovirus; taxonomy; pathosystem; management

Grapevine is one of the economically and culturally important perennial fruit crops. More than 60 viruses infect grapevines and adversely affect their growth and development. Latent infection of most viruses in grapevines leads to chronic modulation of gene expression at transcriptional and post-transcriptional levels. Plant small RNAs (sRNAs) consist of microRNA (miRNA) and small interfering RNA (siRNA). miRNAs are expressed from the plant genome while most siRNAs are derived from double-stranded RNA molecules which are intermediates during virus replication. In a previous study, we constructed four cDNA libraries of sRNAs that were enriched from three virus-infected grapevines and one virus-free grapevine. Majority of siRNAs align most closely with the genomes of DNA viruses in the genus Badnavirus, family Caulimoviridae that led to the discovery of a new Grapevine vein clearing virus in grapevines. In this study, we conducted a comprehensive analysis of miRNAs in the four cDNA libraries and identified novel and stress-related miRNAs. The results indicated that miRNA abundance was influenced by virus infection. A total of 54 new miRNAs were identified and characterized, six of which, VITIS-MIR17, 18, 19, 20, 21, and 22, were detected only in virus-infected samples. One target of VITIS-MIR18 is the gene coding a non-apical meristem protein (GSVIVT00035370001), a transcription factor in the regulation of plant development and stress responses. Among the virus infection-induced known miRNAs, miRNA168 and miRNA3623 likely regulate grapevine's defense response, miRNA319 and miRNA395 modulate the expression of genes that are involved in nutrient metabolisms while miRNA396 plays a role in the regulation of cell division and cell cycle. The abiotic stress-induced miR169 and mi398 were negatively regulated by virus infection in grapevines. In addition, variety-specific miRNAs were discovered and compiled. The newly discovered miRNAs expand the miRNA profiles in the Vitis species. The characteristics of variety-specific and virus infection-associated miRNAs help understand the biology underlying the development and defense response of grapevines.

In this study, we determined the complete sequence of the genomic DNA of a Polish isolate of Blueberry red ringspot virus (BRRSV24) and compared it with a Czech (Darrow 5), and the US isolates of the virus and those of other Caulimoviridae family. The genomic DNA of BRRSV24 consists of 8,265 nucleotides and encodes eight open reading frames (ORFs). The sequence homologies of the eight ORFs of BRRSV24 were from 95 to 98% in respect of Darrow 5 and from 91 to 98% in respect of the US isolates at the amino acid level. This high level of amino acid sequence identity within the coding regions among the Czech, the US and Polish BRRSV isolates is suggestive of their common origin. Electronic supplementary material The online version of this article (doi:10.1007/s11262-011-0679-4) contains supplementary material, which is available to authorized users.

Dahlia mosaic disease of the ornamental flowering plant Dahlia is caused by two caulimoviruses, dahlia mosaic virus (DMV) and dahlia common mosaic virus (DCMV). We used a rolling-circle amplification method to amplify, clone and determine for the first time the full genome sequence of a DCMV isolate from New Zealand (DCMV-NZ). Within the 7949-bp circular double-stranded retro-transcribing DCMV-NZ DNA, we identified six putative open reading frames, typical of all genomes in the family Caulimoviridae. The availability of the complete DCMV sequence provides a reference genome against which all others can be compared.

Genomic DNA of blueberry red ringspot virus (genus Soymovirus, family Caulimoviridae) from highbush blueberry plants growing for years in the Czech Republic and Slovenia was sequenced. The circular dsDNA genomes consist of 8,303 and 8,299 nt, respectively, and contain eight open reading frames (ORFs) longer than 100 amino acids. The European isolates are 90% to 99% identical in aa sequences of distinct proteins. In contrast to the American New Jersey isolate, in-frame initiation codons have been found upstream from AUG codons of ORFs I, IV and V in the European isolates. These and other differences resulted in a longer capsid protein, reverse transcriptase, movement protein and protein the encoded by ORF VII and reduced (75.4% to 93.7%) amino acid identity with corresponding proteins of the New Jersey isolate.

This article introduces the second release of the Gypsy Database of Mobile Genetic Elements (GyDB 2.0): a research project devoted to the evolutionary dynamics of viruses and transposable elements based on their phylogenetic classification (per lineage and protein domain). The Gypsy Database (GyDB) is a long-term project that is continuously progressing, and that owing to the high molecular diversity of mobile elements requires to be completed in several stages. GyDB 2.0 has been powered with a wiki to allow other researchers participate in the project. The current database stage and scope are long terminal repeats (LTR) retroelements and relatives. GyDB 2.0 is an update based on the analysis of Ty3/Gypsy, Retroviridae, Ty1/Copia and Bel/Pao LTR retroelements and the Caulimoviridae pararetroviruses of plants. Among other features, in terms of the aforementioned topics, this update adds: (i) a variety of descriptions and reviews distributed in multiple web pages; (ii) protein-based phylogenies, where phylogenetic levels are assigned to distinct classified elements; (iii) a collection of multiple alignments, lineage-specific hidden Markov models and consensus sequences, called GyDB collection; (iv) updated RefSeq databases and BLAST and HMM servers to facilitate sequence characterization of new LTR retroelement and caulimovirus queries; and (v) a bibliographic server. GyDB 2.0 is available at http://gydb.org. © The Author(s) 2010. Published by Oxford University Press., Centro de Desarrollo Tecnologico Industrial (CDTI) (grant IDI-20100007, partial); Empresa Nacional de Innovacion, S.A (ENISA) (17092008, partial); IMPIVA (IMIDTA/2009/118 and IMDTA/2010/740, partial); European Regional Development Fund (ERDF); Ministerio de Ciencia e Innovacion (MICINN) (Torres-Quevedo grants PTQ-09-01-00020, PTQ-09-01-00670 and PTQ-10-03552, partial). Funding for open access charge: University of Valencia.

In 1999, banana streak disease outbreaks occurred at two locations in Australia in new banana hybrids that were being screened for fusarium wilt resistance. Two different badnaviruses, banana streak GF virus and a newly discovered virus called banana streak IM virus (BSIMV), were detected in these plants. The complete nucleotide sequence of the BSIMV genome was determined and comprised 7768 nt. Three open reading frames were detected, the first beginning with a non-conventional start codon (CUG). A 55-nt repetition in the putative pregenomic RNA promoter was also identified. Phylogenetic analysis suggests that BSIMV is most closely related to banana streak VN virus.

Plant viruses move through plasmodesmata (PD) either as nucleoprotein complexes (NPCs) or as tubule-guided encapsidated particles with the help of movement proteins (MPs). To explore how and why MPs specialize in one mechanism or the other, we tested the exchangeability of MPs encoded by DNA and RNA virus genomes by means of an engineered alfalfa mosaic virus (AMV) system. We show that Caulimoviridae (DNA genome virus) MPs are competent for RNA virus particle transport but are unable to mediate NPC movement, and we discuss this restriction in terms of the evolution of DNA virus MPs as a means of mediating DNA viral genome entry into the RNA-trafficking PD pathway.

Endogenous members of the family Caulimoviridae have now been found in the genomes of many plant species. Although these sequences are usually fragmented and rearranged and show varying degrees of decay, the genomes of the ancestral viruses can often be reassembled in silico, allowing classification within the existing viral taxonomic framework. In this paper, we describe analyses of endogenous members of the family Caulimoviridae in the genomes of Oryza sativa, Nicotiana tabacum and Solanum spp. and on the basis of phylogeny, genome organization and genetic distance within the pol gene, propose two new virus genera called Orendovirus and Solendovirus. A system of nomenclature for endogenous virus sequences in plants is also proposed.

Plant viruses move through plasmodesmata(PD) either as nucleo protein complexes (NPCs) or as tubuleguided encapsidated particles with the help of movement proteins(MPs). Made available in DSpace on 2019-11-02T00:37:11Z (GMT). No. of bitstreams: 1 121332010p.41094112.pdf: 3718141 bytes, checksum: 5ba18a841c6c72b11c3aac5295c4a2e9 (MD5) Previous issue date: 2010

Plant pararetroviruses or Caulimoviridae, Geminiviridae, and Nanoviridae all accumulate as minichromosomes in the nucleus, although their replication strategies differ, with Caulimoviridae using transcription/reverse transcription and Geminiviridae and Nanoviridae rolling circle replication. We give an overview on these viruses and describe their interaction with the host, including silencing and silencing suppression and their occasional integration into the host chromatin. We further describe their replication, transcription, translation and posttranslational modifications, recombination, as well as capsid structure and assembly, movement, and insect transmission.

Schoelz, J. E., Wiggins, B. E., Wintermantel, W. M., and Ross, K. 2006. Introgression of a tombusvirus resistance locus from Nicotiana edwardsonii var. Columbia to N. clevelandii. Phytopathology 96:453-459. A new variety of Nicotiana, N. edwardsonii var. Columbia, was evaluated for its capacity to serve as a new source for virus resistance genes. Columbia was developed from a hybridization between N. glutinosa and N. clevelandii, the same parents used for the formation of the original N. edwardsonii. However, in contrast to the original N. edwardsonii, crosses between Columbia and either of its parents are fertile. Thus, the inheritance of virus resistance genes present in N. glutinosa could be characterized by using Columbia as a bridge plant in crosses with the susceptible parent, N. clevelandii. To determine how virus resistance genes would segregate in interspecific crosses between Columbia and N. clevelandii, we followed the fate of the N gene, a single dominant gene that specifies resistance to Tobacco mosaic virus (TMV). Our genetic evidence indicated that the entire chromosome containing the N gene was introgressed into N. clevelandii to create an addition line, designated N. clevelandii line 19. Although line 19 was homozygous for resistance to TMV, it remained susceptible to Tomato bushy stunt virus (TBSV) and Cauliflower mosaic virus (CaMV) strain W260, indicating that resistance to these viruses must reside on other N. glutinosa chromosomes. We also developed a second addition line, N. clevelandii line 36, which was homozygous for resistance to TBSV. Line 36 was susceptible to TMV and CaMV strain W260, but was resistant to other tombusviruses, including Cucumber necrosis virus, Cymbidium ringspot virus, Lettuce necrotic stunt virus, and Carnation Italian ringspot virus.

Arabidopsis thaliana ecotypes vary in their responses to viruses. In this study, we analyzed the variation in response of A. thaliana ecotype Tsu-0 to Cauliflower mosaic virus (CaMV). This ecotype was previously reported to be resistant to two CaMV isolates (CM1841 and CM4-184), but susceptible to W260. In this study, we show that Tsu-0 is resistant to four additional CaMV isolates. CaMV propagated within the rosette leaves of Tsu-0 plants, but did not appear to spread systemically into the inflorescence. However, virus viability in rosette leaves of Tsu-0 plants apparently was not compromised because infectious CaMV could be recovered from these organs. W260 overcomes Tsu-0 resistance by a passive mechanism (i.e., this virus avoids activating plant defenses). The portion of the viral genome responsible for W260 resistance breakage was mapped to the 5′ third of gene VI, which we have termed RBR-1. This region is also responsible for controlling the ability of CaMV to infect different types of solanaceous plants. Hence, the pathways by which plants of different families interact with CaMV may be conserved through evolution.

Amplification and cloning of dahlia mosaic virus promoter were carried out for the first time. Sequence analysis showed homology between this promoter and the promoters of other caulimoviruses. In addition, amplification and cloning of the carnation etched ring virus promoter was performed.

We analysed the sequence diversity in the reverse transcriptase (RT)/ribonuclease H (RNaseH) coding region of 19 badnavirus isolates infecting yam (Dioscorea spp.) in Ghana, Togo, Benin, and Nigeria. Phylogenetic analysis of the deduced amino acid sequences revealed that the isolates are broadly divided into two distinct species, each clustering with Dioscorea alata bacilliform virus (DaBV) and Dioscorea sansibarensis bacilliform virus (DsBV). Fourteen isolates had 90-96% amino acid identity with DaBV, while four isolates had 83-84% amino acid identity with DsBV. One isolate from Benin, BN4Dr, was distinct and had 77 and 75% amino acid identity with DaBV and DsBV, respectively, and may be a member of a new badnavirus species infecting yam in West Africa. Viruses of the two main species were present in Ghana, Togo and Benin and were observed to infect both D. alata and D. rotundata indiscriminately. This is the first confirmed report of DsBV infection in yam in Ghana and Togo. The results of this study demonstrate that members of two distinct species of badnaviruses infect yam in the West African yam zone and suggest a putative new species, BN4Dr. We also conclude that these species are not confined to limited geographic regions or specific for yam host species. However, the three badnavirus species are serologically related. The sequence information obtained from this study can be used to develop PCR-based diagnostics to detect members of the various species and/or strains of badnaviruses infecting yam in West Africa.

A previously published partial sequence of pineapple bacilliform virus was shown to be from a retrotransposon (family Metaviridae) and not from a badnavirus as previously thought. Two newly discovered sequence groups isolated from pineapple were associated with bacilliform virions and were transmitted by mealybugs. Phylogenetic analyses indicated that they were members of new badnavirus species. A third caulimovirid sequence was also amplified from pineapple, but available evidence suggests that this DNA is not encapsidated, but more likely derived from an endogenous virus.

In this study, we compared the transcriptional activities between Cauliflower mosaic virus (CaMV)35S promoter and polyubiquitin (Ljubq1) promoter from Lotus japonicus using β-glucuronidase (gus) reporter gene in transgenic plants of L. japonicus. The promoter analysis demonstrated that the Ljubq1 promoter possessed higher activity than the CaMV35S promoter in leaves, stems, roots, nodules, and pollen. Finally, we created GATEWAY conversion technology-compatible binary vectors for over-expression and RNA interference under the Ljubq1 promoter. These materials could provide alternative choice for studies in L. japonicus.

The non-enveloped bacilliform viruses are the second group of plant viruses known to possess a genome consisting of circular double-stranded DNA. We have characterized the viral transcript and determined the complete sequence of the genome of Commelina mellow mottle virus (CoYMV), a member of this group. Analysis of the viral transcript indicates that the virus encodes a single terminally-redundant genome-length plus 120 nucleotide transcript. A fraction of the transcripts is polyadenylated, although the majority of the transcript is not polyadenylated. Analysis of the genome sequence indicates that the genome is 7489 bp in size and that the transcribed strand contains three open reading frames capable of encoding proteins of 23, 15 and 216 kd. The function of the 25 and 15 kd proteins is unknown. Similarities between the 216 kd polypeptide and the cauliflower mosaic virus coat protein and protease/reverse transcriptase polyprotein suggest that the 216 kd polypeptide is a polyprotein that is proteolytically processed to yield the virion coat protein, a protease, and replicase (reverse transcriptase and ribonuclease H). Each strand of the CoYMV genome is interrupted by site-specific discontinuities. The locations of the 5'-ends of these discontinuities, and the presence and location of a region on the CoYMV transcript capable of annealing with the 3'-end of cytosolic initiator methionine tRNA are consistent with replication by reverse transcription. We have demonstrated that a construct containing 1.3 CoYMV genomes is infective when introduced into Commelina diffusa, the host for CoYMV, using Agrobacterium-mediated infection.

The hypersensitive response (HR) is a common feature of plant disease resistance reactions and a type of programmed cell death (PCD). Many pathogens are able to modulate pathways involved in cell death. In contrast to animal viruses, inhibitors of PCD activity have not been identified for plant-infecting viruses. Previously, we have reported that the nuclear shuttle protein (NSP) of Tomato leaf curl New Delhi virus (ToLCNDV) induces an HR in Nicotiana tabacum and Lycopersicon esculentum plants when expressed under the control of the Cauliflower mosaic virus 35S promoter. However, HR is not evident in plants infected with ToLCNDV, suggesting that the virus encodes a factor (or factors) that counters this response. Analysis of all ToLCNDV-encoded genes pinpointed the transcriptional activator protein (TrAP) as the factor mediating the anti-HR effect. Deletion mutagenesis showed the central region of TrAP, containing a zinc finger domain and nuclear localization signal, to be important in inhibiting the HR. These results demonstrate that TrAP counters HR-induced cell death, the first such activity identified for a plant-infecting virus.

Cauliflower mosaic virus (CaMV) is transmitted by aphids. For acquisition by the vector, a transmissible complex must form, composed of the virus particle, the viral coat-associated protein P3 and the helper protein P2. However, the components of the transmissible complex are largely separated in infected plant cells: most P3 virions are confined in electron-dense inclusion bodies, whereas P2 is sequestered in electron-lucent inclusion bodies (elIBs). This spatial separation controls virus acquisition by favouring the binding of virus-free P2 to the vector first, rendering the vector competent for later uptake of P3 virions. Consequently, sequential acquisition of virus from different cells or tissues is possible, with important implications for the biology of CaMV transmission. CaMV strains Campbell and CM1841 contain a single amino acid mutation (G94R) in the helper protein P2, rendering them non-transmissible from plant to plant. However, the mutant P2-94 protein supports aphid transmission when expressed heterologously and supplied to P3–CaMV complexes in vitro. The non-transmissibility of P2-94 was re-examined in vivo and it is shown here that the non-transmissibility of this P2 mutant is not due to low accumulation levels in infected plants, as suggested previously, but more specifically to the failure to form elIBs within infected plant cells. This demonstrates that elIBs are complex viral structures specialized for aphid transmission and suggests that viral inclusion bodies other than viral factories, most often considered as ‘garbage cans’, can in fact exhibit specific functions.

Yam (Dioscorea spp.) samples (n = 690) from seven South Pacific Islands were screened for badnavirus infection by ELISA using two antisera to African badnaviruses. Positive readings were obtained for 26.4–34.6% of samples representing both known (D. bulbifera, D. nummularia and D. pentaphylla) and unreported host species (D. alata, D. esculenta, D. rotundata and D. trifida) in this region. Total DNAs were extracted from 25 ELISA-positive plants and 4 ELISA-negative controls and subjected to PCR amplification with badnavirus-specific primers targeting the reverse transcriptase (RT)–RNaseH genes. All 29 samples yielded the expected size PCR-product for badnaviruses, which were cloned and sequenced. Phylogenetic analyses of the resulting 45 partial (500–527 bp) RT–RNaseH sequences revealed 11 new sequence groups with