Identification and distribution of novel badnaviral sequences integrated in the genome of cacao (Theobroma cacao).

Theobroma cacao is one of the most economically important tropical trees, being the source of chocolate. As part of an ongoing study to understand the diversity of the badnavirus complex, responsible for the cacao swollen shoot virus disease in West Africa, evidence was found recently of virus-like sequences in asymptomatic cacao plants. The present study exploited the wealth of genomic resources in this crop, and combined bioinformatic, molecular, and genetic approaches to report for the first time the presence of integrated badnaviral sequences in most of the cacao genetic groups. These sequences, which we propose to name eTcBV for endogenous T. cacao bacilliform virus, varied in type with each predominating in a specific genetic group. A diagnostic multiplex PCR method was developed to identify the homozygous or hemizygous condition of one specific insert, which was inherited as a single Mendelian trait. These data suggest that these integration events occurred before or during the species diversification in Central and South America, and prior to its cultivation in other regions. Such evidence of integrated sequences is relevant to the management of cacao quarantine facilities and may also aid novel methods to reduce the impact of such viruses in this crop.

Badnaviruses and banana genomes: a long association sheds light on Musa phylogeny and origin.

Badnaviruses are double-stranded DNA pararetroviruses of the family Caulimoviridae. Badnaviral sequences found in banana are distributed over three main clades of the genus Badnavirus and exhibit wide genetic diversity. Interestingly, the nuclear genome of many plants, including banana, is invaded by numerous badnaviral sequences although badnaviruses do not require an integration step to replicate, unlike animal retroviruses. Here, we confirm that banana streak viruses (BSVs) are restricted to clades 1 and 3. We also show that only BSVs from clade 3 encompassing East African viral species are not integrated into Musa genomes, unlike BSVs from clade 1. Finally, we demonstrate that sequences from clade 2 are definitively integrated into Musa genomes with no evidence of episomal counterparts; all are phylogenetically distant from BSVs known to date. Using different molecular approaches, we dissected the coevolution between badnaviral sequences of clade 2 and banana by comparing badnavirus integration patterns across a banana sampling representing major Musa speciation events. Our data suggest that primary viral integrations occurred millions of years ago in banana genomes under different possible scenarios. Endogenous badnaviral sequences can be used as powerful markers to better characterize the Musa phylogeny, narrowing down the likely geographical origin of the Musa ancestor.

ICTV Virus Taxonomy Profile: Caulimoviridae.

Caulimoviridae is a family of non-enveloped reverse-transcribing plant viruses with non-covalently closed circular dsDNA genomes of 7.1-9.8 kbp in the order Ortervirales. They infect a wide range of monocots and dicots. Some viruses cause economically important diseases of tropical and subtropical crops. Transmission occurs through insect vectors (aphids, mealybugs, leafhoppers, lace bugs) and grafting. Activation of infectious endogenous viral elements occurs in Musa balbisiana, Petunia hybrida and Nicotiana edwardsonii. However, most endogenous caulimovirids are not infectious. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Caulimoviridae, which is available at ictv.global/report/caulimoviridae.

Geographical Distribution of Cacao swollen shoot virus Molecular Variability in Ghana.

Cacao (Theobroma cacao L.) was introduced into West Africa from South America during the nineteenth century. However, cacao swollen shoot disease (CSSD) was first observed in Ghana in 1936 and, later, discovered in Nigeria, Côte d'Ivoire, Togo, and Sierra Leone. The objectives of this work were to assess the genetic diversity and spatial distribution of the Cacao swollen shoot virus (CSSV) in Ghana and investigate the origin and spread of the virus by identifying alternative host plants. Results obtained from polymerase chain reaction amplifications and phylogenetic relationship analyses of infected cacao and alternative host plants collected from the cacao-growing regions in Ghana revealed the existence of nine CSSV groups, A, B, C, E, G, J, K, L and M, with six groups detected for the first time in Ghana. The CSSV groups in Ghana are very divergent and correspond to at least five different putative species, according to the International Committee on Taxonomy of Viruses recommendations (A, B-C complex, G, E, and M), with the M species only being detected in the alternate host Ceiba pentandra. The spatial distribution of the different molecular groups in Togo, Côte d'Ivoire, and Ghana makes it difficult to predict a single origin for CSSV among the West African cacao-growing countries.

The genome of African yam (Dioscorea cayenensis-rotundata complex) hosts endogenous sequences from four distinct Badnavirus species.

Several endogenous viral elements (EVEs) have been identified in plant genomes, including endogenous pararetroviruses (EPRVs). Here, we report the first characterization of EPRV sequences in the genome of African yam of the Dioscorea cayenensis-rotundata complex. We propose that these sequences should be termed 'endogenous Dioscorea bacilliform viruses' (eDBVs). Molecular characterization of eDBVs shows that they constitute sequences originating from various parts of badnavirus genomes, resulting in a mosaic structure that is typical of most EPRVs characterized to date. Using complementary molecular approaches, we show that eDBVs belong to at least four distinct Badnavirus species, indicating multiple, independent, endogenization events. Phylogenetic analyses of eDBVs support and enrich the current taxonomy of yam badnaviruses and lead to the characterization of a new Badnavirus species in yam. The impact of eDBVs on diagnosis, yam germplasm conservation and movement, and breeding is discussed.

A possible scenario for the evolution of Banana streak virus in banana.

Outbreaks of Banana streak virus (BSV) have been recorded worldwide where Musa spp. is grown during the last 20 years with no convincing evidence of epidemics. Epidemics were previously reported in Uganda where BSV is currently endemic. BSV is a plant pararetrovirus of the family Caulimoviridae, genus Badnavirus it causes chlorosis leaf streak disease. The information currently available on banana streak disease makes it possible to identify a complex of distinct BSV species each causing the same disease. BSV exists in two states: one as an episomal form, infecting plant cells; the other as viral DNA integrated within the B genome of banana (endogenous BSV-eBSV) forming a viral genome for de novo viral particles. Both forms can be infectious in banana plants. The BSV phylogeny is polyphyletic with BSV distributed in two clades. Clade 1 clusters BSV species that occur worldwide and may have an eBSV counterpart, whereas Clade 3 only comprises BSV species from Uganda. Clearly, two distinct origins explain such BSV diversity. However, the epidemiology/outbreaks of BSV remains unclear and the role of eBSV needs to be clarified. In this review, the biodiversity of BSV is explained and discussed in the light of field and molecular epidemiology data. A scheme is proposed for the co-evolution of BSV and banana based on old or recent infection hypotheses related to African domestication sites and banana dissemination to explain the disease context.

The prevalence of badnaviruses in West African yams (Dioscorea cayenensis-rotundata) and evidence of endogenous pararetrovirus sequences in their genomes.

Yam (Dioscorea spp.) is an important vegetatively-propagated staple crop in West Africa. Viruses are pervasive in yam worldwide, decreasing growth and yield, as well as hindering the international movement of germplasm. Badnaviruses have been reported to be the most prevalent in yam, and genomes of some other badnaviruses are known to be integrated in their host plant species. However, it was not clear if a similar scenario occurs in Dioscorea yam. This study was conducted to verify the prevalence of badnaviruses, and determine if badnavirus genomes are integrated in the yam genome. Leaf samples (n=58) representing eight species of yam from global yam collections kept at CIRAD, France, and 127 samples of D. rotundata breeding lines (n=112) and landraces (n=15) at IITA, Nigeria, were screened using generic badnavirus PCR primers. Positive amplification of an expected ca. 579bp fragment, corresponding to a partial RT-RNaseH region, was detected in 47 (81%) of 58 samples analysed from CIRAD collections, and 100% of the 127 IITA D. rotundata samples. All the D. cayenensis and D. rotundata samples from the CIRAD and IITA collections tested PCR-positive, and sequencing of a selection of the PCR products confirmed they were typical of the genus Badnavirus. A comparison of serological and nucleic acid techniques was used to investigate whether the PCR-positives were sequences amplified from badnavirus particles or putative endogenous badnavirus sequences in the yam genome. Protein A sandwich-enzyme-linked immunosorbent assay (PAS-ELISA) with badnavirus polyclonal antisera detected cross-reacting viral particles in only 60% (92 of 153) of the CIRAD collection samples analysed, in contrast to the aforementioned 81% by PCR. Immunosorbent electron microscopy (ISEM) of virus preparations of a select set of 16 samples, representing different combinations of positive and negative PCR and PAS-ELISA results, identified bacilliform particles in 11 of these samples. Three PCR-positive yam samples from Burkina Faso (cv. Pilimpikou) were identified in which no viral particles were detected by either PAS-ELISA or ISEM. Southern hybridisation results using a yam badnavirus RT-RNaseH sequence (Gn155Dr) as probe, supported a lack of badnavirus particles in the cv. Pilimpikou and identified their equivalent sequences to be of plant genome origin. Probe Gn155Dr, however, hybridised to viral particles and plant genomic DNA in three D. rotundata samples from Guinea. These results represent the first data demonstrating the presence of integrated sequences of badnaviruses in yam. The implications of this for virus-indexing, breeding and multiplication of seed yams are discussed.

The common evolutionary history of badnaviruses and banana.

Recent plant genome sequencing efforts have revealed myriad viral sequences suggesting a cryptic interaction between both partners. Interestingly, no integration step has ever been reported as an obligatory step in the life cycle of plant viruses. Circular dsDNA viruses belonging to the family Caulimoviridae are the most abundant among integrated plant viral sequences. In this review, we describe how this hitherto hidden interaction could inform the evolutionary history of both partners badnaviruses and banana plants.

Geographical differentiation of the molecular diversity of cacao swollen shoot virus in Togo.

The variability of cacao swollen shoot virus (CSSV) was studied in two areas of Togo with distinct epidemiological backgrounds, Kloto and Litimé. This molecular study was based on 120 sequence comparisons of the first part of ORF3 of the viral genome. The phylogenetic study distinguished three groups of CSSV isolates, A, B, and C, with clear geographical differentiation between the Kloto and Litimé areas. The only group detected in Kloto was group C, whereas the Litimé area only contained isolates from groups A and B, which most probably originated from Ghana. The divergence between the three groups strongly suggests a different origin for each of the groups.

High molecular variability of sugarcane bacilliform viruses in Guadeloupe implying the existence of at least three new species.

Thirty-five unique partial sugarcane bacilliform virus (SCBV) sequences extending over 529 bp were identified in sugarcane samples from Guadeloupe diagnosed by Immunocapture-PCR (IC-PCR) using specific badnavirus primers. Phylogenetic analysis of these sequences along with the two known genome sequences of Sugarcane bacilliform Mor virus (SCBMV) and Sugarcane bacilliform IM virus (SCBIMV) revealed high molecular variability in the SCBV genome. Seven phylogenetic groups, named A to G, were characterized: virus isolates from groups A-B, C and D are proposed to be members of three additional SCBV species. The two (7446 and 7444 bp) and one (7317 bp) complete sequences of SCBV isolates from groups A and D, respectively, likely represented the genome of two new species. Phylogenetic analysis of the complete genome and RT/RNase H sequences confirmed the polyphyletic structure of SCBV isolates and the absence of a clear separation between SCBV and Banana streak virus (BSV) isolates within badnavirus group 1. These results showed that reconsideration of taxonomy and classification of SCBV and BSV are necessary.