Banana Streak OL Virus Research Articles

Genetic diversity analysis reveals new badnaviruses infecting banana in South China

Banana streak virus (BSV) is the causal agent of the most prevalent diseases in banana and causes serious economic losses to banana production worldwide. To examine variability in BSV, 65 isolates were obtained from 99 samples collected from Guangdong, Hainan, Guangxi and Yunnan provinces in South China. The 65 BSV isolates shared 62.8–97.8% nt identity over the RT/RNase H regions when compared to eight BSV species with published complete genomes. Sequence and phylogenetic analyses based on the RT/RNase H region indicated that there was great genetic diversity among BSV isolates in South China, and some isolates belonged to species of badnavirus that had never before been reported in China, including banana streak OL virus (BSOLV) (DN6–2), banana streak IM virus (BSIMV) (DN1–7 and DN5–1), and banana streak VN virus (BSVNV) (YLJ-8). The full genomes of two isolates (DN5–1and DN5–4) were cloned and sequenced. The genetic characteristics of both isolates were very close to those of typical badnaviruses. Based on percentage nucleotide sequence identity and phylogenetic relationships, DN5–1 was categorized as BSIMV. Interestingly, while DN5–4 was similar to both sugarcane bacilliform viruses (SCBVs) and BSVs based on sequence identities of the RT/RNase H region, analysis of its complete sequence and genome organization revealed that it can be further classified as sugarcane bacilliform Guadeloupe A virus-2 (SCBGAV-2). To our knowledge, this is the first report of natural infection of banana by SCBGAV-2 in China. In addition, our results suggest that considering the complete genomic sequence rather than the RT/RNase H region alone is important when classifying badnavirus isolates.

Prevalence and diversity of Banana streak viruses in the Dominican Republic

An extensive survey was carried out in the Dominican Republic in order to monitor the prevalence of the three most widespread BSV species: Banana streak OL virus (BSOLV), Banana streak GF virus (BGFV) and Banana streak IM virus (BSIMV), in Macho x Hembra (MxH) and FHIA-21 plantains and in Cavendish dessert banana throughout all banana-producing areas. This survey led to the first detection of BSVs in the Dominican Republic. BSGFV was the most prevalent of the three targeted BSV species in both FHIA-21 and MxH varieties. Prevalence of BSGFV and BSOLV were substantially higher in FHIA-21 than in MxH. Molecular analysis of the eBSV pattern of sampled FHIA-21 and MxH plants showed that both varieties harbor infectious alleles OL1 (eBSOLV) and GF7 (eBSGFV) and are devoid of eBSIMV. Very low prevalence was recorded for both BSOLV and BSGFV in Cavendish banana, which is devoid of infectious eBSVs. BSIMV was not detected at all. Our results point to a marginal role of insect-borne transmission in the epidemiology of BSVs in the Dominican Republic.

Multiplex detection of three banana viruses by reverse transcription loop-mediated isothermal amplification (RT-LAMP)

Banana bunchy top virus (BBTV), banana streak viruses (BSVs) and cucumber mosaic virus (CMV) are frequently reported infecting bananas globally. Effective control of their spread depends on robust detection of these viruses in propagation stock, planting material, infected nursery plants, and through strict quarantine. We developed single reverse transcription loop-mediated isothermal amplification (RT-LAMP) assays for BBTV, banana streak OL virus (BSV-OL) and CMV that were sensitive, specific, efficient, and completed in less than 60 min. RNA-based RT-LAMP minimized false positives that arose from banana genomes harboring endogenous viral genomes, such as BSVs. RT-LAMP was also more sensitive than RT-PCR in detecting the DNA viruses, BBTV and BSV-OL, in infected plants. We also developed a multiplex assay using three sets of primers specific for each virus to simultaneously detect BBTV, BSV-OL and CMV in a sample of RNA from the same plant. The reliability and convenience of this assay makes it useful for plant quarantine and indexing plants for propagation.

Molecular characterization of a new badnavirus associated with streak symptoms on enset (Ensete ventricosum, Musaceae)

AbstractElectron microscopy of leaf samples displaying streak symptoms from enset (Ensete ventricosum), a banana‐like plant widely cultivated in Ethiopia, showed the presence of bacilliform shaped virions as known for badnaviruses. DNA extracts subjected to rolling circle amplification (RCA), polymerase chain reaction (PCR) and cloning and sequence analysis revealed that the virus has a circular double‐stranded DNA genome of 7,163 nucleotides encoding predicted proteins of 21.5 kDa, 14.5 kDa and 202.5 kDa, a genome organization known for badnaviruses. The virus is phylogenetically most closely related to Sugarcane bacilliform Guadeloupe D virus with a nucleotide sequence identity of 77.2% at the conserved RT/RNase‐H region and 73.6% for the whole genome. Following the current species demarcation criteria, the virus should be considered as an isolate of a new species in the genus Badnavirus for which the name Enset leaf streak virus (ELSV) is suggested. Leaf samples from enset and banana were screened using virus‐specific primers, and ELSV was detected in six of 40 enset but not found in any of 61 banana samples. On the other hand, Banana streak OL virus (BSOLV) was detected from the majority (60%) of symptomatic banana samples but not from enset samples. This paper reports the first full‐genome sequence of a putative new badnavirus species infecting plants in the genus Ensete. In addition, this is the first report of the occurrence of BSOLV in Ethiopia.

Lessons learned from the virus indexing of Musa germplasm: insights from a multiyear collaboration

AbstractThe Bioversity International Transit Center (ITC) for banana hosts more than 1500 accessions largely covering the genetic diversity of the genus Musa. Its objective is to conserve this genetic diversity and to supply plant materials to users worldwide. All the Musa accessions must be tested for virus presence and, if infected, virus elimination must be attempted, to enable the supply of virus‐free plant material. An international collaborative effort launched under the auspices of Bioversity International (2007–2013) finally led to the implementation of a two‐step process to test the accessions. The first step, called pre‐indexing, involved only molecular tests and was designed as a pre‐screen of new germplasm lines or existing accessions to reduce the need for post‐entry virus therapy and repeated virus indexing. The second step, called full indexing, was performed on either older existing accessions or newer accessions which tested negative during pre‐indexing, and involved molecular tests, transmission electron microscopy (TEM) and symptom observation. In total, 270 germplasm lines (434 samples) were pre‐indexed; while full indexing was carried out on 243 accessions (68 of which had been pre‐indexed). A significant proportion of the samples tested during pre‐indexing was infected with at least one virus (68%), showing the utility of this early pre‐screening step. Banana streak OL virus and Banana mild mosaic virus were the most commonly detected viruses during both pre‐ and full indexing. For 22 accessions, viral particles were observed by TEM in full indexing while the molecular tests were negative, underlining the importance of combining various detection techniques. After full indexing, viruses were not detected in 166 accessions, which were then released for international distribution from the ITC. This publication exemplifies how the practical application of diagnostic protocols can raise fundamental questions related to their appropriate use in routine practice and the need for their continuous monitoring and improvement after their first publication.

Characterization of the banana streak virus capsid protein and mapping of the immunodominant continuous B-cell epitopes to the surface-exposed N terminus.

This study identified the structural proteins of two badnavirus species, Banana streak MY virus (BSMYV) and Banana streak OL virus (BSOLV), and mapped the distribution of continuous B-cell epitopes. Two different capsid protein (CP) isoforms of about 44 and 40 kDa (CP1 and CP2) and the virion-associated protein (VAP) were consistently associated with purified virions. For both viral species, the N terminus of CP2 was successfully sequenced by Edman degradation but that of CP1 was chemically blocked. De novo peptide sequencing of tryptic digests suggested that CP1 and CP2 derive from the same region of the P3 polyprotein but differ in the length of either the N or the C terminus. A three-dimensional model of the BSMYV-CP was constructed, which showed that the CP is a multi-domain structure, containing homologues of the retroviral capsid and nucleocapsid proteins, as well as a third, intrinsically disordered protein region at the N terminus, henceforth called the NID domain. Using the Pepscan approach, the immunodominant continuous epitopes were mapped to the NID domain for five different species of banana streak virus. Anti-peptide antibodies raised against these epitopes in BSMYV were successfully used for detection of native virions and denatured CPs in serological assays. Immunoelectron microscopy analysis of the virion surface using the anti-peptide antibodies confirmed that the NID domain is exposed on the surface of virions, and that the difference in mass of the two CP isoforms is due to variation in length of the NID domain.

Subpopulation level variation of banana streak viruses in India and common evolution of banana and sugarcane badnaviruses.

Genome sequences of three episomal Banana streak MY virus (BSMYV) isolates sampled from triploid banana hybrids (Chini Champa: AAB; Malbhog: AAB and Monthan: ABB), grown in North-East and South India are reported in this study by sequence-independent improved rolling circle amplification (RCA). RCA coupled with restriction fragment length polymorphism revealed diverse restriction profiles of five BSMYV isolates. Nucleotide substitution rates of BSMYV subpopulation and Banana streak OL virus subpopulation was 7.13 × 10(-3) to 1.59 × 10(-2) and 2.65 × 10(-3) to 5.49 × 10(-3), respectively, for the different coding regions. Analysis of the genetic diversity of banana and sugarcane badnaviruses revealed a total of 32 unique recombination events among banana and sugarcane badnaviruses (inter BSV-SCBV), in addition to the extensive recombination with in banana streak viruses and sugarcane bacilliform viruses (intra-BSV and intra-SCBV). Many unique fragments were shown to contain similar ruminant sequence fragments which indicated the possibility that the two groups of badnaviruses or their ancestors to colonise same host before making the host shift. The distribution of recombination events, hot-spots (intergenic region and C-terminal of ORF3) as well as cold-spots (distributed in ORF3) displayed the mirroring of recombination traces in both group of badnaviruses. These results support the hypothesis of relatedness of banana and sugarcane badnaviruses and the host and geographical shifts that followed the fixation of the species complex appear to be a recent event.

Occurrence and distribution of banana streak disease and standardization of a reliable detection procedure for routine indexing of banana streak viruses in India

In a survey for banana streak disease during 2010–2013, occurrence of typical streak symptoms was recorded with an incidence of 20–50% in different banana orchards at different locations in India. Serological and molecular studies provided evidence for the presence of episomal Banana streak MY virus (BSMYV) and Banana streak OL virus (BSOLV) in the field samples collected from different banana growing regions. Various methods of virus detection for banana streak viruses (BSVs) were employed. Direct PCR and direct binding-PCR (DB-PCR) did not give accurate and confirmatory results for the presence of episomal virus infection. Rolling circle amplification (RCA) was found to be highly reliable but was time consuming and difficult to use for indexing of a large number of samples. The duplex-immunocapture-PCR (D-IC-PCR) employing polyclonal antiserum with an immunocapture time of 3h was found to be a sensitive, reliable and accurate procedure for detection of episomal BSV infection. For definite detection of BSMYV, the widely occurring banana streak virus species in India D-IC-PCR using BSMYV specific primers and Musa specific internal primers was found to be most reliable procedure which can be used for routine indexing of tissue culture plantlets at different stages in the test labs as well as for germplasm exchange at quarantine stations. Based on the immuno- and immuno-nucleo detection procedures up to 46% of samples collected from different banana growing regions of India were found positive for BSV infection. Present study for the first time reported the widespread occurrence and distribution of BSV in India.

Episomal detection of Banana streak OL virus in single and mixed infection with Cucumber mosaic virus in banana 'Nanicão Jangada'

Banana streak virus (BSV) and Cucumber mosaic virus (CMV) are commonly found in all banana growing-areas of the world. These viruses cause diseases that lead to yield reduction and constrain plant breeding and distribution of Musa germplasm. Most of the diagnostic methods targeting BSV can generate dubious results because of the considerable genetic and serological diversity among BSV isolates and the presence of integrated BSV sequences in the banana plant. Both viruses are usually detected in single and mixed infections in banana plantations in the north region of Parana state using DAS-ELISA and PCR. A rolling-circle amplification protocol tested in this study allowed specific detection and identification of an episomal BSV isolate infecting Nanicao Jangada cultivar, thus confirming the occurrence of Banana streak OL virus in Brazil.

Occurrence, prevalence and molecular diversity of banana streak viruses in Cuba

Banana streak viruses (BSV) belong to the genus Badnavirus of the family Caulimoviridae. They cause banana streak disease on banana and plantains worldwide. The recent detection of BSV in Cuba has prompted a nationwide research effort focused on the occurrence, prevalence and diversity of BSV species on dessert type banana on the island. Indexing by multiplex immunocapture-PCR (M-IC-PCR) performed on samples collected throughout the country showed that the overall prevalence of Banana streak OL virus, Banana streak GF virus and Banana streak IM virus is low in Musa acuminata genotypes in Cuba. However, the prevalence of BSV species Mysore (BSMYV) was surprisingly high in samples of cv Yangambi km5 collected from distinct and distant locations. The presence in Cuba of an as yet unreported BSV species was also investigated, showing that Banana steak VN virus is also present in Musa acuminata genotypes.

Sequence analysis of shorter than genome length episomal Banana streak OL virus like sequences isolated from banana in India

Electron microscopy and sequencing of reverse transcriptase and ribonuclease H (RT/RNase H) region of Badnavirus genome from two banana cultivars: Poovan (triploid: AAB) and Safed velchi (diploid: AB), exhibiting leaf streak symptoms, confirmed the association of Banana streak OL virus (BSOLV). As per ICTV species demarcation threshold of 80 % identity in RT/RNase H region, both the isolates were identified as BSOLV. Rolling circle and end-to-end amplification showed the association of two short episomal BSOLV variants: BSOLV-IN1 and BSOLV-IN2 from Poovan and Safed velchi banana, respectively. The genome sizes of both isolates were 6,950 nucleotides long, but shorter than the typical BSOLV genome of 7,389 bp. Open reading frames (ORFs) 1 and 2 of shorter BSOLV isolates shared almost complete nucleotide identity (>99 %) to that of BSOLV. However, the ORF 3 (5,130 bp) and intergenic region (IGR), 886 bp, showed deletions compared with ORF 3 (5,499 bp) and IGR (956 bp) of BSOLV. In phylogenetic analysis for ORF 3 polyprotein, both the isolates clustered with BSOLV, Banana streak CA virus (BSCAV), and Sugarcane bacilliform GA virus (SCBGAV). Identical ORF 1, ORF 2, and the presence of all the conserved domains in short ORF 3 and promoter elements in IGR indicated that these isolates represent replicationally competent shorter variants of BSOLV. These two shorter-than-BSOLV genome sequences and two other identical banana streak virus sequences in GenBank (BSV-TRY; DQ859899 and BSV-GD; DQ451009) might have evolved due to error-prone reverse transcription and splicing or excision from the integrated sequences by homologous recombination in natural banana hybrids under field conditions.

Development of a Novel Rolling-Circle Amplification Technique to Detect Banana streak virus that also Discriminates Between Integrated and Episomal Virus Sequences.

Banana plants are hosts to a large number of Banana streak virus (BSV) species. However, diagnostic methods for BSV are inadequate because of the considerable genetic and serological diversity among BSV isolates and the presence of integrated BSV sequences in some banana cultivars which leads to false positives. In this study, a sequence-nonspecific, rolling-circle amplification (RCA) technique was developed and shown to overcome these limitations for the detection and subsequent characterization of BSV isolates infecting banana. This technique was shown to discriminate between integrated and episomal BSV DNA, specifically detecting the latter in several banana cultivars known to contain episomal or integrated sequences of Banana streak Mysore virus (BSMyV), Banana streak OL virus (BSOLV), and Banana streak GF virus (BSGFV). Using RCA, the presence of BSMyV and BSOLV was confirmed in Australia, while BSOLV, BSGFV, Banana streak Uganda I virus (BSUgIV), Banana streak Uganda L virus (BSUgLV), and Banana streak Uganda M virus (BSUgMV) were detected in Uganda. This is the first confirmed report of episomally-derived BSUglV, BSUgLV, and BSUgMV in Uganda. As well as its ability to detect BSV, RCA was shown to detect two other pararetroviruses, Sugarcane bacilliform virus in sugarcane and Cauliflower mosaic virus in turnip.

Clonagem e purificação de fragmento da proteína capsidial de Banana streak OL virus

O objetivo deste trabalho foi clonar e induzir a expressão de fragmento da proteína capsidial de Banana streak OL virus (BSOLV-CP) em Escherichia coli, bem como purificar a proteína recombinante obtida. Empregou-se um par de iniciadores específicos para amplificar, em PCR, um fragmento de aproximadamente 390 pb, da região codificadora da porção central da BSOLV-CP. O fragmento obtido foi clonado em vetor pGEM-T Easy, subclonado em vetor pQE-30 e transformado em células de E. coli M15 (pREP4) por choque térmico. A expressão da proteína foi induzida por tiogalactopiranosídeo de isopropila (IPTG), e a proteína recombinante BSOLV-rcCP de 14 kDa foi detectada em Western blot e Dot blot. A expressão da proteína BSOLV-rcCP abre novas possibilidades para a obtenção de antígenos para a produção de antissoros contra o BSOLV.

DETECTION, INCIDENCE AND DIVERSITY OF BANANA STREAK VIRUSES, BANANA MILD MOSAIC VIRUS AND BANANA VIRUS X IN GUADELOUPE

Banana streak viruses (BSVs) infect banana worldwide. BSV sequences present in the genome of Musa balbisiana hamper the detection of cognate episomal viruses by PCR, since PCR results in the amplification of both episomal viral DNA and integrated viral sequences, leading to false positives. A simple single-step multiplex immunocapture PCR (M-IC-PCR) assay was developed for the detection of only episomal BSV. This technique was applied in a study of the occurrence of BSV in Guadeloupe. Leaf samples from the main banana genotypes were indexed for the presence of major BSV species. Although no symptoms could be observed, BSV was found to be common in plantain (AAB), probably as a result of the widespread use of contaminated suckers. In contrast, BSV was uncommon on dessert banana cultivars (AAA), showing that the use of virus-free certified in vitro plants is an efficient strategy for controlling the spread of BSV, and that vector-borne transmission of BSV from plantain to dessert banana is very low in Guadeloupe. Our study also showed that Banana streak GF virus is the most prevalent species in Guadeloupe, with species Banana streak OL virus and Banana streak Mysore virus present at much lower levels. Using the same leaf samples, a study on the occurrence and diversity of Banana virus X (BVX), a member of the Flexiviridae family, which has been recently described and characterised in Guadeloupe, was undertaken. Indexing by direct-binding reverse transcription PCR showed that BVX is very uncommon in Guadeloupe and has a low level of molecular diversity. This is in contrast to Banana mild mosaic virus, which is a closely related member of the Flexiviridae family, and for which an immunocapture reverse transcription nested PCR detection method was established. (Resume d'auteur)