Resistance of Musa balbisiana Accessions of the Philippines to Banana Bunchy Top Virus.

Banana bunchy top disease (BBTD) is caused by banana bunchy top virus (BBTV), the most important virus affecting banana. Currently, no cultivar or accession of banana has complete resistance to BBTD. A total of 36 wild Musa spp. accessions, including 34 Musa balbisiana and 2 M. acuminata subsp. errans ("Agutay"), were screened for resistance against BBTV. In greenhouse tests using viruliferous banana aphids (Pentalonia nigronervosa), all M. balbisiana accessions remained symptomless, and BBTV was not detected in any of these plants by PCR at 3 and 6 months postinoculation. In contrast, 100% disease incidence was recorded in M. acuminata subsp. errans and in cv. Lakatan susceptible control plants. The PCR-negative M. balbisiana plants were then transferred to a field with high BBTV inoculum pressure where they remained symptomless and PCR-negative for up to 5 years, while all cv. Lakatan developed BBTD. Wild M. balbisiana accessions showed a high level of resistance and possibly immunity to BBTV and are expected to provide a resource for conventional and marker-assisted breeding.[Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Independent modulation of individual genomic component transcription and a cis-acting element related to high transcriptional activity in a multipartite DNA virus.

BACKGROUND: The genome of Banana bunchy top virus (BBTV) consists of at least six circular, single-stranded DNA components of ~ 1 kb in length. Some BBTV isolates may also carry satellite DNA molecules that are not essential for BBTV infection. The relation between multipartite DNA virus replication and their transcriptional levels and the underlying mechanism remain unclear. RESULTS: To understand the coordinated replication and transcription of the multiple genomic components, the absolute amounts of each BBTV DNA component were measured by real-time PCR (qPCR), and their transcriptional levels were determined by RNAseq and reverse transcription-qPCR (qRT-PCR). Significant differences were found in the absolute amounts of individual BBTV genomic components. Transcriptional levels of each BBTV genomic component obtained from the RNAseq data matched closely to those obtained from qRT-PCR, but did not correspond to the absolute amount of each DNA component. The ratio of transcript over DNA copies ranged from 46.21 to 1059.44%, which was possibly regulated by the promoter region in the intergenic region of each component. To further determine this speculation, the promoter region of the DNA-S, -M or -N was constructed to the upstream of green fluorescent protein (GFP) gene for transient expression by agrobacterium-mediated transformation method. The qRT-PCR showed the highest transcriptional activity was promoted by DNA-N promoter, about 386.58% activity comparing with CaMV 35S promoter. Confocal microscopy observation showed that the intensity of green fluorescence was corresponding to that of qRT-PCR. CONCLUSIONS: Our data clearly showed that BBTV was able to control the transcriptional level of each DNA component independently by through the promoter sequences in the intergenic region. Moreover, a cis-acting element from DNA-N component had a high transcriptional activity.

Genetic diversity of banana bunchy top virus isolates from China.

Banana bunchy top virus (BBTV) (the genus Babuvirus, the family Nanoviridae) is a single-stranded circular DNA virus with a genome composed of six components designated as DNA-R, -U3, -S, -M, -C, and -N. This study analyzed the nucleotide identities of the DNA-R of 23 isolates from banana-producing provinces of China, including Guangdong, Hainan, Guangxi, and Yunnan. Results showed that the nucleotide identity of DNA-R was 72.3-100%. Phylogenetic analysis indicated that these BBTV isolates were clustered in different subgroups within the Asian group (AG). Sequence analysis of the five other components (DNA -U3, -S, -M, -C, and -N) of the five isolates from China confirmed the results established for DNA-R of these BBTV isolates. This study suggested that the variation of DNA-R from Chinese BBTV isolates was considerably higher than the variation of other AG isolates, but their genetic diversity was low.

Identification and in silico characterisation of defective molecules associated with isolates of banana bunchy top virus.

Banana bunchy top virus (BBTV) is a multi-component single-stranded DNA virus. From 267 potentially infected Musa plants, 24 apparently 'defective' BBTV components have been identified. Interestingly, 23/24 of these defective molecules were apparently derived from DNA-R. All of the identified defective molecules had retained at least part of the CR-SL and CR-M but had insertions and/or deletions that in most cases resulted in open reading frame disruptions. Our detection of three monophyletic but diverse (and therefore likely circulating) defective DNA-R lineages suggests that, in many cases, defective DNA-R molecules might remain associated with BBTV genomes for prolonged periods.

A new genus of alphasatellites associated with banana bunchy top virus in Southeast Asia.

Autonomously replicating alphasatellites (family Alphasatellitidae) are frequently associated with plant single-stranded (ss)DNA viruses of the families Geminiviridae, Metaxyviridae, and Nanoviridae. Alphasatellites encode a single replication-initiator protein (Rep) similar to Rep proteins of helper viruses and depend on helper viruses for encapsidation, movement, and transmission. Costs versus benefits of alphasatellite-helper virus association are poorly understood. Our surveys in Southeast Asia (SEA) for wild and cultivated banana plants infected with banana bunchy top virus (BBTV, Nanoviridae) and Illumina sequencing reconstruction of their viromes revealed, in addition to a six-component BBTV genome, one to three distinct alphasatellites present in sixteen of twenty-four BBTV-infected plants. Comparative nucleotide and Rep protein sequence analyses classified these alphasatellites into four distinct species: two known species falling into the genus Muscarsatellite (subfamily Petromoalphasatellitinae) previously identified in SEA and two novel species falling into the tentative genus Banaphisatellite (subfamily Nanoalphasatellitinae) so far containing a single species recently identified in Africa. The banaphisatellites were found to be most related to members of the genus Fabenesatellite of subfamily Nanoalphasatellitinae and the genus Gosmusatellite of subfamily Geminialphasatellitinae, both infecting dicots. This suggests a dicot origin of banaphisatellites that got independently associated with distinct strains of monocot-infecting BBTV in Africa and SEA. Analysis of conserved sequence motifs in the common regions driving replication and gene expression of alphasatellites and BBTV strains revealed both differences and similarities, pointing at their ongoing co-evolution. An impact of alphasatellites on BBTV infection and evasion of RNA interference-based antiviral defences was evaluated by measuring relative abundance of BBTV genome components and alphasatellites and by profiling BBTV- and alphasatellite-derived small interfering RNAs. Taken together, our findings shed new light on the provenance of alphasatellites, their co-evolution with helper viruses, and potential mutual benefits of their association.

Evidence for Dicot Plants as Alternative Hosts of Banana Bunchy Top Virus and Its Alphasatellites in South-East Asia.

Banana bunchy top virus is a multicomponent circular ssDNA virus (family Nanoviridae) that causes one of the most devastating diseases of cultivated bananas and plantains (family Musaceae). It is transmitted by the aphids Pentalonia nigronervosa and P. caladii among host plants of Musaceae and some other families of monocots. Our Illumina sequencing reconstruction of virome components of BBTV-infected banana plants and their neighbor non-banana plants sampled in Vietnam and Laos revealed the monocot Commelina sp. (Commelinaceae) and the dicots Bidens pilosa and Chromolaena odorata (both Asteraceae) as hosts of BBTV and circular ssDNA alphasatellites (family Alphasatellitidae). Counting the proportions and relative abundances of Illumina reads representing BBTV genome components and alphasatellites suggested that Chromolaena and Commelina are poor hosts for BBTV and one to three alphasatellite species, whereas Bidens is a permissive host for BBTV and four alphasatellite species representing two genera of Alphasatellitidae. Our findings provide evidence for the dicot plants of family Asteraceae as alternative hosts of BBTV and its alphasatellites, which warrants further investigation of these and other dicots as a potential refuge and source of BBTV and multiple alphasatellites that become associated with this virus and likely affect its replication, transmission, and host range.

Detection and in silico characterization of banana bunchy top virus in West Bengal, India: relevance to global genetic diversity and population structure.

Banana bunchy top disease is one of the major prevailing virus diseases associated with banana cultivation, spreading rapidly within a small scale of time. Till date there are only few extensive reports of completely sequenced isolates in India. A study was conducted to detect BBTV infection across 12 districts in West Bengal (WB) where extensive prevalence of the disease was ascertained. In silico characterization of the six genome components were accomplished which showed 84.90-99.86% similarity with other BBTV isolates reported worldwide. The phylogenetic analysis based upon DNA R and DNA S suggested formation of monophyletic cluster of majority of the WB isolates and its close association with Tripura, Manipur, Australia and Africa isolates indicating diversion from geographical differentiation. Dynamics of evolutionary pattern such as genetic diversity including Tajima's D test and Fu Li's Fs test, average number of nucleotide differences (K), Polymorphic sites (S); Fst distance; Mismatch distribution plot; Haplotype network, and selection pressure were performed based upon geographical distribution of the virus. Population genetics analysis of both Pacific Indian Ocean group and South East Asian group of the global BBTV population revealed low nucleotide diversity, high haplotype diversity, high gene flow within the group, and negative or purifying selection constraint indicating recent population expansion. Hence, this study portrays Indian subcontinent as the possible hotspot for rapid demographic expansion from a small virus population size, contributing valuable addition to the currently available information on BBTV worldwide. Supplementary Information: The online version contains supplementary material available at 10.1007/s13337-023-00815-0.

Colorimetric detection platform for banana bunchy top virus (BBTV) based on closed-tube loop mediated isothermal amplification (LAMP) assay.

A colorimetric closed-tube Loop mediated isothermal amplification (LAMP) assay was developed for rapid and sensitive detection of banana bunchy top virus (BBTV) from leaf and sucker tissues of infected banana plants. Six LAMP primers were designed targeting BBTV coat protein gene. Isothermal amplification was set at 65 °C and end point detection made by including hydroxy naphthol blue dye in the reaction where the positive samples showed colour change from violet to sky blue. Molecular characterization of LAMP amplicon was made with restriction digestion and sequencing. Restriction digestion with Sau3AI having single cut site within the LAMP internal primer flanking region yielded two fragments of expected size. Sequence analysis confirmed that amplification corresponded to BBTV coat protein gene. Comparison of LAMP assay with conventional PCR showed that LAMP assay was 1000 times more sensitive than conventional PCR in BBTV detection with a detection limit of 0.05 ng total DNA per reaction. Supplementary Information: The online version contains supplementary material available at 10.1007/s13337-022-00784-w.

Elimination of BBTV via a systemic in vitro electrotherapy approach.

Banana bunchy top virus (BBTV) is the most destructive etiological agent limiting banana cultivation areas globally. This study attempted BBTV elimination by traditional shoot-tip culture (control) and alternative shoot-tip + electrotherapy (treated) techniques. Shoot-tip culture from Musa acuminata cv. 'Grand Naine' infected sources were exposed to 100 mA electric current for different time intervals (20-60 min). Virus indexing (via PCR) and genetic fidelity (by ISSR assay) from the cultures were tested, alongside the physio-biochemical parameters. Exposure of electric current for less than 50 min was ineffective for BBTV elimination. Still, a rise in the duration from 50 min or more led to eradicating the virus from some explants. Elimination of BBTV was complete from 100 % of explants exposed to 100 mA for 60 min, as confirmed by lack of BBTV detection even at six months after acclimatization. In the control treatment, the maximum efficiency of BBTV elimination was 28 % after eight subcultures. On the other hand, improved survival % was observed in the treated culture. Moreover, homogenous ISSR patterns were there between the treated and the mother plant and similar physio-biochemical activities were seen in electro-exposed cultures and healthy ones. Thus, the study reports complete BBTV-elimination from banana with international compliances, for the first time, via electrotherapy while maintaining genomic template and biochemical stability.

Development of a bead-based assay for detection of three banana-infecting viruses.

Background: Banana bunchy top virus (BBTV), cucumber mosaic virus (CMV) and banana streak virus (BSV) are important banana viruses, there are possible infections frequently with several viruses in field. Since the viruses are readily trasmitted in vegetative propagules, which pose a threat to banana production in banana-growing areas. Methods: A multiplex polymerase chain reaction (PCR) protocol combined with LiquiChip analysis to identify BSV, BBTV, and CMV, with consistent amplification of plant ubiquitin (UBQ), the banana plant messenger RNA used as a procedural control. Multiplex reverse transcription (RT)-PCR amplicons were extended by allele-specific primers, followed by hybridization with carboxylated microspheres containing unique fluorescent oligonucleotides, which were detected using the LiquiChip 200 workstation. Results: In this study, we aimed to develop a rapid, sensitive, and simultaneous detection method for BSV, BBTV, and CMV using a bead-based multiplex assay that can be applied in routine diagnosis. We demonstrated that this detection system was extremely efficient and highly specialized for differentiating individual in a mixture of viruses while being ten times more sensitive than traditional RT-PCR. The development of this method makes it feasible to detect banana viruses in field collected leaf samples.

Restriction-free cloning for molecular manipulation and augmented expression of banana bunchy top viral coat protein.

Banana bunchy top virus (BBTV) causing bunchy top disease, is one of the most devastating diseases of banana and plantain. All the six genomic components of isolates from different parts of the world have been well characterised, with most of the studies focusing on replicase gene and coat protein gene. Overexpression of coat protein (CP) in Escherichia coli system can contribute significantly in structural as well as immunological studies. In the present investigation, the full length BBTV CP was cloned to pGEX-4T-2 expression vector and overexpressed in various Escherichia coli strains to obtain high quality and quantity of the CP. An augmented overexpression and stability of recombinant coat protein was achieved by molecular manipulation of the clone by restriction-free (RF) cloning platform. The RF cloning was employed to replace the thrombin cleavage site in the vector backbone, which was also present in the protein of interest, and to incorporate TEV protease site to cleave fusion protein at this specific site, and separate the affinity tag. The RF method allows direct transformation of the PCR product to undergo ligation in vivo and obtain the transformants thereby avoiding the restriction digestion and ligation of the product to the linearized plasmid. From a litre culture, 1.084 mg/ml of fusion protein with GST tag was obtained after GSH sepharose affinity column chromatography. The fluorescence spectra indicated partial disordered tertiary structure of the fusion protein. Cleavage of tag was attempted using TEV protease overexpressed and purified in the laboratory. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-03017-x.

RNAi technology for management of banana bunchy top disease.

Banana bunchy top disease (BBTD) is one of the world's most destructive viral diseases of banana and plantain, causing up to 100% yield loss in severe cases. The disease is vectored by banana aphids (Pentalonia nigronervosa) and carried long distances through the movement of infected plant materials. The banana aphids harboring banana bunchy top virus (BBTV) present in banana producing regions are the sole vector and the most efficient method of transmitting the virus to the healthy plants. Controlling the spread of BBTD has been very challenging since no known banana germplasm is immune to BBTV. The disease can be managed with the use of virus-free planting material and roguing. However, once BBTD is established in the field, it is very difficult to eradicate or manage it. Therefore, a more sustainable way of controlling the disease is developing host plant resistance against the virus and the vector. Biotechnological strategies via RNA interference (RNAi) could be used to target the banana aphid as well as BBTV to reduce virus-associated yield losses of banana and plantain, which feed over 500 million people around the world. This review discusses the status of BBTD and perspectives on effective RNAi technologies for controlling BBTV and the vector, banana aphid, transmitting the virus as sustainable management of the disease.

Cleavage of the Babuvirus Movement Protein B4 into Functional Peptides Capable of Host Factor Conjugation is Required for Virulence.

Banana bunchy top virus (BBTV) poses a serious danger to banana crops worldwide. BBTV-encoded protein B4 is a determinant of pathogenicity. However, the relevant molecular mechanisms underlying its effects remain unknown. In this study, we found that a functional peptide could be liberated from protein B4, likely via proteolytic processing. Site-directed mutagenesis indicated that the functional processing of protein B4 is required for its pathogenic effects, including dwarfism and sterility, in plants. The released protein fragment targets host proteins, such as the large subunit of RuBisCO (RbcL) and elongation factor 2 (EF2), involved in protein synthesis. Therefore, the peptide released from B4 (also a precursor) may act as a non-canonical modifier to influence host-pathogen interactions involving BBTV and plants.

Banana bunchy top virus (BBTV) nuclear shuttle protein interacts and re-distributes BBTV coat protein in Nicotiana benthamiana.

Banana bunchy top virus (BBTV) is a circular single-stranded DNA virus with multi-components. The knowledge about interaction between viral proteins and pathogenesis mechanism of BBTV remains unclear. In this study, the coat protein gene (CP, ORF 516 bp) and nuclear shuttle protein gene (NSP, ORF 465 bp) from BBTV B2 isolate of the Southeast-Asia group were cloned. The intracellular localization analysis showed the CP locates in the cell nucleus of tobacco cells, while the NSP distributes in the cell nucleus and cytoplasm. Co-localization analysis indicated the NSP itself does not change distribution, but CP re-distributes to the cell nucleus and cytoplasm, suggesting that NSP interacts with CP and re-locates the CP in the cell. The interaction between CP and NSP was further verified by co-immunoprecipitation (Co-IP) in tobacco protoplasts. The study will help us to understand the interaction between viral proteins and pathogenesis mechanism of BBTV in host plants.

Virus titre determines the efficiency of Pentalonia nigronervosa (Aphididae: Hemiptera) to transmit banana bunchy top virus.

Banana bunchy top disease (BBTD) caused by banana bunchy top virus (BBTV) is one of the most serious viral diseases of banana and plantains. BBTV is transmitted by Pentalonia nigronervosa (Hemiptera, Aphididae) in a persistent circulative manner. Better knowledge of vector-virus-host relationship and the mechanism of transmission is essential for developing an effective control strategy. In this study, the viral copies in single to group of aphids with different acquisition access period (AAP) were quantified using SYBR green-based quantitative polymerase chain reaction (qPCR). The result indicated that a single aphid was able to acquire 861.04 copies of the virus after 24 h of AAP from the infected banana plant and transmitted the virus to 16.6% tissue culture plants, whereas 50 viruliferous aphids (15,066.94 viral copies) were necessary to achieve 100% transmission in a shortest time of 21.6 days. The number of viral copies acquired by the aphids were gradually increased with increased AAP. Hundred percent transmission was observed with 20 aphids in 48 h of AAP or 30-50 aphids in 24 h of AAP. The inoculated plants expressed typical bunchy top symptoms quickly when higher number of aphids (30 and above) were used with 24 h of AAP. Further, we report that the tissue culture banana plants are highly prone or vulnerable to BBTV infection compared to sucker grown plants. We conclude that higher the number of viral copies in the vector, higher the percent transmission and quicker the symptom expression and the results will contribute to a better understanding of vector-BBTV interactions and useful for epidemiological studies.

Development of a gold-nano particle based novel dot immunobinding assay for rapid and sensitive detection of Banana bunchy top virus.

An improved gold nanoparticle based Dot immunobinding assay (DIBA) was developed for the detection of Banana bunchy top virus (BBTV), that is more efficient, sensitive, rapid and simpler than conventional DIBA and ELISA. Instead of enzyme conjugates, gold nanoparticles were used as reporters owing to their unique optical properties. Antibody was raised against expressed recombinant coat protein of BBTV. The gold nanoparticles were conjugated to primary / detection antibody raised following immunization with recombinant coat protein, making it highly specific for the virus. Gold nanoparticle conjugated primary antibody (GCPab) based DIBA developed in this study has a detection efficiency comparable to ELISA. The results of using this assay format for detection of BBTV in banana plants from four geographical regions of India are also presented in this report. The test could detect the virus at sap dilution up-to 10-2. Using this improved DIBA, any lab with basic amenities can perform indexing on large numbers of samples.

Distribution, molecular characterization and diversity of banana bunchy top virus in Tripura, India.

Banana bunchy top virus (BBTV) is a serious threat to banana (Musa spp.) production in India. Generally, BBTV isolates within the country share very low genetic diversity. However, in India, relatively greater diversity has been observed between isolates from north-eastern (NE) region (Meghalaya) and rest of India. Tripura is situated in the south-west corner of NE India and shares international border with Bangladesh. During 2014-2015, diagnostic surveys were conducted in seven districts of Tripura and polymerase chain reaction based detection established that BBTD is widely prevalent in all parts of Tripura showing an average incidence of 22.02%. Among the cultivars, maximum BBTV infection (27.03%) was recorded in 'Chini Champa', followed by plantain (24.29%). A representative population (31 isolates) of BBTV from Tripura was characterized based on DNA R and DNA S. Phylogenetic analysis based on BBTV DNA R and DNA S generated two broad clusters of Pacific-Indian Oceans (PIO) and south-east Asian groups including all Tripura isolates within PIO cluster. The clustering pattern and genetic diversity of BBTV population from Tripura suggested monophyletic origin of majority of representative isolates from a common ancestor of PIO group. The exchange of vegetative propagules within and in between countries could have contributed to the geographical expansion of PIO isolates in Tripura. However, four variant BBTV isolates has been identified from North Tripura and Khowai districts possessing somewhat unique variability than that of distinct isolate (BBTV-Umiam) reported from NE India (Meghalaya).

Cloning of BBTV (Banana Bunchy Top Virus) components and screening of BBTV using functionalized gold nanoparticles.

Banana bunchy top virus (BBTV) affects all varieties of banana plants and causes heavy economic loss in most of the banana cultivating areas. The BBTV genome comprises of six DNA components; in this study, we have cloned the six BBTV-DNA components from one of the BBTV-infected plants (Tri-8) and were submitted to GenBank. Analysis of the BBTV DNA-R component showed that it belonged to south Pacific group. Resistance against BBTV has not been observed so far in banana plants and removal and killing of the infected plants has been routinely practiced. Hence, early detection of BBTV infection would be desirable and various detection methods routinely employed include enzyme linked immunosorbent assay (antigen-antibody based) and molecular-based methods such as polymerase chain reaction (PCR), qPCR, or LAMP PCR. Most of these methods require enzymes or antibodies for detection and hence are expensive. Here, we report a visual detection method (AuNP probe assay) using gold nanoparticles (AuNPs) functionalized with an ssDNA-thiolated probe (CR1). This method is based on the hybridization of the functionalized AuNPs with the target DNA (BBTV). In the AuNP probe assay, the functionalized AuNPs retains red colour when BBTV DNA is present, and in the absence of BBTV DNA, the colour of the functionalized AuNPs changes to purple when salt is added. The AuNP probe assay was compared with PCR for the detection of banana plants and it was found that AuNP probe assay was better than PCR in detecting BBTV infection (86.5% for AuNP probe assay and 65% for PCR). The AuNP probe assay was found to be highly specific to BBTV and was found to detect up to 1 pg/µl of the plasmid (pTZBBTri 4, BBTV DNA) mixed with healthy banana DNA.

The antagonistic effect of Banana bunchy top virus multifunctional protein B4 against Fusarium oxysporum.

The viral-induced banana bunchy top disease and the fungal-induced banana blight are two major causes of concern for industrial scale production of bananas. Banana blight is particularly troublesome, affecting ∼80% of crops worldwide. Strict guidelines and protocols are in place in order to ameliorate the effects of this devastating disease, yet little success has been achieved. From the data presented here, we have found that Banana bunchy top virus (BBTV)-infected bananas are more resistant to Fusarium oxysporum f. sp. cubense (Foc). BBTV appears to be antagonistic towards Foc, thus improving the survivability of plants against blight. The BBTV suppressor of RNA silencing, namely protein B4, displays fungicidal properties in vitro. Furthermore, transgenic tomatoes expressing green fluorescent protein (GFP)-tagged protein B4 demonstrate enhanced resistance to F. oxysporum f. sp. lycopersici (Fol). Differential gene expression analysis indicates that increased numbers of photogenesis-related gene transcripts are present in dark-green leaves of B4-GFP-modified tomato plants relative to those found in WT plants. Conversely, the transcript abundance of immunity-related genes is substantially lower in transgenic tomatoes compared with WT plants, suggesting that plant defences may be influenced by protein B4. This viral-fungal interaction provides new insights into microbial community dynamics within a single host and has potential commercial value for the breeding of transgenic resistance to Fusarium-related blight/wilt.

The global distribution of Banana bunchy top virus reveals little evidence for frequent recent, human-mediated long distance dispersal events.

Banana bunchy top virus (BBTV; family Nanoviridae, genus Babuvirus) is a multi-component single-stranded DNA virus, which infects banana plants in many regions of the world, often resulting in large-scale crop losses. We analyzed 171 banana leaf samples from fourteen countries and recovered, cloned, and sequenced 855 complete BBTV components including ninety-four full genomes. Importantly, full genomes were determined from eight countries, where previously no full genomes were available (Samoa, Burundi, Republic of Congo, Democratic Republic of Congo, Egypt, Indonesia, the Philippines, and the USA [HI]). Accounting for recombination and genome component reassortment, we examined the geographic structuring of global BBTV populations to reveal that BBTV likely originated in Southeast Asia, that the current global hotspots of BBTV diversity are Southeast Asia/Far East and India, and that BBTV populations circulating elsewhere in the world have all potentially originated from infrequent introductions. Most importantly, we find that rather than the current global BBTV distribution being due to increases in human-mediated movements of bananas over the past few decades, it is more consistent with a pattern of infrequent introductions of the virus to different parts of the world over the past 1,000 years.