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Parsley severe stunt-associated virus (PSSaV) is a recently identified nanovirus first reported in Germany. During a survey for identification of nanoviruses infecting apiaceous plants in south-eastern Iran, PSSaV was identified and characterized using a combination of rolling circle amplification (RCA) and high-throughput sequencing. Parsley plant samples were collected from vegetable production farms in Kerman province. From two symptomatic samples (39Ba and 40Ba), seven PSSaV components (DNA-C, -S, -M, -R, -N, -U1 and -U2) with two phylogenetically distinct variants of DNA-R (R1 and R2) were identified. In common with the German isolate of PSSaV, no DNA-U4 component was identified. In addition, associated alphasatellite molecules were identified in samples 39Ba [n = 6] and 40Ba [n = 5]. Sequence analyses showed that concatenated component sequences of the two Iranian PSSaVs share 97.2% nucleotide identity with each other and 82% to the German isolate. The coat proteins (CPs) of the PSSaV Iranian sequences share 97.2% amino acid identity and ~ 84% identity with that of the German isolate. Sequence and phylogenetic analyses of a total of 11 recovered alphasatellites from the two samples can be classified into the genera Fabenesatellite [n = 2], Milvetsatellite [n = 1], Mivedwarsatellite [n = 2], Subclovsatellite [n = 2], Sophoyesatellite [n = 4] in the family Alphasatellitidae. Identification of PSSaV and other nanoviruses in wild and cultivated plants in Iran reveals that nanoviruses could be causing yield reduction in crops plants in this country.

Banana bunchy top virus (BBTV, genus Babuvirus, family Nanoviridae) causes the most destructive viral disease of banana worldwide but is restricted to some countries in Africa and the Asia-Pacific region. Hosts of the virus are predominantly restricted to the plant family Musaceae, and it is transmitted through vegetative planting material and in a circulative, non-propagative manner by the banana aphid (Pentalonia nigronervosa) and P. caladii. In banana, disease symptoms include stunting, leaf chlorosis and rosetting, and a cessation of fruiting, and no natural source of immunity has been confirmed. Cytopathological effects and virus replication are restricted to the phloem tissue. The virus has isometric virions, 18–20 nm in diameter, separately encapsidating each of the six integral circular ssDNA genome components. All components are monocistronic, except for DNA-R, which encodes the master replication initiation protein, and a smaller internal ORF of unknown function. Replication is via a rolling circle mechanism, and the genome components share two conserved areas in their respective untranslated regions; the stem loop common region that contains the origin of replication and the major common region which binds a heterogeneous population of DNA primers, around 80 nucleotides in length, that prime complementary strand synthesis. Some virus isolates also support alphasatellite molecules.

Multipartite viruses package their genomic segments independently and mainly infect plants; few target animals. Nanoviridae is a family of multipartite single-stranded DNA plant viruses that individually encapsidate single-stranded DNAs of approximately 1 kb and transmit them through aphids without replication in the aphid vectors, thereby causing important diseases of leguminous crops and banana. Significant findings regarding nanoviruses have recently been made on important features, such as their multicellular way of life, the transmission of distinct encapsidated genome segments through the vector body, evolutionary ambiguities, mode of infection, host range and geographical distribution. This review deals with all the above-mentioned features in view of recent advances with special emphasis on the emergence of new species and recognition of new host range of nanoviruses and aims to shed light on the evolutionary linkages, the potentially devastating impact on the world economy, and the future challenges imposed by nanoviruses.

Stress granules (SGs) are structures within cells that regulate gene expression during stress response, e.g. viral infection. In mammalian cells assembly of SGs is dependent on the Ras-GAP SH3-domain-binding protein (G3BP). The C-terminal domain of the viral nonstructural protein 3 (nsP3) of Semliki Forest virus (SFV) forms a complex with mammalian G3BP and sequesters it into viral RNA replication complexes in a manner that inhibits the formation of SGs. The binding domain of nsP3 to HsG3BP was mapped to two tandem 'FGDF' repeat motifs close to the C-terminus of the viral proteins. It was speculated that plant viruses employ a similar strategy to inhibit SG function. This study identifies an Arabidopsis thaliana NTF2-RRM domain-containing protein as a G3BP-like protein (AtG3BP), which localizes to plant SGs. Moreover, the nuclear shuttle protein (NSP) of the begomovirus abutilon mosaic virus (AbMV), which harbors a 'FVSF'-motif at its C-terminal end, interacts with the AtG3BP-like protein, as does the 'FNGSF'-motif containing NSP of pea necrotic yellow dwarf virus (PNYDV), a member of the Nanoviridae family. We therefore propose that SG formation upon stress is conserved between mammalian and plant cells and that plant viruses may follow a similar strategy to inhibit plant SG function as it has been shown for their mammalian counterparts.

Single-stranded DNA (ssDNA) plant viruses belong to the families Geminiviridae and Nanoviridae. They are transmitted by Hemipteran insects in a circulative, mostly non-propagative, manner. While geminiviruses are transmitted by leafhoppers, treehoppers, whiteflies and aphids, nanoviruses are transmitted exclusively by aphids. Circulative transmission involves complex virus&ndash, vector interactions in which epithelial cells have to be crossed and defense mechanisms counteracted. Vector taxa are considered a relevant taxonomic criterion for virus classification, indicating that viruses can evolve specific interactions with their vectors. Thus, we predicted that, although nanoviruses and geminiviruses represent related viral families, they have evolved distinct interactions with their vector. This prediction is also supported by the non-structural Nuclear Shuttle Protein (NSP) that is involved in vector transmission in nanoviruses but has no similar function in geminiviruses. Thanks to the recent discovery of aphid-transmitted geminiviruses, this prediction could be tested for the geminivirus alfalfa leaf curl virus (ALCV) and the nanovirus faba bean necrotic stunt virus (FBNSV) in their common vector, Aphis craccivora. Estimations of viral load in midgut and head of aphids, precise localization of viral DNA in cells of insect vectors and host plants, and virus transmission tests revealed that the pathway of the two viruses across the body of their common vector differs both quantitatively and qualitatively.

The recent identification of a new nanovirus, pea necrotic yellow dwarf virus, from pea in Germany prompted us to survey wild and cultivated legumes for nanovirus infections in several European countries. This led to the identification of two new nanoviruses: black medic leaf roll virus (BMLRV) and pea yellow stunt virus (PYSV), each considered a putative new species. The complete genomes of a PYSV isolate from Austria and three BMLRV isolates from Austria, Azerbaijan and Sweden were sequenced. In addition, the genomes of five isolates of faba bean necrotic yellows virus (FBNYV) from Azerbaijan and Spain and those of four faba bean necrotic stunt virus (FBNSV) isolates from Azerbaijan were completely sequenced, leading to the first identification of FBNSV occurring in Europe. Sequence analyses uncovered evolutionary relationships, extensive reassortment and potential remnants of mixed nanovirus infections, as well as intra- and intercomponent recombination events within the nanovirus genomes. In some virus isolates, diverse types of the same genome component (paralogues) were observed, a type of genome complexity not described previously for any member of the family Nanoviridae. Moreover, infectious and aphid-transmissible nanoviruses from cloned genomic DNAs of FBNYV and BMLRV were reconstituted that, for the first time, allow experimental reassortments for studying the genome functions and evolution of these nanoviruses.

Plant viruses of the families Luteoviridae and Geminiviridae rely on hemipteran vectors for the infection of their hosts. Several lines of evidence have revealed that these viruses are transmitted by competent vectors in a circulative manner, involving entry into the vector’s body and the crossing of epithelial tissues forming the alimentary tract and the salivary glands. Similar to luteovirids and geminiviruses, a third family of plant viruses, the family Nanoviridae, have also been reported to be transmitted by aphids in a circulative manner. However, there is limited direct evidence of a possible path of translocation through the aphid vectors. Here, we used time-course experiments and transmission assays coupled with real-time PCR and immunofluorescence assays on dissected tissues to examine the translocation, compartmentalization and retention of banana bunchy top virus (BBTV) into the aphid vector Pentalonia nigronervosa. Our results indicate that BBTV translocates rapidly through the aphid vector; it is internalized into the anterior midgut in which it accumulates and is retained at concentrations higher than either the haemolymph or the principal salivary glands. Despite the large increase in viral concentration, we have failed to detect BBTV transcripts with RT-PCR. When tissues were not permeabilized, BBTV localized as distinct puncta in the proximity of the basal surface of the cells forming the anterior midgut and principal salivary glands, suggesting an on-going process of virion escape and internalization, respectively. Interestingly, we document that those organs can have direct contact within the aphid body, suggesting a possible haemolymph-independent translocation path.

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.

Banana bunchy top disease (BBTD) caused by Banana bunchy top virus (BBTV) is one of the most devastating diseases of banana and poses a serious threat for cultivars like Hill Banana (Syn: Virupakshi) and Grand Naine in India. In this study, we have cloned and sequenced the complete genome comprised of six DNA components of BBTV infecting Hill Banana grown in lower Pulney hills, Tamil Nadu State, India. The complete genome sequence of this hill banana isolate showed high degree of similarity with the corresponding sequences of BBTV isolates originating from Lucknow, Uttar Pradesh State, India, and from Fiji, Egypt, Pakistan, and Australia. In addition, sixteen coat protein (CP) and thirteen replicase genes (Rep) sequences of BBTV isolates collected from different banana growing states of India were cloned and sequenced. The replicase sequences of 13 isolates showed high degree of similarity with that of South Pacific group of BBTV isolates. However, the CP gene of BBTV isolates from Shervroy and Kodaikanal hills of Tamil Nadu showed higher amino acid sequence variability compared to other isolates. Another hill banana isolate from Meghalaya state had 23 nucleotide substitutions in the CP gene but the amino acid sequence was conserved. This is the first report of the characterization of a complete genome of BBTV occurring in the high altitudes of India. Our study revealed that the Indian BBTV isolates with distinct geographical origins belongs to the South Pacific group, except Shervroy and Kodaikanal hill isolates which neither belong to the South Pacific nor the Asian group.

Nanoviruses are multipartite single-stranded DNA (ssDNA) plant viruses that cause important diseases of leguminous crops and banana. Little has been known about the variability and molecular evolution of these viruses. Here we report on the variability of faba bean necrotic stunt virus (FBNSV), a nanovirus from Ethiopia. We found mutation frequencies of 7.52 × 10 −4 substitutions per nucleotide in a field population of the virus and 5.07 × 10 −4 substitutions per nucleotide in a laboratory-maintained population derived thereof. Based on virus propagation for a period of more than 2 years, we determined a nucleotide substitution rate of 1.78 × 10 −3 substitutions per nucleotide per year. This high molecular evolution rate places FBNSV, as a representative of the family Nanoviridae , among the fastest-evolving ssDNA viruses infecting plants or vertebrates.

A virus disease of faba bean (Vicia faba L.) in China, characterized by leaf yellowing and rolling and plant stunting, was shown to be caused by a virus of the genus Nanovirus based on serological reactions to nanovirus-specific monoclonal antibodies and the generation of polymerase chain reaction amplicons using nanovirus-specific primers. To identify the faba bean-infecting nanovirus, regions of the DNA components encoding the master replication initiator protein and capsid protein of two nanovirus isolates from China were cloned, sequenced and compared with those of other members of the genus Nanovirus. The two Chinese virus isolates shared nucleotide sequence identities ranging from 95 to 98% with the type isolate of Milk vetch dwarf virus (MDV) from Japan. They were thus identified as isolates of MDV, a virus so far known to cause important diseases of legumes in Japan. This is the first record of MDV-infecting faba bean in China.

Fu, H.-C., Hu, J.-M., Hung, T.-H., Su, H.-J., and Yeh, H.-H. 2009. Unusual events involved in Banana bunchy top virus strain evolution. Phytopathology 99:812-822. Banana bunchy top virus (BBTV) can be transmitted by aphids and consists of at least six integral components (DNA-R, -U3, -S, -M, -C, and -N). Several additional replication-competent components (additional Reps) are associated with some BBTV isolates. A collected BBTV strain (TW3) that causes mild symptoms was selected to study the processes in BBTV evolution. Southern blot hybridization, polymerase chain reaction (PCR), and real-time PCR did not detect DNA-N in TW3. Real-time PCR quantification of BBTV components revealed that, except for the copy number of TW3 DNA-U3, each detected integral component of BBTV TW3 was at least two orders lower than that of the severe strains. No infection was observed in plants inoculated with aphids, which were first given acquisition access to the TW3-infected banana leaves. Recombination analysis revealed recombination between the integral component TW3 DNA-U3 and the additional Rep DNA-Y. All BBTV integral components contain a replication initiation region (stem-loop common region) that share high sequence identity. Sequence alignment revealed that TW3 DNA-R, -S, -M, and -C all have a stem-loop common region containing a characteristic 9-nucleotide deletion found only in all reported DNA-N. Our data suggest that the additional Rep DNAs can serve as sources of additional genetic diversity for integral BBTV components.

Field and laboratory experiments were carried out on the island of Oahu, HI, to compare the susceptibility of the two most commonly grown banana (Musa sp.) cultivars in the state ('Dwarf Brazilian' or Santa Catarina [locally known as dwarf apple] and 'Williams') to the aphid-borne Banana bunchy top virus (genus Babuvirus, family Nanoviridae, BBTV). Several morphological and physiological features of the two cultivars were monitored to determine whether the banana aphid, Pentalonia nigronervosa Coquerel (Hemiptera: Aphididae), transmits BBTV to both cultivars at a similar rate; and whether after successful inoculation, does each cultivar respond similarly to viral infection. Results from the laboratory experiment showed that a similar percentage of both cultivars were infected with BBTV by aphid vectors (90% for both cultivars). However, field results showed a significantly lower percentage of dwarf apple (39%) infected with BBTV compared with Williams (79%). We also found that all physiological and morphological features measured (i.e., plant height, leaf area, canopy, chlorophyll level, and moisture content) for both cultivars were impacted similarly by BBTV. The incubation period, or the time between plant infection and initial appearance of disease symptoms, was similar for both cultivars. Results also showed that BBTV transmission efficiency was lower in the field than in the laboratory, despite that more aphids per plant were used for field than laboratory inoculation tests. The results highlight the potential use of less susceptible cultivars to help manage BBTV and the importance of screening banana varieties in the field to determine their response to vectors and associated diseases.

Field and laboratory studies were conducted to determine the impact of using a herbicide as a bananacide on aphid transmission of Banana bunchy top virus (family Nanoviridae, genus Babuvirus, BBTV) to healthy banana (Musa spp.) plants. BBTV-infected banana plants in a commercial orchard were treated with Roundup Weathermax herbicide. Using polymerase chain reaction, the time after herbicide treatment that BBTV could no longer be detected in the infected plants was determined. The impact of the herbicide treatment on Pentalonia nigronervosa Coquerel (Hemiptera: Aphididae) virus acquisition and ability to inoculate healthy banana plants with BBTV also were determined. Generally, banana plants were dead beyond 42 d after herbicide injection (DAI), and BBTV was detected in a similar high percentage of treated plants from 0 up to 21 DAI. During two field trials, 0 and 32% of P. nigronervosa acquired the virus from treated plants at 42 DAI, respectively, but none successfully inoculated a healthy banana plant beyond 35 DAI. Finally, 22% of P. nigronervosa colonies collected directly from the pseudostem of injected plants at the final sample date (42 DAI) tested positive for BBTV and infected 9.5% of the healthy banana plants. The findings indicate that banana plants may remain a potential source of virus inoculum 6 wk after injection with a bananacide. The implications of these findings with respect to BBTV management are discussed.

Field experiments were conducted in Oahu, Hawaii, to investigate the effects of banana bunchy top virus (BBTV) infection on growth and morphology of banana (Musa acuminata). The time interval between aphid inoculation of BBTV and the initial appearance of disease symptoms (i.e. incubation period) was also determined. Plants infected with BBTV showed a significant reduction in petiole size (i.e. length and distance), plant canopy and height, leaf area, pseudostem diameter and chlorophyll content compared with control plants. Growth differences between virus-infected and control plants were not observed until 40–50 days after the plants were inoculated with viruliferous aphids. Other growth parameters such as petiole width and leaf production were not statistically different between infected and control plants. The incubation period of banana bunchy top disease or appearance of symptoms ranged from 25 to 85 days after aphid inoculation. However, PCR assays provided earlier detection of BBTV in banana plants compared with visual symptoms.

In the plant virus world, there are six genera of plant viruses with dsDNA genomes and six genera with ssDNA (Fauquet et al., 2005). The dsDNA viruses are comprised of 4 genera in the Caulimoviridae, the genus Badnavirus and the genus Tungrovirus. The ssDNA viruses are comprised of four genera in Geminiviridae, and the two genera Nanovirus and Babuvirus in the Nanoviridae. The genera Babuvirus and Badnavirus are not well studied in Asia. However, we recognized the significance of two species, Banana bunchy top virus (BBTV) in the genus Babuvirus and Banana streak virus (BSV) in the genus Badnavirus, during the survey of banana viruses in Asia. Their main characters will be introduced in this mini-review.

Banana bunchy top disease is a major constraint to banana production in most regions where this crop is grown. The disease is caused by Banana bunchy top virus (BBTV), a multicomponent, single-stranded DNA virus of the family Nanoviridae. We have designed primers to a conserved region of the master replication-associated protein that are useful for the polymerase chain reaction (PCR)-mediated detection of BBTV. In addition, primers to banana genomic sequence are used as an internal control, overcoming the uncertainty (owing to false-negatives) inherent in PCR diagnostics. Together these primer sets are a valuable tool in the effort to control BBTV, particularly in screening micropropagated banana plantlets for the absence of virus before release to farmers.

Species of plant viruses within the Luteoviridae, Geminiviridae, and Nanoviridae are transmitted by phloem-feeding insects in a circulative, nonpropagative manner. The precise route of virus movement through the vector can differ across and within virus families, but these viruses all share many biological, biochemical, and ecological features. All share temporal and spatial constraints with respect to transmission efficiency. The viruses also induce physiological changes in their plant hosts resulting in behavioral changes in the insects that optimize the transmission of virus to new hosts. Virus proteins interact with insect, endosymbiont, and plant proteins to orchestrate, directly and indirectly, virus movement in insects and plants to facilitate transmission. Knowledge of these complex interactions allows for the development of new tools to reduce or prevent transmission, to quickly identify important vector populations, and to improve the management of these economically important viruses affecting agricultural and natural plant populations.

Faba bean necrotic yellows virus (FBYNV), Milk vetch dwarf virus (MDV), and Subterranean clover stunt virus (SCSV) are nanoviruses that infect leguminous plants. From MDV- and SCSV-infected tissue we identified viral DNAs that encode a replication initiator protein (Rep), essential for replication of the multiple circular single-stranded DNAs of these viruses. These previously undescribed Rep proteins of MDV and SCSV are strikingly similar in sequence and functionally equivalent to the master Rep protein of FBYNV. Moreover, we demonstrated that the master Rep proteins of the three viruses are able to trigger replication of heterologous nanovirus DNAs. Such cross-species replication may reflect a considerable potential for genetic reassortment among nanoviruses in nature and be of significance for their evolution.

Summary Banana bunchy top virus (BBTV) is a ssDNA virus transmitted by the banana aphid, (Pentalonia nigronervosa). A polymerase chain reaction (PCR) assay was used to study BBTV transmission efficiency, to determine the minimum acquisition-access period, the minimum inoculation-access period, the retention time, and to examine the possibility of transovarial transmission in this vector. BBTV was acquired by banana aphids within 4 h and was transmitted within 15 min feeding. On average, more than 65% of single viruliferous adult aphids transmitted BBTV. The aphids retained BBTV for their adulthood of 15–20 days. None of the 131 offspring from adult aphids reared on infected bananas were BBTV positive. Aphid transmission experiments were conducted to determine if taro and gingers are hosts of BBTV. None of the 87 taro and ginger plants exposed to aphid inoculation were infected by BBTV. The BBTV-free status of these plants was verified by PCR assay for 6 months post-inoculation. In addition, none of the taro and ginger samples collected from fields adjacent to BBTV-infected banana plants tested positive for BBTV.

The nucleotide sequences of seven circular, single-stranded DNA components of one isolate of subterranean clover stunt virus (SCSV) have been determined. Each component, of about 1 kb, appears to encode a single open reading frame in the same sense as the encapsidated DNA. Notably, the proteins encoded by two SCSV components are related Each has a consensus nucleotide binding motif and shares about 40% amino acid identity with the other and with the putative replication proteins of banana bunchy top virus and coconut foliar decay virus. The noncoding regions of the five other SCSV components share a highly conserved noncoding sequence of about 160 nucleotides. All seven components were found to contain a sequence capable of forming a stable stem-loop structure in the noncoding region which contains a conserved 9-nucleotide sequence in the loop, very similar to that of the geminiviruses. In addition, the putative replication proteins of SCSV are similar to those of the geminiviruses. We suggest that the SCSV-like viruses and the geminiviruses share a common ancestor and that this ancestor was more like SCSV in particle structure and genome organisation.

SUMMARY A disease showing chlorosis, leaf rolling and stunting in Vicia faba and other legumes was observed in West Asia and North Africa during 1987–1988. The putative causal agent could not be transmitted mechanically, but could be transmitted by aphids, most efficiently by Acyrthosiphon pisum, in the persistent manner. Further studies revealed isometric virus-like particles (VLPs) closely associated with the disease, although their infectivity could not be demonstrated by membrane feeding. These particles, measuring c. 18 nm in diameter and containing a capsid protein of about 22 kDa and ssDNA of about 1 kb, are hereafter designated faba bean necrotic yellows virus (FBNYV). A high proportion of circular nucleic acid molecules of about 0.9 kb were visualised by electron microscopy. Hybridisation analysis of cloned viral DNA suggests that the circular genome is larger than 1 kb and consists of several components of similar size. An antiserum produced against FBNYV was used in ELISA, immunoelectron microscopy (IEM) and Western blot experiments for virus detection in aphids and field samples and for serological comparison with other viruses. Weak heterologous reactions between FBNYV and subterranean clover stunt virus (SCSV) were detected in IEM, but could not be confirmed in ELISA or Western blots. No serological relationship to banana bunchy top virus (BBTV) was detected. Using a direct tissue blot immunoassay (TBIA), FBNYV was detected in vascular tissue of infected faba bean leaves and stems.

Nanoviridae is a family of single stranded DNA viruses which infect the plants through their phloem tissues. The few members of these viruses are now turning towards animals. It includes two genera, Nanovirus and Babuvirus. Nanovirus includes three species namely Faba beans necrotic yellows virus (FBNYV), Milk vetch dwarf virus (MVDV) and Subterranean clover stunt virus (SCSV) while Babuvirus have two species accounted yet, namely Abaca bunchy top virus (ABTV) and Banana bunchy top virus (BBTV). The viral coat proteins are likely to be responsible for many diseases in plants as well as animals. The intra-genomic changes or post translational modifications with in SCSV have decreased the length of the sequence, but increased the antigenic potential and numbers of antigenic binding regions on the surface of the protein. We have predicted the most probable responsible antigenic determinants and MHC binders within the viruses in Nanoviridae family. MHC molecules play a crucial role in host immune response. The nonamers of antigenic determinants in Nanoviridae family viral coat proteins are highly sensitive to H-2Kd and I-Ag7 molecules. The species within particular genera shows their common epitopes along with diverse colleague epitopes. These common antigenic peptides can be used as their identifi ers. These identifi ers along with their diverse colleagues could be most informative for antidotes production against themselves. Also, these are important for synthetic peptide vaccine production against the relevant viruses.

We describe a new plant single-stranded DNA (ssDNA) virus, a nanovirus isolate originating from the faba bean in Ethiopia. We applied rolling circle amplification (RCA) to extensively copy the individual circular DNAs of the nanovirus genome. By sequence analyses of more than 208 individually cloned genome components, we obtained a representative sample of eight polymorphic swarms of circular DNAs, each about 1 kb in size. From these heterogeneous DNA populations after RCA, we inferred consensus sequences of the eight DNA components of the virus genome. Based on the distinctive molecular and biological properties of the virus, we propose to consider it a new species of the genus Nanovirus and to name it faba bean necrotic stunt virus (FBNSV). Selecting a representative clone of each of the eight DNAs for transfer by T-DNA plasmids of Agrobacterium tumefaciens into Vicia faba plants, we elicited the development of the typical FBNSV disease symptoms. Moreover, we showed that the virus thus produced was readily transmitted by two different aphid vector species, Aphis craccivora and Acyrthosiphon pisum . This represents the first reconstitution of a fully infectious and sustainably insect-transmissible nanovirus from its cloned DNAs and provides compelling evidence that the genome of a legume-infecting nanovirus is typically comprised of eight distinct DNA components.

Anhalt, M. D., and Almeida, R. P. P. 2008. Effect of temperature, vector life stage, and plant access period on transmission of Banana bunchy top virus to banana. Phytopathology 98:743-748. The study of the transmission biology of insect-borne plant viruses is important to develop disease control practices. We characterized the transmission of a nanovirus, Banana bunchy top virus (BBTV), by its aphid vector Pentalonia nigronervosa Coquerel (Hemiptera, Aphididae) with respect to temperature, vector life stage, and plant access time. Adult aphids transmitted BBTV more efficiently than third instar nymphs at all temperatures tested. Adult aphids transmitted the virus more efficiently at 25 and 30°C than at 20°C, but temperature had no impact on transmission efficiency by nymphs. By decoupling the relationship between temperature and aphid BBTV acquisition or inoculation, we determined that temperature affected inoculation events more strongly than acquisition. Longer plant access periods increased viral acquisition and inoculation efficiencies in a range of 60 min to 24 h. Both BBTV acquisition and inoculation efficiencies peaked after 18 h of plant access period. We also show that BBTV transmission by P. nigronervosa requires a latent period. Our results demonstrate that vector transmission of BBTV is affected by temperature, vector life stage, and plant access period.

The majority of described plant viruses are transmitted by insects of the Hemipteroid assemblage that includes aphids, whiteflies, leafhoppers, planthoppers, and thrips. In this review we highlight progress made in research on vector interactions of the more than 200 plant viruses that are transmitted by hemipteroid insects beginning a few hours or days after acquisition and for up to the life of the insect, i.e., in a persistent-circulative or persistent-propagative mode. These plant viruses move through the insect vector, from the gut lumen into the hemolymph or other tissues and finally into the salivary glands, from which these viruses are introduced back into the plant host during insect feeding. The movement and/or replication of the viruses in the insect vectors require specific interactions between virus and vector components. Recent investigations have resulted in a better understanding of the replication sites and tissue tropism of several plant viruses that propagate in insect vectors. Furthermore, virus and insect proteins involved in overcoming transmission barriers in the vector have been identified for some virus-vector combinations.

The multicomponent single-stranded DNA plant nanoviruses encode unique master replication initiator (Rep) proteins. We have mapped the 5′ and 3′ termini of the corresponding polyadenylated mRNAs from faba bean necrotic yellows virus (FBNYV) and subterranean clover stunt virus and found that these are terminally redundant by up to about 160 nt. Moreover, the origin of viral DNA replication is transcribed into RNA that is capable of folding into extended secondary structures. Other nanovirus genome components, such as the FBNYV DNA encoding the protein Clink or an FBNYV DNA encoding a non-essential para-Rep protein, are not transcribed in such a unique fashion. Thus, terminally redundant mRNAs and the resulting transcription of the replication origin appear to be restricted to nanovirus master Rep DNAs. We speculate that this may be a way to regulate the expression of the essential master Rep protein.

Banana bunchy top virus (BBTV, genus Babuvirus, family Nanoviridae) causes the most destructive viral disease of banana worldwide, but is restricted to some countries in Africa and the Asia-Pacific region. Hosts of the virus are found only in the plant family Musaceae, and it is transmitted through vegetative planting material and in a circulative, nonpropagative manner by its sole vector the banana aphid (Pentalonia nigronervosa). In banana, disease symptoms include stunting, leaf chlorosis, and rosetting, and a cessation of fruiting, and no natural source of immunity is known. Histological effects and virus replication are restricted to the phloem tissue. The virus has isometric virions, 18–20 nm in diameter, separately encapsidating each of the six integral circular single-stranded DNA (ssDNA) genome components. All components are monocistronic, except for DNA-R, which encodes the master replication initiation protein, and a smaller internal open reading frame (ORF) of unknown function. Replication is via a rolling-circle mechanism, and the genome components share two conserved areas in their respective untranslated regions; the stem loop common region that contains the origin of replication and the major common region which binds a heterogeneous population of DNA primers, around 80 nt in length, that prime complementary strand synthesis.

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. Weanalyzed 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.

Circumstantial evidence suggests that the genome of Faba bean necrotic yellows virus (FBNYV), a nanovirus, consists of eight distinct, circular, single-stranded DNAs, each of about 1 kb and encoding only one protein. Here, the use of cloned full-length FBNYV DNAs for reproducing FBNYV-like symptoms in Vicia faba, the principal natural host of FBNYV, is reported. Characteristic symptoms of FBNYV infection were obtained in faba bean plants following biolistic DNA delivery or agroinoculation with all eight FBNYV DNAs. Although the eight different DNAs have been invariably detected in field samples infected with the various geographical FBNYV isolates, experimental infection with different combinations of fewer than eight DNAs also led to typical FBNYV symptoms. Even only five genome components, DNA-R, DNA-S, DNA-M, DNA-U1 and DNA-U2, were sufficient for inducing disease symptoms in V. faba upon agroinoculation. Symptomatic plants agroinoculated or bombarded with eight DNAs contained typical FBNYV virions; however, the virus was not transmitted by Aphis craccivora or Acyrthosiphon pisum, two efficient aphid vectors of FBNYV.

Recent reports have suggested that cotton leaf curl virus (CLCuV), a geminivirus of the genus Begomovirus, may be responsible for cotton leaf curl disease in Pakistan. However, the causal agent of the disease remains unclear as CLCuV genomic components resembling begomovirus DNA A are unable to induce typical disease symptoms when reintroduced into plants. All attempts to isolate a genomic component equivalent to begomovirus DNA B have been unsuccessful. Here, we describe the isolation and characterisation of a novel circular single-stranded (ss) DNA associated with naturally infected cotton plants. In addition to a component resembling DNA A, purified geminate particles contain a smaller unrelated ssDNA that we refer to as DNA 1. DNA 1 was cloned from double-stranded replicative form of the viral DNA isolated from infected cotton plants. Blot hybridisation using probes specific for either CLCuV DNA or DNA 1 was used to demonstrate that both DNAs co-infect naturally infected cotton plants from different geographical locations. DNA 1 was detected in viruliferous Bemisia tabaci and in tobacco plants infected under laboratory conditions using B. tabaci, indicating that it is transmitted by whiteflies. Sequence analysis showed that DNA 1 is approximately half the size of CLCuV DNA but shares no homology, indicating that it is not a defective geminivirus component. DNA 1 has some homology to a genomic component of members of Nanoviridae, a family of DNA viruses that are normally transmitted by aphids or planthoppers. DNA 1 encodes a homologue of the nanovirus replication-associated protein (Rep) and has the capacity to autonomously replicate in tobacco. The data suggest that a nanovirus-like DNA has become whitefly-transmissible as a result of its association with a geminivirus and that cotton leaf curl disease may result from a mutually dependent relationship that has developed between members of two distinct DNA virus families that share a similar replication strategy.

Purified faba bean necrotic yellows virus (FBNYV; genus Nanovirus) alone is not transmissible by its aphid vector, Acyrthosiphon pisum, regardless of whether it is acquired from artificial diets or directly microinjected into the aphid's hemocoel. The purified virus contains all of the genetic information required for its infection cycle as it readily replicated in cowpea protoplasts and systemically infected Vicia faba seedlings that were biolistically inoculated using gold particles coated with intact virions or viral DNA. The bombarded plants not only developed the typical disease syndrome, thus indicating that FBNYV is the sole causal agent of the disease, but also served as a source from which the virus was readily acquired and transmitted by A. pisum. The defect of the purified virus in aphid transmissibility suggests that FBNYV requires a helper factor (HF) for its vector transmission that is either nonfunctional or absent in purified virus suspensions. The requirement for an HF was confirmed in complementation experiments using two distinct isolates of the virus. These experiments revealed that aphids transmitted the purified virus isolate from artificial diets only when they had fed previously on plants infected with the other FBNYV isolate. Also, microinjected FBNYV, which persisted to the same extent in A. pisum as naturally acquired virus, was transmissible when aphids had acquired the HF from infected plants. This suggests that one of the functions of the HF in the transmission process is to facilitate virus transport across the hemocoel–salivary gland interface.

Publisher Summary This chapter explores the possible reasons for the negligence of banana bunchy top virus (BBTV), the way in which it has been controlled in Australia, and the possible directions for future research that could reduce the often devastating effects of the disease. The economic effects of the disease were as dramatic as its spread. In New South Wales, practically no plantation was free of BBTV in the Tweed and Brunswick areas. Most attempts to control or eradicate banana bunchy top virus have been made in commercial banana-producing countries. In Australia, a control program has successfully contained the disease at a very low level in southeast Queensland and the north coast of New South Wales for about 50 years. However, the disease still requires continual monitoring. The basis for control came from the discovery that (1) banana bunchy top was caused by a virus, (2) this virus was transmitted by the aphid, P. nigronervosa, (3) the virus could be disseminated in infected vegetative planting material, and (4) there were no known non-musaceous alternate hosts. For the campaign to be successful, little more information about the virus was required and in the ensuing 60 years little more has been discovered. Most research has been confined to verification of the identity of the disease in particular areas, varietal reactions to infection, some epidemiology and more recently isolation of the dsRNAs associated with infection.