The Partial Characterization of a Badnavirus Infecting the Greater Asiatic or Water Yam (Dioscorea alata)

A bacilliform virus from Dioscorea alata, designated Dioscorea alata bacilliform virus (DaBV), from Barbados and West Africa and from other Dioscorea spp. from West African, Carribean, Asian and South American countries, has been characterized. The virus was transmitted by the mealybug, Planococcus citri and by mechanical transmission of partially purified preparations to several Dioscorea spp. DaBV was serologically related to a distinct bacilliform virus from Dioscorea bulbifera, to one isolate of sugarcane bacilliform badnavirus and two isolates of banana streak badnavirus (BSV) but was not related to another isolate of BSV or to Kalanchoe top spotting or cacao swollen shoot badnaviruses. The coat protein of DaBV was about 56 kDa and the nucleic acid was double-stranded DNA of about 7.5 kbp, part of which showed distant homology with other badnaviruses. Thus, DaBV is a distinct hitherto uncharacterized badnavirus.     

Molecular characterization of <Emphasis Type="Italic">Banana streak virus</Emphasis> isolate from <Emphasis Type="Italic">Musa Acuminata</Emphasis> in China

Banana streak virus (BSV), a member of genus Badnavirus, is a causal agent of banana streak disease throughout the world. The genetic diversity of BSVs from different regions of banana plantations has previously been investigated, but there are relatively few reports of the genetic characteristic of episomal (non-integrated) BSV genomes isolated from China. Here, the complete genome, a total of 7722bp (GenBank accession number DQ092436), of an isolate of Banana streak virus (BSV) on cultivar Cavendish (BSAcYNV) in Yunnan, China was determined. The genome organises in the typical manner of badnaviruses. The intergenic region of genomic DNA contains a large stem-loop, which may contribute to the ribosome shift into the following open reading frames (ORFs). The coding region of BSAcYNV consists of three overlapping ORFs, ORF1 with a non-AUG start codon and ORF2 encoding two small proteins are individually involved in viral movement and ORF3 encodes a polyprotein. Besides the complete genome, a defective genome lacking the whole RNA leader region and a majority of ORF1 and which encompasses 6525bp was also isolated and sequenced from this BSV DNA reservoir in infected banana plants. Sequence analyses showed that BSAcYNV has closest similarity in terms of genome organization and the coding assignments with an BSV isolate from Vietnam (BSAcVNV). The corresponding coding regions shared identities of 88% and ∼95% at nucleotide and amino acid levels, respectively. Phylogenetic analysis also indicated BSAcYNV shared the closest geographical evolutionary relationship to BSAcVNV among sequenced banana streak badnaviruses.     

Unusual filamentous virus-like particles in symptomless blackberry associated with severe leaf curling in the virus indicator Rubus macraei, and anomalous data using a degenerate badnavirus primer in PCR of Rubus species

Electron microscopy of ultrathin sections of leaves of symptomless Himalaya Giant blackberry and of the virus indicator species, Rubus macraei, showing severe leaf curl symptoms following graft inoculation with scions from this blackberry, detected highly flexuous virus‐like particles with an unusual ‘beaded’ structure. Such particles were restricted to a few vascular cells and were distinct from P‐protein common in some such cells. This virus, provisionally named Hawaiian rubus leaf curl virus (HRLCV), symptomlessly infected a wide range of Rubus species and cultivars. Badnavirus‐like bacilliform particles were observed in some cells of a single R. macraei plant showing leaf curl symptoms following graft inoculation with the causal agent of this disease symptom from Himalaya Giant blackberry after passage through red raspberry, but not in any other material. PCR with primer sets for the badnaviruses Rubus yellow net virus and Gooseberry veinbanding associated virus, showed that no Rubus sources studied contained these viruses. However, using a sequence‐specific primer set designed from the sequence of the product generated with a badnavirus degenerate primer set, a specific product was amplified from healthy plants of all of 16 raspberry cultivars and two Rubus species, but not from 16 blackberry cultivars (including cv. Himalaya Giant). All of these sources were free from viruses known to occur in Rubus. Sequence analysis of this product showed no homology with any known badnavirus, or with any other published sequences. It seems most likely therefore that a region of the raspberry genome has been amplified using the degenerate badnavirus primer set and that it is absent from the blackberry genome.     

Molecular Characterization and Detection of African Oil Palm Ringspot Virus

African oil palm ringspot virus (AOPRV) had been previously described as a fovea‐like virus associated with a lethal disease of African oil palm (Elaeis guineensis) in South America. The original report was based on partial sequence and a distant relationship between AOPRV and Apple stem pitting virus, Apricot latent virus and Grapevine rupestris stem pitting‐associated virus, definitive species of the genus Foveavirus, family Flexiviridae. We report the full sequence of the RNA genome of AOPRV, and demonstrate that this virus is more closely related to two unassigned virus species of the family Flexiviridae (Cherry green ring mottle virus and Cherry necrotic rusty mottle virus) than to any definitive species of the genus Foveavirus. Thus, AOPRV should be considered as a new species of the Flexiviridae until the International Committee on Taxonomy of Viruses (ICTV) resolves the taxonomic status of the increasing number of unassigned species in this family. The molecular characterization of AOPRV has provided a highly sensitive and reliable RT‐PCR assay for the early detection of AOPRV in different genotypes of African, American (E. oleifera) and hybrid oil palms.     

Highly Efficient Immunodiagnosis of Episomal Banana streak MY virus Using Polyclonal Antibodies Raised Against Recombinant Viral‐Associated Protein

Banana streak MY virus (BSMYV) is the causal agent of viral leaf streak disease of banana, which leads to considerable losses in banana production in most of the banana‐growing regions worldwide. Developing high‐throughput virus detection system is essential for managing viral diseases especially in vegetatively propagated crops like banana. In this study, viral‐associated protein (VAP) coded by ORF II of BSMYV was expressed in Escherichia coli, and polyclonal antibodies were raised against purified recombinant VAP (rVAP) fusion protein in rabbits. Specificity and sensitivity of resulting antibodies were tested in Western blot, immunosorbent electron microscopy (ISEM) and enzyme‐linked immunosorbent assays (ELISAs). In direct antigen‐coated (DAC)‐ELISA, antibodies reacted specifically to BSMYV in crude sap, up to 1 : 8000 dilutions, but not to healthy leaf extracts. Using this antiserum, an immunocapture polymerase chain reaction (IC‐PCR) assay was developed and compared with DAC‐ELISA. VAP antibody‐based IC‐PCR is highly specific and could differentiate episomal virus infection from the integrated endogenous BSV (eBSV) sequences. The recombinant antibodies were validated by testing with a large number of banana germplasm conserved in the field gene bank. Field samples collected during surveys and mother cultures used in tissue culture propagation suggest that antibodies generated against rVAP are sensitive and useful for large‐scale detection of BSMYV. To the best of our knowledge, this is the first report on the production of polyclonal antiserum against recombinant VAP of BSMYV and its suitability for serology‐based testing by ELISA and IC‐PCR. This VAP‐based immunodiagnosis can be applied in quarantine, germplasm exchange and certification programmes.     

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

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

Simultaneous CRISPR/Cas9‐mediated editing of cassava eIF4E isoforms nCBP‐1 and nCBP‐2 reduces cassava brown streak disease symptom severity and incidence

Cassava brown streak disease (CBSD) is a major constraint on cassava yields in East and Central Africa and threatens production in West Africa. CBSD is caused by two species of positive‐sense RNA viruses belonging to the family Potyviridae, genus Ipomovirus: Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV). Diseases caused by the family Potyviridae require the interaction of viral genome‐linked protein (VPg) and host eukaryotic translation initiation factor 4E (eIF4E) isoforms. Cassava encodes five eIF4E proteins: eIF4E, eIF(iso)4E‐1, eIF(iso)4E‐2, novel cap‐binding protein‐1 (nCBP‐1), and nCBP‐2. Protein–protein interaction experiments consistently found that VPg proteins associate with cassava nCBPs. CRISPR/Cas9‐mediated genome editing was employed to generate ncbp‐1, ncbp‐2, and ncbp‐1/ncbp‐2 mutants in cassava cultivar 60444. Challenge with CBSV showed that ncbp‐1/ncbp‐2 mutants displayed delayed and attenuated CBSD aerial symptoms, as well as reduced severity and incidence of storage root necrosis. Suppressed disease symptoms were correlated with reduced virus titre in storage roots relative to wild‐type controls. Our results demonstrate the ability to modify multiple genes simultaneously in cassava to achieve tolerance to CBSD. Future studies will investigate the contribution of remaining eIF4E isoforms on CBSD and translate this knowledge into an optimized strategy for protecting cassava from disease.     

Complete Genome Sequence of a Novel Wild tomato mosaic virus Isolate Infecting Nicotiana tabacum in China

Virus particles of approximately 740–760 nm in length and 13 nm in diameter were observed from a diseased Nicotiana tabacum (tobacco) plant in Sichuan Province, China. The complete genomic sequence of the virus isolate XC1 was determined to contain 9659 nucleotides without 3′ terminal poly(A) tail. XC1 has a genome typical of members of the genus Potyvirus, encoding a large polyprotein of 3075 amino acids. Putative proteolytic cleavage sites and a number of well characterized functional motifs were identified by sequence comparisons with those of known potyviruses. Sequence comparison revealed that XC1 shared the highest level of nucleotide sequence identity (76.5%) with Wild tomato mosaic virus (WTMV). Phylogenetic analysis showed that XC1 was closely related to the WTMV Guangdong isolate with an identity of 94.3% between CP gene sequence of the two viruses. We thus named XC1 WTMV‐XC‐1 as a novel isolate of WTMV. The full sequence of WTMV‐XC‐1 may serve as a basis for future investigations on the gene diversity of WTMV.     

Identification of Rubus yellow net virus as a distinct badnavirus and its detection by PCR in Rubus species and in aphids

Rubus yellow net virus (RYNV) infects Rubus species and cultivars worldwide and is an essential component of raspberry veinbanding mosaic (RVBMD), a virus disease complex that causes serious decline in plant vigour and productivity. The virus is transmitted, probably in a semi‐persistent manner, by the large raspberry aphid, Amphorophora idaei in Europe, and A. agathonica in North America. The particles of RYNV are bacilliform in shape and measure 80–150 × 25–30 nm, similar to those of badnaviruses. A1.7 kb fragment of the viral DNA was amplified by PCR and then directly sequenced. Analysis of this sequence suggests that RYNV is possibly a distinct species in the genus Badnavirus and is most closely related to Gooseberry vein banding associated virus (GVBAV) and Spiraea yellow leaf spot virus, two other badnaviruses described recently. Using the sequence derived from the PCR‐amplified viral DNA fragment, RYNV‐specific primers were designed and used in PCR to assay for RYNV in a range of Rubus germplasm infected with RYNV, with other unrelated viruses and virus‐like diseases found in Rubus, and in healthy plants. RYNV was detected in all glasshouse cultures of RYNV‐infected plants, whether alone or in complex infections with other viruses, but not from healthy Rubus plants, nor from plants infected with other viruses. It was also detected in field‐grown raspberry plants with and without symptoms of RVBMD and in raspberry plants infected with RYNV by viruliferous A. idaei. RYNV was also detected by PCR in A. idaei following access feeds on RYNV‐infected plants of 1 h or more. PCR failed to amplify DNA from gooseberry infected with GVBAV confirming the specificity of the RYNV analysis. PCR detection of RYNV in dormant raspberry buds allows assays to be made outside the natural growing season, providing a useful application for plant introduction and quarantine programmes.     

Characterisation and genetic diversity of pepper leafroll virus,a new bipartite begomovirus infecting pepper,bean and tomato in Peru

The complete genome of a novel bipartite begomovirus (genus Begomovirus, family Geminiviridae) was cloned from a severely diseased yellow Peruvian chili pepper (Capsicum baccatum cv. Pendulum) plant collected in the department of La Libertad, Northern Peru and full‐length sequenced. The two genomic components share a common region of 156 nucleotides with a 100% sequence identity. Analysis of the genome organisation and phylogenetic comparisons revealed that the virus is a typical New World begomovirus. The closest related begomovirus, an isolate of Tomato yellow vein streak virus (ToYVSV), shared only 76.8% nucleotide sequence identity for the DNA‐A component. Therefore, following species demarcation criteria of the International Committee on Taxonomy of Viruses, this virus isolate belongs to a new begomovirus species for which the name pepper leafroll virus (PepLRV) is proposed. Pepper plants infected with the cloned PepLRV isolate developed leaf roll symptoms similar to those observed in field‐infected plants suggesting this virus as the causal agent of the disease syndrome observed in the field. Widespread occurrence of PepLRV throughout Peru was demonstrated, infecting plants of diverse cultivated species such as tomato, pepper, common and pallar beans, and of the weed species Nicandra physaloides. Low genetic diversity was observed among PepLRV isolates present in this country with no evident geographical or temporal structure of the population, typical of a recent founder effect. This is the first report of a begomovirus infecting pepper and bean crops in Peru.     

Phylogeny of <Emphasis Type="Italic">Banana Streak Virus</Emphasis> Reveals Recent and Repetitive Endogenization in the Genome of Its Banana Host (<Emphasis Type="Italic">Musa</Emphasis> sp.)

Banana streak virus (BSV) is a plant dsDNA pararetrovirus (family Caulimoviridae, genus badnavirus). Although integration is not an essential step in the BSV replication cycle, the nuclear genome of banana (Musa sp.) contains BSV endogenous pararetrovirus sequences (BSV EPRVs). Some BSV EPRVs are infectious by reconstituting a functional viral genome. Recent studies revealed a large molecular diversity of episomal BSV viruses (i.e., nonintegrated) while others focused on BSV EPRV sequences only. In this study, the evolutionary history of badnavirus integration in banana was inferred from phylogenetic relationships between BSV and BSV EPRVs. The relative evolution rates and selective pressures (d_N/d_S ratio) were also compared between endogenous and episomal viral sequences. At least 27 recent independent integration events occurred after the divergence of three banana species, indicating that viral integration is a recent and frequent phenomenon. Relaxation of selective pressure on badnaviral sequences that experienced neutral evolution after integration in the plant genome was recorded. Additionally, a significant decrease (35%) in the EPRV evolution rate was observed compared to BSV, reflecting the difference in the evolution rate between episomal dsDNA viruses and plant genome. The comparison of our results with the evolution rate of the Musa genome and other reverse-transcribing viruses suggests that EPRVs play an active role in episomal BSV diversity and evolution.     

Begomoviruses Infecting Tomato Crops in Panama

The key regions in Panama involved in open field‐ and greenhouse‐grown commercial tomato production, including the Chiriquí, Veraguas, Herrera, Los Santos, Coclé and Panama Oeste provinces, were surveyed for the incidence and distribution of begomoviruses in the growing seasons of 2011 and 2012. The surveys took place in 14 of the 51 districts of the above‐mentioned provinces and comprised all relevant tomato production areas of the provinces. A total of 28 tomato plots were surveyed. The exact location of each plot was geo‐referenced using a hand‐held Global Positioning System unit. In total, 319 individual tomato plants (181 in 2011 and 138 in 2012) were sampled. Plants displayed diverse combinations of virus‐like symptoms of different severity, including necrosis, yellowing, mosaic, mottling, rolling, curling, distortion and puckering of leaves, reduced leaf size, and stunted growth. DNA was extracted from each plant for a subsequent polymerase chain reaction (PCR) analysis, using two sets of degenerate primers able to detect members of the genus Begomovirus. The samples displaying a positive reaction were subsequently analysed with specific primer pairs to identify the affecting begomoviruses. A total of 42.3% of all collected samples showed a positive signal to PCRs. Three begomovirus species were detected with the species‐specific set of primers; in particular, in the samples obtained in 2011, Potato yellow mosaic Panama virus (PYMPV), Tomato leaf curl Sinaloa virus (ToLCSiV) and Tomato yellow mottle virus (TYMoV) were detected, while in the 2012 samples, only PYMPV and ToLCSiV were found. To our knowledge, this is the first reported incidence of ToLCSiV and TYMoV in Panamanian tomato crops.     

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

The 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.     

Rapid Detection of Banana Streak Virus by Loop‐mediated Isothermal Amplification Assay in South China

Banana streak virus (BSV) is a significant constraint to banana production and genetic improvement. It is necessary to develop and use BSV detection strategies that are both reliable and sensitive for the management of the virus. A loop‐mediated isothermal amplification (LAMP) assay was developed and evaluated for the detection of BSV. Four primers matching a total of six sequences of the conserved ORF III polyprotein genes were synthesized for developing a specific and sensitive LAMP for DNA extracts from field‐infected banana plants. LAMP assay could detect as low as 1 pg/μl template DNA. Test results of all field samples collected from different regions of South China showed that LAMP is more sensitive than PCR. This relatively simple and sensitive technique showed excellent potential with field‐collected samples and for routine screening of tissue culture materials in South China.     

Biological Characterization and Complete Genome Sequence of a Possible Strain of Indian cassava mosaic virus from Jatropha curcas in India

The outbreak of a severe mosaic disease with a significant incidence was noticed on Jatropha curcas plants growing in Lucknow, Northern India. The causal virus was successfully transmitted by whiteflies (Bemisia tabaci) and grafting from naturally infected to healthy J. curcas plants. The association of Begomovirus with the mosaic disease of J. curcas was detected by PCR using primers specific to DNA‐A of Begomoviruses. Further, full‐length DNA‐A genome of ～2.7 kb was amplified by RCA followed by digestion with Bam HI restriction enzyme. Cloning and sequencing of obtained amplicons resulted in 2740 nucleotides of complete DNA‐A consisting of six ORFs and IR region (GenBank Accession HM230683 ). The sequence analysis revealed highest 85% similarities with Jatropha curcas mosaic virus, 77–84% with Indian cassava mosaic virus and 73–76% with Sri Lankan cassava mosaic virus isolates. Phylogenetic analysis of the Begomovirus isolate also showed a clear‐cut distinct relationship with earlier reported Begomoviruses from Jatropha curcas and other Begomoviruses. On the basis of the guidelines of the International Committee on Taxonomy of Viruses (ICTV‐2008), our virus isolate was identified as a possible strain of Indian cassava mosaic virus, and its name Jatropha mosaic India virus (JMIV) is proposed.