Complete genome sequence and phylogenetic analysis of a newly discovered fusagra-like virus infecting Carica papaya in Ecuador.

A new fusagra-like virus infecting papaya (Carica papaya L.) was genetically characterized. The genome of the virus, provisionally named "papaya sticky fruit-associated virus" (PSFaV), is a single molecule of double-stranded RNA, 9,199 nucleotides (nt) in length, containing two discontinuous open reading frames. Pairwise sequence comparisons based on complete RNA-dependent-RNA-polymerase (RdRp) sequences revealed identity of 79.4% and 83.3% at the nt and amino acid (aa) level, respectively, to babaco meleira-like virus (BabMelV), an uncharacterized virus sequence discovered in babaco (Vasconcellea x heilbornii) in Ecuador. Additional plant-associated viruses with sequence identity in the 50% range included papaya meleira virus (PMeV) isolates from Brazil. Phylogenetic analysis based on the amino acid sequences of the capsid protein (CP), RdRp, and CP-RdRp fusion protein genes placed PSFaV in a group within a well-supported clade that shares a recent ancestor with Sclerotium rolfsii RNA virus 2 and Phlebiopsis gigantea mycovirus dsRNA 2, two fungus-associated fusagraviruses. Genomic features and phylogenetic relatedness suggest that PSFaV, along with its closest relative BabMelV, represent a species of novel plant-associated virus classified within the recently established family Fusagraviridae.

Occurrence, Distribution, and Population Structure of Schlumbergera Virus X in Dragon Fruit in Ecuador.

The occurrence of Schlumbergera virus X (SchVX) in commercial dragon fruit fields in three provinces of Ecuador has been identified in this study. The virus was found in symptomatic and asymptomatic cladodes of the two major species (Hylocereus undatus and H. megalanthus) cultivated in the country. Symptoms in H. undatus included irregular and ring-shaped chlorotic spots that coalesce into large chlorotic patches along the cladodes, whereas small chlorotic spot symptoms on the cladodes were observed in H. megalanthus. Phylogenetic inferences based on 27 partial nucleotide sequences of the RNA-dependent RNA polymerase (RdRp) and three whole genome comparisons showed that Ecuadorean isolates from H. undatus and H. megalanthus share a most recent ancestor with isolates from Spain and Portugal. In addition, an SchVX isolate with a distinct genomic lineage was found in symptomatic H. polyrhizus plants from a single location, suggesting two independent virus introductions into the country.

First Report of Beet Western Yellows Virus Infecting Vasconcellea x heilbornii (Caricaceae) in Ecuador.

Vasconcellea x heilbornii, known as babaco, is a hybrid native to Ecuador grown in small orchards in sub-tropical highland regions. Over the last decade, several viruses have been identified in babaco using high-throughput sequencing (HTS) (Cornejo-Franco et al. 2020, (Reyes-Proaño et al. 2023). In 2021, total RNA from a babaco plant showing distinctive leaf yellowing was extracted using the PureLink RNA Mini Kit (Thermo Fischer Scientific, USA) and subjected to HTS on an Illumina NovaSeq6000 system as 150 paired-end reads (Macrogen Inc., South Korea). Library construction was done using the TruSeq Stranded Total RNA Sample kit with Plant Ribo-Zero, as described (Villamor et al. 2022). Reads were processed using BBDuk and de novo assembled using SPAdes 3.15. both implemented in Geneious 2022. Contig analysis was done by BLASTx using the NCBI viral sequence database (as of November 2022). HTS generated 54 million reads, of which 12% assembled into contigs corresponding to genomes of previously reported babaco viruses including babaco virus Q (BabVQ), babaco nucleorhabdovirus 1 (BabRV1) and babaco ilarvirus 1 (BabIV1). Interestingly, 144 reads (0.0003%) assembled into seven contigs ranging from 100 to 480 nucleotides (nt) in length. These contigs showed homology, with 97% amino acid (aa) identity (100% query coverage), to regions of the RNA-dependent-RNA-polymerase (RdRp) of beet western yellows virus (BWYV, Acc. No. NC_004756), a member of the Polerovirus genus. To confirm the occurrence of BWYV in babaco, double-stranded RNA (dsRNA) was extracted from 15 g of leaf tissue from the original sample as described (Dodds et al. 1984) and used as template for reverse-transcription (RT)-PCR using overlapping primers designed to span all short contigs. RT-PCR amplified fragments were cloned into a pGEM®T-easy vector (Promega, USA) and sequenced by the Sanger method (Macrogen Inc., South Korea). The sequences were assembled into a single 2.7 kbp BWYV genome fragment comprising the complete protein 1 (P1) and partial RdRp gene (GenBank Acc. No. PP480670). Sequence alignments between the partially sequenced genome of the babaco isolate and its corresponding fragment from the closest BWYV isolate (NC_004756) revealed 94% and 97% identities at the nt and aa levels, respectively. To assess the prevalence of BWYV in babaco, 30 leaf samples showing yellowing symptoms from Pichincha (n=15) and Azuay (n=15) provinces were tested by RT-PCR using total RNA. Total RNA extraction and reverse transcription were done using the methodology described by Halgren et al. (2007). For RT-PCR, the primer set BWYV_Bab_F: 5'-CAGTGTCCTCCAAGTGCAACAT-3' / BWYV_Bab_R: 5'GGTTCCTTCCCAGTTTGGTGGT-3', which amplifies a 235 nt-long P1 region, was used. Three RT-PCR products from each positive sample were purified using the GeneJET PCR clean-up kit (Thermo Scientific, USA) and sequenced. BWYV was confirmed in 9 out of 15 samples (60%) from Pichincha, and in 10 out of 15 samples (64%) from Azuay. Samples were also tested for additional babaco viruses as described (Reyes-Proaño et al. 2023). All BWYV-infected plants turned out positive for papaya ringspot virus (PRSV), babaco mosaic virus (BabMV), BabVQ, and BabIV1. Hence, the impact of BWYV infection on babaco plants in single and mixed infections warrants further investigation. To the best of our knowledge, this is the first report of BWYV in a crop in Ecuador, and the first time it has been found in a Caricaceae species.

Identification and genetic characterization of a new totivirus from Bursera graveolens in western Ecuador.

The complete genomic sequence of a previously uncharacterized virus provisionally named "Bursera graveolens associated totivirus 1" (BgTV-1) was obtained from Bursera graveolens (Kunth) Triana & Planch., a tree known as "palo santo" in Ecuador. The BgTV-1 genome is a monopartite double-stranded RNA (dsRNA) that is 4794 nucleotides (nt) long (GenBank accession number ON988291). Phylogenetic analysis of the capsid protein (CP) and RNA-dependent RNA polymerase (RdRp) placed BgTV-1 in a clade with other plant-associated totiviruses. Amino acid (aa) sequence comparisons of putative BgTV-1 proteins showed the highest sequence similarity to those of taro-associated totivirus L (QFS21890.1-QFS21891.1) and Panax notoginseng virus A (YP_009225664.1- YP_009225665.1), with 51.4% and 49.8% identity, respectively, in the CP and 56.4% and 55.2% identity, respectively, in the RdRp. BgTV-1 was not detected in total RNA from either of the two endophytic fungi cultured from BgTV-1-positive B. graveolens leaves, suggesting that BgTV-1 may be a plant-infecting totivirus. Based on its distinct host and the low aa sequence similarity between the CP of BgTV-1 and its counterparts from the closest relatives, the virus described in this study should be assigned as a new member of the genus Totivirus.

Papaya Sticky Disease Caused by Virus "Couples": A Challenge for Disease Detection and Management.

Papaya sticky disease (PSD) is a major virus disorder of papaya (Carica papaya). The disease is characterized by fruit damage caused by the oxidation of spontaneously exuded latex. In Brazil, PSD is caused by the coinfection of two viruses, papaya meleira virus (PMeV), a toti-like virus, and papaya meleira virus-2 (PMeV-2), an umbra-like virus. The disorder has also been reported in Mexico and, more recently, in Australia, but the presence of both PMeV and PMeV-2 in symptomatic plants has been documented only in Brazil. In 2021, 2-year-old papaya plants (cultivar Passion Red) exhibiting PSD-like symptoms were observed in Santa Elena Province, Ecuador. Molecular tests of leaf tissue and fruit latex from symptomatic plants failed to detect PMeV. However, papaya virus Q (PpVQ), an umbra-like virus related to but distinct from PMeV-2, and a novel virus, tentatively named papaya sticky fruit-associated virus (PSFaV), were found in the symptomatic samples. PSFaV shares 56% nucleotide identity with the genome of PMeV, suggesting that PSD symptoms can be caused by "couples" of viruses related to but distinct from PMeV (a toti-like virus) and PMeV-2 (an umbra-like virus). This review discusses the history and epidemiology of PSD and the genomic features of newly discovered virus couples involved in this syndrome. Given the unusual etiology of PSD, which involves distinct virus species, the importance of implementing proper diagnostic approaches for PSD is highlighted.

Characterization of a new potyvirus infecting Thevetia ahouai in Ecuador.

A new potyvirus was found in Thevetia ahouai L. (Fam. Apocynaceae) plants exhibiting white spots on leaves and fruit discoloration in Ecuador. The complete genome sequences of two isolates of this virus, tentatively named "thevetia white spot virus" (ThWSV), were determined and found to be 9,912 (isolate 1) and 9,904 (isolate 2) nucleotides (nt) in length, each encoding a polyprotein of 363 kDa. Sequence comparisons between the two isolates showed 80 and 87% identity at the nt and amino acid (aa) level, respectively, whereas the overall sequence identity between ThWSV and its closest relative was 69% and 71% at the nt and aa level, respectively.

Two new umbravirus-like associated RNAs (ulaRNAs) discovered in maize and johnsongrass from Ecuador.

Two new umbravirus-like associated RNAs (ulaRNAs) were found, respectively, in maize and Johnsongrass samples from Ecuador. The complete sequences consist of 3,053 and 3,025 nucleotides, respectively, and contain four open reading frames (ORFs). Their genome sequences were 58% identical to each other and 28 to 60% identical to the most closely related viruses. Phylogenetic analysis using full genome sequences and amino acid sequence of the RNA-dependent-RNA polymerase (RdRp) placed both sequences in a clade sharing the most recent common ancestor with ulaRNAs from sugarcane and maize, suggesting that they belong to a monophyletic grass-infecting lineage. Their terminal regions exhibit features common to umbraviruses and ulaRNAs.

Genome Characterization and Pathogenicity of Two New Hyptis pectinata Viruses Transmitted by Distinct Insect Vectors.

Two newly described viruses belonging to distinct families, Rhabdoviridae and Geminiviridae, were discovered co-infecting Hyptis pectinata from a tropical dry forest of Ecuador. The negative-sense RNA genome of the rhabdovirus, tentatively named Hyptis latent virus (HpLV), comprises 13,765 nucleotides with seven open reading frames separated by the conserved intergenic region 3'-AAUUAUUUUGAU-5'. Sequence analyses showed identities as high as 56% for the polymerase and 38% for the nucleocapsid to members of the genus Cytorhabdovirus. Efficient transmission of HpLV was mediated by the pea aphid (Acyrthosiphon pisum) in a persistent replicative manner. The single-stranded DNA genome of the virus tentatively named Hyptis golden mosaic virus (HpGMV) shared homology with members of the genus Begomovirus with bipartite genomes. The DNA-A component consists of 2,716 nucleotides (nt), whereas the DNA-B component contains 2,666 nt. Pairwise alignments using the complete genomic sequence of DNA-A of HpGMV and closest relatives showed identities below the cutoff (<91% shared nt) established by the ICTV as species demarcation, indicating that HpGMV should be classified in a distinct begomovirus species. Transmission experiments confirmed that the whitefly Bemisia tabaci Middle East-Asia Minor 1 (MEAM1) is a vector of HpGMV.

First report of Neoscytalidium dimidiatum causing stem canker on yellow dragon fruit (Hylocereus megalantus) in Ecuador.

Dragon fruit cultivation is an emerging industry in Ecuador. In August of 2020, yellow dragon fruit plants (Hylocereus megalantus) showing brown hardened scabs on cladodes and fruits were observed in a field in Guayas, a coastal province of Ecuador (Fig. 1A). Symptoms were observed in ~ 40% of the assessed plants (n=100) with damage varying from mild (necrotic spots) to severe (canker). Ten cladode sections of ~ 10 cm2 with signs of canker were collected from five affected plants (two from each plant). Symptomatic cladode sections were sliced, surface-sterilized with a solution of 1% sodium hypochlorite, rinsed with sterile water, transferred into potato dextrose agar (PDA) media, and incubated at 28°C for five days in the dark. Three fungal isolates recovered from the cladodes produced colonies with dense dark aerial mycelia that matched the morphological description for Neoscytalidium dimidiatum (Crous et al 2006) (Fig 1B, 1C). Arthric chains of cylindrical conidia were observed under the microscope and presented zero or one septum with sizes between 10.9 ± 0.27 x 4.97 ± 0.36 µm (n=50). Pycnidia produced ellipsoid-shaped conidia, and sizes ranged from 4.5 ± 0.3 x 11.02 ± 0.5 µm (n=50). Cultured isolates were subjected to DNA extractions using the fungal DNA mini kit (Omega, Bio-Tek, Inc) for molecular identification by amplifying the 5.8S rDNA and adjacent internal transcriber spacer (ITS) 1 and 2 regions using primers (ITS1 / ITS4) as described (White et al., 1990). In addition, the ß-tubulin and elongation factor 1-α targets were amplified by primers Bt2a / Bt2b (Glass and Donaldson, 1995) and EF1-728 F / EF1-986R (Carbone and Kohn, 1999), respectively, following the recommended PCR conditions. Amplified products were cloned using a pGEMT-easy kit (Promega, USA) and sequenced. Sequence comparisons for each target revealed that the three isolates were 100% identical to each other. Sequences obtained from a single plant were submitted to NCBI Genbank and assigned acc. Numbers OP377444, OP381216, and OP381217, for the ITS, ß-tubulin, and elongation factor, respectively. BLAST analyses of the three amplified targets confirmed homology to counterparts from N. dimitiatum, with 99-100 % identities to isolates from China (JX524168), United Arab Emirates (MN447201), and Israel (KF020895). To fulfill Koch´s postulates, thirteen 4-month-old healthy plants were inoculated either with the pathogen (n=10) or mock-inoculated (n=3). In addition, two detached yellow dragon fruits and a mock were inoculated following the same protocol as stems. Inoculation was performed by making small wounds on cladodes or fruits using a sterile needle and placing a 2-mm agar plug containing mycelia from a 7-day-old colony. For mock inoculations, 2-mm plugs with clean PDA media were used. The inoculated area was wrapped with black plastic film for seven days at 27 to 32°C. At ten days post-inoculation, brown scab lesions with an orange halo were observed in the inoculated plants but not in the mock-inoculated plants and fruit (Fig. 1D, 1E). N. dimidiatum was re-isolated from experimentally-induced canker lesions, and morphologically identified. Stem canker caused by N. dimidiatum is a severe disease affecting dragon fruit production worldwide (Chuang et al., 2021). In Ecuador, this pathogen was found affecting yellow dragon fruit (H. megalantus) under natural conditions; however, under experimental conditions, red dragon fruit (H. undatus), which is widely produced in several coastal provinces of the country, was also found to be susceptible. Further studies are needed to investigate epidemiological aspects of this important pathogen, which threatens dragon fruit production in Ecuador.

Transmission and Pathogenicity of Papaya Virus E: Insights from an Experimental Papaya Orchard.

A study was conducted to investigate epidemiological aspects of papaya virus E (PpVE), a cytorhabdovirus commonly found in papaya (Carica papaya L.) plantings in Ecuador. Besides papaya, PpVE was found in three Fabaceae weeds, including Rhynchosia minima, Centrosema plumieri, and Macroptilium lathyroides, the latter being the species with the highest virus prevalence. Greenhouse experiments showed that in M. lathyroides, single infections of PpVE induce only mild leaf mosaic, whereas in mixed infections with cowpea severe mosaic virus, PpVE contributes to severe mosaic. In papaya, PpVE did not induce noticeable symptoms in single or mixed infections with papaya ringspot virus. Transmission experiments confirmed that whiteflies (Bemisia tabaci) transmit PpVE in a semipersistent, nonpropagative manner.

An umbra-related virus found in babaco (Vasconcellea × heilbornii).

The complete sequence of a new viral RNA from babaco (Vasconcellea × heilbornii) was determined. The genome consisted of 4,584 nucleotides, containing two open reading frames (ORFs 1 and 2), a 9-nt-long noncoding region (NCR) at the 5' terminus, and an unusually long (1,843 nt) NCR at the 3' terminus. The presence of a potential heptameric slippery signal located 12 nt upstream the stop codon of ORF 1 suggests a -1 ribosomal frameshift mechanism for the translation of ORF 2. Sequence comparisons of ORF 2 revealed similarity to the RNA-dependent RNA polymerase (RdRp) of several umbra- and umbra-like viruses. Phylogenetic analysis of the RdRp placed the new virus in a well-supported and cohesive clade that includes umbra-like viruses reported in papaya, citrus, opuntia, maize, and sugarcane hosts. Viruses of this clade share a most recent ancestor with the umbraviruses but have different genomic features. The creation of a new genus within the family Tombusviridae is proposed for the classification of these novel viruses.

Potato Virus Y (PVY) Isolates from Solanum betaceum Represent Three Novel Recombinants Within the PVYN Strain Group and Are Unable to Systemically Spread in Potato.

Tamarillo, or tree tomato (Solanum betaceum), is a perennial small tree or shrub species cultivated in subtropical areas for fresh fruit and juice production. In Ecuador, tamarillo orchards are affected by several viruses, with one previously identified as potato virus Y (PVY); however, the specific strain composition of PVY in tamarillo was not determined. In 2015 and 2016, eight tamarillo plants exhibiting symptoms of leaf drop, mosaic, and mottled fruit were sampled near Tumbaco and Quito, Ecuador. These tamarillo PVY isolates were able to systemically infect tobacco, Nicotiana benthamiana, naranjilla, and tamarillo. Seven of the eight PVY isolates from tamarillo exhibited N-serotype, while one of the PVY isolates studied, Tam15, had no identifiable serotype. One isolate, Tam17, had N-serotype but produced asymptomatic systemic infection in tobacco. In tamarillo, four tamarillo isolates induced mosaic and slight growth retardation and were unable to systemically infect pepper or potato. Tamarillo, on the other hand, was unable to support systemic infection of PVY isolates belonging to the PVYO and PVYEu-N strains. The whole genomes of eight PVY isolates were sequenced from a series of overlapping RT-PCR fragments. Phylogenetically, tamarillo PVY isolates were found to belong to the large PVYN lineage, in a new tamarillo clade. Recombination analysis revealed that these tamarillo PVY isolates represent at least three novel recombinant types not reported before. The combination of the biological and molecular properties found in these eight PVY isolates suggested the existence of a new tamarillo strain of PVY that may have coevolved with S. betaceum.

A New Tymovirus Isolated From Solanum quitoense: Characterization and Prevalence in Two Solanaceous Crops in Ecuador.

Naranjilla (Solanum quitoense Lam.) and tamarillo (S. betaceum Cav.) are two important perennial solanaceous crops grown in Ecuador for the fresh market and juice production. Viruses infecting tamarillo and naranjilla are currently poorly studied, and no clean stock program exists in Ecuador. Here, we report a new virus, provisionally named as naranjilla mild mosaic virus (NarMMV) (genus Tymovirus, family Tymoviridae), isolated from naranjilla grown in an orchard in Pichincha Province, Ecuador. The complete genome of the virus consists of 6,348 nucleotides and encodes three open reading frames typical for members of the genus Tymovirus. Phylogenetically, Chiltepin yellow mosaic virus, Eggplant mosaic virus, and the recently characterized naranjilla chlorotic mosaic virus (NarCMV) were found to be the closest relatives of NarMMV. Unlike NarCMV, the new virus induced mild mosaic in naranjilla and more severe symptoms in tamarillo. Similar to NarCMV, NarMMV was unable to systemically infect potato. Virus surveys found NarMMV prevalent in naranjilla production areas of two provinces of Ecuador, especially where hybrid cultivars of naranjilla were cultivated. NarMMV was also found in field-grown tamarillo. The new virus cross-reacted with antibodies developed against NarCMV. Hence, this antibody will be useful for its field diagnosis using enzyme-linked immunosorbent assay or immunocapture reverse transcription polymerase chain reaction in future virus-free certification programs.

Characterization of a New Tymovirus Causing Stunting and Chlorotic Mosaic in Naranjilla (Solanum quitoense).

Naranjilla ("little orange"), also known as lulo (Solanum quitoense Lam.), is a perennial shrub species cultivated in the Andes for fresh fruit and juice production. In 2015, a naranjilla plant exhibiting stunting, mosaic, and chlorotic spots was sampled in the Pastaza province of Ecuador and maintained under greenhouse conditions. An infectious agent was mechanically transmitted to indicator plants and was subjected to biological and molecular characterization. Spherical particles approximately 30 nm in diameter, composed of a single 20-kDa capsid protein, were observed under an electron microscope in infected naranjilla plants. High-throughput sequencing conducted on inoculated Nicotiana benthamiana plants produced a single sequence contig sharing the closest relationship with several tymoviruses. The entire 6,245-nucleotide genome of a new tymovirus was amplified using reverse-transcription polymerase chain reaction and resequenced with the Sanger methodology. The genome had three open reading frames typical of tymoviruses, and displayed a whole-genome nucleotide identity level with the closest tymovirus, Eggplant mosaic virus, at 71% (90% coverage). This tymovirus from naranjilla was able to systemically infect eggplant, tamarillo, N. benthamiana, and naranjilla. In naranjilla, it produced mosaic, chlorotic spots, and stunting, similar to the symptoms observed in the original plant. The virus was unable to infect potato and tobacco and unable to systemically infect pepper plants, replicating only in inoculated leaves. We concluded that this virus represented a new tymovirus infecting naranjilla, and proposed the tentative name Naranjilla chlorotic mosaic virus (NarCMV).

Complete genome sequence of a variant of maize-associated totivirus from Ecuador.

The complete genomic sequence of a variant of the recently reported maize-associated totivirus (MATV) from China was obtained from commercial maize in Ecuador. The genome of MATV-Ec (Ecuador) (4,998 bp) is considerably longer than that of MATV-Ch (China) (3,956 bp), the main difference due to a ≈ 1-kb-long capsid-protein-encoding fragment that is completely absent from the Chinese genome. Sequence alignments between MATV-Ec and MATV-Ch showed an overall identity of 82% at the nucleotide level, whereas at the amino acid level, the viruses exhibited 95% and 94% identity for the putative capsid protein and the RNA-dependent RNA polymerase (RdRp), respectively. Phylogenetic analysis of the viral RdRp domain indicated that MATV-Ec and MATV-Ch share a common ancestor with other plant-associated totiviruses, with Panax notoginseng virus A as the closest relative. MATV-Ec was detected in 46% (n = 80) of maize plants tested in this study, but not in endophytic fungi isolated from plants positive for the virus.

Effect of Raspberry bushy dwarf virus, Raspberry leaf mottle virus, and Raspberry latent virus on Plant Growth and Fruit Crumbliness in 'Meeker' Red Raspberry.

Raspberry crumbly fruit in red raspberry (Rubus idaeus), widespread in the Pacific Northwest of the United States and British Columbia, Canada, is most commonly caused by a virus infection. Raspberry bushy dwarf virus (RBDV) has long been attributed as the causal agent of the disease. Recently, the identification of two additional viruses, Raspberry leaf mottle virus (RLMV) and Raspberry latent virus (RpLV), in northern Washington and British Columbia, suggested the existence of a possible new virus complex responsible for the increased severity of the disease. Virus testing of crumbly fruited plants from five fields in northern Washington revealed the presence of RLMV and RpLV, in addition to RBDV. Plants with less severe crumbly fruit symptoms had a much lower incidence of RLMV or RpLV. Field trials using replicated plots of 'Meeker' plants containing single and mixed infections of RBDV, RLMV, or RpLV, along with a virus-free control, were developed to determine the role of RLMV and RpLV in crumbly fruit. Field evaluations during establishment and two fruiting years revealed that plants infected with the three viruses or the combinations RBDV+RLMV and RBDV+RpLV had the greatest reduction in cane growth, or fruit firmness and fruit weight, respectively. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) showed that the titer of RBDV was increased ~400-fold when it occurred in mixed infections with RLMV compared to RBDV in single infections. In addition, a virus survey revealed that RLMV and RpLV are present at high incidence in northern Washington; whereas the incidence in southern Washington and Oregon, where crumbly fruit is not as serious a problem, was considerably lower.

Expanding the Plant Virome: Umbra-Like Viruses Use Host Proteins for Movement.

Before the very recent discovery of umbra-like viruses (ULVs), the signature defining feature of all plant RNA viruses was the encoding of specialized RNA-binding movement proteins (MPs) for transiting their RNA genomes through gated plasmodesmata to establish systemic infections. The vast majority of ULVs share umbravirus-like RNA-dependent RNA polymerases and 3'-terminal structures, but they differ by not encoding cell-to-cell and long-distance MPs and by not relying on a helper virus for trans-encapsidation and plant-to-plant transmission. The recent finding that two groups of ULVs do not necessarily encode MPs is expanding our understanding of the minimum requirements for modern plant RNA viruses. ULV CY1 from citrus uses host protein PHLOEM PROTEIN 2 (PP2) for systemic movement, and related ULVs encode a capsid protein, thereby providing an explanation for the lack of helper viruses present in many ULV-infected plants. ULVs thus resemble the first viruses that infected plants, which were likely deposited from feeding organisms and would have similarly required the use of host proteins such as PP2 to exit initially infected cells.

Analysis of a new negevirus-like sequence from Bemisia tabaci unveils a potential new taxon linking nelorpi- and centiviruses.

This study presents the complete genome sequence of a novel nege-like virus identified in whiteflies (Bemisia tabaci MEAM1), provisionally designated as whitefly negevirus 1 (WfNgV1). The virus possesses a single-stranded RNA genome comprising 11,848 nucleotides, organized into four open reading frames (ORFs). These ORFs encode the putative RNA-dependent-RNA-polymerase (RdRp, ORF 1), a glycoprotein (ORF 2), a structural protein with homology to those in the SP24 family, (ORF 3), and a protein of unknown function (ORF 4). Phylogenetic analysis focusing on RdRp and SP24 amino acid sequences revealed a close relationship between WfNgV1 and Bemisia tabaci negevirus 1, a negevirus sequence recently discovered in whiteflies from Israel. Both viruses form a clade sharing a most recent common ancestor with the proposed nelorpivirus and centivirus taxa. The putative glycoprotein from ORF 2 and SP24 (ORF 3) of WfNgV1 exhibit the characteristic topologies previously reported for negevirus counterparts. This marks the first reported negevirus-like sequence from whiteflies in the Americas.

Genetic characterization of Blueberry necrotic ring blotch virus, a novel RNA virus with unique genetic features.

A new disorder was observed on southern highbush blueberries in several south-eastern states in the USA. Symptoms included irregularly shaped circular spots or blotches with green centres on the upper and lower surfaces of leaves. Double-stranded RNA was extracted from symptomatic leaves suggesting the presence of virus(es) possibly involved in the disease. Sequencing revealed the presence of a novel RNA virus with a ~14 kb genome divided into four RNA segments. Sequence analyses showed that the virus, for which we propose the name Blueberry necrotic ring blotch virus (BNRBV), possesses protein domains conserved across RNA viruses in the alpha-virus-like supergroup. Phylogenetic inferences using different genes placed BNRBV in a clade that includes the Bromoviridae, the genus Cilevirus (CiLV) and the recently characterized Hibiscus green spot virus (HGSV). Despite the strong genetic relationships found among BNRBV, Cilevirus and HGSV, the genome of BNRBV contains three features that distinguish it significantly from its closest relatives: (i) the presence of two helicase domains with different evolutionary pathways, (ii) the existence of three conserved nucleotide stretches located at the 3' non-coding regions of each RNA segment and (iii) the conservation of terminal nucleotide motifs across each segment. Furthermore, CiLV and HGSV possess poly(A)-tailed bipartite and tripartite genomes, respectively, whereas BNRBV has a quadra-partite genome lacking a poly(A) tail. Based on these genetic features a new genus is proposed for the classification of BNRBV.

In Vitro-Based Production of Virus-Tested Babaco (Vasconcellea x heilbornii, syn. Carica pentagona) in Ecuador: An Integrated Approach to an Endangered Crop.

Babaco (Vasconcellea x heilbornii), a fruit-bearing vegetatively propagated crop native to Ecuador, is appreciated for its distinctive flavor and nutritional properties. The aim of this research was to determine a functional protocol for tissue culture propagation of virus-free babaco plants including in vitro establishment, multiplication, rooting, and acclimation. First, symptomless babaco plants from a single commercial nursery were analyzed for virus detection and cared for using different disinfection treatments in the greenhouse to reduce contamination during the in vitro establishing step, and three cytokinins, 6-(γ,γ-Dimethylallylamino) purine (2IP), 6-Benzylaminopurine (BAP), and Thidiazuron (TDZ), were used to determine the best hormone for multiplication. The best treatment for plant disinfection was the weekly application of copper sulfate at the greenhouse and a laboratory disinfection using ethanol (EtOH) (70%), Clorox (2%), and a solution of povidone iodine (2.5%), with an 80% survival during in vitro plant establishment. TDZ showed a better multiplication rate when compared with other hormones, and 70% of the rooted plants were successfully acclimated at the greenhouse. Generated plants were virus-free when tested against babaco mosaic virus (BabMV) and papaya ringspot virus (PRSV), two of the most important viruses that can affect babaco. An efficient protocol to produce virus-free babaco plants was elaborated with an integrated use of viral diagnostic tools to ensure the production of healthy start material to farmers.