Complete genome sequence of vaccinium-associated virus C, a new member of the family Totiviridae from Vaccinium floribundum.

Blueberries (Vaccinium sp.) are a major crop grown in the Pacific Northwest region. Currently, there are at least 17 known viruses that infect blueberry plants, and some of them cause a wide range of symptoms and economic losses. A new virus, vaccinium-associated virus C (VaVC) (family Totiviridae, genus Totivirus) was identified in an imported blueberry accession from the USDA-ARS National Clonal Germplasm Repository in Corvallis, Oregon. The complete genomic sequence of VaVC was determined, but the biological significance of VaVC is unknown and requires further study. Additional Vaccinium sp. accessions should be screened to investigate the incidence of this new virus.

Complete genome sequence of a new mitovirus associated with walking iris (Trimezia northiana).

The complete genome sequence of a new member of the family Mitoviridae was obtained from walking iris (Trimezia northiana (Schneev.) Ravenna by high-throughput sequencing. This is the first putative mitovirus identified in a monocotyledonous plant. The new mitovirus was tentatively named "walking iris virus 1" (WIV1). The complete genome of WIV1 is 2,858 nt in length with a single ORF encoding a viral replicase (RdRp). The highest level of amino acid sequence identity was 45% to Beta vulgaris mitovirus 1. In the viral replicase, a conserved protein domain for mitovirus RNA-dependent RNA polymerase and six highly conserved motifs were detected, consistent with other members of the family Mitoviridae. Phylogenetic inferences placed WIV1 among members of the genus Duamitovirus (family Mitoviridae) in a monophyletic clade with other plant mitoviruses. Sequence comparison and phylogenetic analysis support the classification of WIV1 as a new member of the genus Duamitovirus (family Mitoviridae).

Evidence that an Unnamed Isometric Virus Associated with Potato Rugose Disease in Peru Is a New Species of Genus Torradovirus.

A previously uncharacterized torradovirus species infecting potatoes was detected by high-throughput sequencing from field samples from Peru and in customs intercepts in potato tubers that originated from South America in the United States of America and the Netherlands. This new potato torradovirus showed high nucleotide sequence identity to an unidentified isometric virus (SB26/29), which was associated with a disease named potato rugose stunting in southern Peru characterized over two decades ago. Thus, this virus is tentatively named potato rugose stunting virus (PotRSV). The genome of PotRSV isolates sequenced in this study were composed of two polyadenylated RNA segments. RNA1 ranges from 7,086 to 7,089 nt and RNA2 from 5,228 to 5,230 nt. RNA1 encodes a polyprotein containing the replication block (helicase-protease-polymerase), whereas RNA2 encodes a polyprotein cleaved into a movement protein and the three capsid proteins (CPs). Pairwise comparison among PotRSV isolates revealed amino acid identity values greater than 86% in the protease-polymerase (Pro-Pol) region and greater than 82% for the combined CPs. The closest torradovirus species, squash chlorotic leaf spot virus, shares amino acid identities of ∼58 and ∼41% in the Pro-Pol and the combined CPs, respectively. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

First report of tobacco rattle virus infecting Weigela florida in the USA.

Weigela (Weigela florida (Bunge) A. DC., Family: Caprifoliaceae) are woody shrubs native to North China, Korea, and Japan. In the U.S., weigela are commonly used as landscape ornamental plants (McNamara et al. 2010). Two viruses have been reported in weigela: tomato spotted wilt orthotospovirus and impatiens necrotic spot orthotospovirus (Sastry et al. 2019). Ten weigela plants, originating from commercial nurseries in Minnesota, exhibiting chlorosis, chlorotic line patterns, and necrosis (e-Xtra) were submitted for virus diagnostics as potted plants at the University of Minnesota Plant Disease Clinic and the Virology Lab in 2019 and 2020 (five plants each year). Under greenhouse conditions, symptoms progressed from chlorosis to necrosis and even plant death in two of the five plants in 2019. Electron microscopy revealed rod-shaped particles of ≈20 nm in diameter and lengths between 40-200 nm with similar morphology to members of the genus Tobravirus (e-Xtra). Virus-like particles were enriched by ultracentrifugation and total nucleic acids were extracted from partial purifications using a phenol:chloroform extraction method (Lockhart et al. 1997). Tobacco rattle virus (TRV) was identified by cloning and sequencing of the 463bp amplicon obtained with the TRV detection primers described in Robinson, 1992. High-throughput sequencing (HTS) was done to confirm the TRV detection. A cDNA library was prepared from purified viral RNA using the TruSeq Stranded mRNA kit and sequenced on Illumina NovaSeq 6000 platform as 150 bp-paired end reads. A total of 44,316,446 raw data reads were obtained, preprocessed using the BBDuk plugin, and de novo assembled using SPAdes assembler. Viral contigs were identified using the NCBI BLASTX tool. The assembly of TRV RNA1 was 6,842 nt with 20,627,348 reads (47% of total reads) mapped to it and an average coverage per nucleotide at 323,639X. The assembly of TRV RNA2 was 3,033 nt with 22,769,253 reads (52% of total reads) mapped to it and an average coverage per nucleotide at 798,660X. NCBI GenBank accession numbers for the assemblies representing RNA1 and RNA2 are OQ408335 and OQ408336, respectively. NCBI BLASTn analysis showed the highest level of nucleotide identity to TRV genomic RNA segments 1 and 2, with 97% and 99% identity to the TRV isolate RNA1 (GQ903771) and RNA2 (GQ903772), respectively, that originated from Michigan potato. No other viral contigs were detected from the virion nucleic acid extraction by HTS, however this enrichment method doesn't exclude other viruses. In addition to using the detection primers by Robinson 1992, we designed primers based on our HTS data: TRV-WG-DetF3 5'- GACGAAGGAGGCTGTCATTGC-3' and TRV-WG-DetR3 5'-CGGACTATCGTGATGCCCATGC- 3'. RT-PCR amplicons from each of the 10 symptomatic plants were cloned and sequenced. Among these clones, Sanger sequence identities ranged between 96-100% compared to the HTS data and 98-99% to the TRV potato isolate from MI. To our knowledge, this is the first report of TRV infecting the ornamental host W. florida worldwide. TRV is a nematode-transmitted viral pathogen of economic importance, most notably in potatoes (Sastry et al. 2019). In the US, TRV has been reported on several landscape ornamentals, horticultural crops, and native habitats. Further research is needed to investigate the impact of TRV on the ornamental industry and the role of ornamentals as reservoirs for cultivated crops like potatoes.

The Virome of Babaco (Vasconcellea × heilbornii) Expands to Include New Members of the Rhabdoviridae and Bromoviridae.

Babaco (Vasconcellea × heilbornii) is a subtropical species in the Caricaceae family. The plant is native to Ecuador and represents an important crop for hundreds of families. The objective of this study was to characterize, at the genomic level, two new babaco viruses identified by high-throughput sequencing. The viruses, an ilarvirus and a nucleorhabdovirus, were found in a symptomatic babaco plant from a commercial nursery in the Azuay province of Ecuador. The tripartite genome of the new ilarvirus, provisionally named babaco ilarvirus 1 (BabIV-1), is related to subgroup 3 ilarviruses, including apple mosaic virus, apple necrotic mosaic virus, and prunus necrotic ringspot virus as the closest relatives. The genome of the nucleorhabdovirus, provisionally named babaco nucleorhabdovirus 1 (BabRV-1), showed the closest relation with joa yellow blotch-associated virus and potato yellow dwarf nucleorhabdovirus. Molecular-based detection methods found BabIV-1 and BabRV-1 in 21% and 36%, respectively, of plants surveyed in a commercial babaco nursery, highlighting the importance of enforcing virus testing and nursery certification programs for babaco.

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.

Complete genome sequence of a Moroccan isolate of cereal chlorotic mottle virus.

Cereal chlorotic mottle virus (CCMoV) is a cicadellid-transmitted plant rhabdovirus associated with chlorotic and necrotic streaks on several gramineous hosts and weeds. The virus was initially described in 1979 in Australia, but its genome has never been sequenced. In this study, the complete genome sequence of a Moroccan isolate of CCMoV was generated by high-throughput sequencing from infected oat leaves (Avena sativa). The genome is 13,800 nt long, containing seven open reading frames (ORFs) arranged in the canonical organization of rhabdoviruses: 3'-nucleocapsid (N), phosphoprotein (P), unknown protein (p3), unknown protein (p4), matrix (M), glycoprotein (G), viral polymerase (L)-5'. Pairwise analysis showed that maize fine streak virus (MFSV, genus Gammanucleorhabdovirus) was the closest relative. The amino acid identity values between homologous proteins from CCMoV and MFSV are as follows: 59.27% (N), 36.7% (P), 24% (P3), 62% (P4), 43.70% (M), 49.15% (G), 60.93% (L). Based on its phylogenetic relationship and analogous genome architecture, CCMoV should be assigned as member of the genus Gammanucleorhabdovirus. The low sequence similarity observed between CCMoV and MFSV suggests that CCMoV is a member of a distinct virus species.

Genomic characterization of a new torradovirus from common fleabane (Erigeron annuus).

A new virus was detected in common fleabane (Erigeron annuus) showing virus-like symptoms including leaf yellowing, mosaic, and mottling. This virus is tentatively named "fleabane yellow mosaic virus" (FbYMV). The complete genome sequence consists of two RNA segments of 7,133 nt (RNA 1) and 4,810 nt (RNA 2), excluding the poly(A) tract. Sequence analysis showed a genome organization comparable to that of members of the genus Torradovirus. The level of sequence identity between FbYMV and known members of the genus Torradovirus was below the cutoff established by the ICTV for species demarcation. Therefore, FbYMV should be classified as a new member of the genus Torradovirus.

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.

Genome characterization and complete sequence of a new badnavirus from Pandanus amaryllifolius.

A new badnavirus was sequenced from fragrant pandan grass (Pandanus amaryllifolius) displaying mosaic and chlorosis on the leaves. The complete genome sequence was determined by high-throughput sequencing. The new badnavirus was tentatively named "pandanus mosaic associated virus" (PMaV). Similar to those of other members of the genus Badnavirus, the genome of PMaV consists of a circular DNA molecule of 7,481 bp with three open reading frames (ORF) potentially coding for three proteins. ORF3 encodes a polyprotein with conserved protein domains including zinc finger, trimeric dUTPase, aspartic protease, reverse transcriptase (RT), and RNase H domains. Pairwise comparisons of the highly conserved RT + RNase H region revealed the highest nucleotide (nt) sequence identity (70.71%) to taro bacilliform CH virus-Et17 (MG017324). In addition to PMaV, viral sequences corresponding to orchid fleck dichorhavirus (OFV) were detected in the same plant sample. The complete sequence of the OFV coding region shared >98% nt sequence identity with other isolates of OFV available in the GenBank database. Disease symptoms could not be attributed exclusively to PMaV or OFV, as both viruses were present in the pandan grass exhibiting mosaic and chlorosis.

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.

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).

Characterization of a not so new potexvirus from babaco (Vasconcellea x heilbornii).

A new member of the genus Potexvirus was fully sequenced and characterized. The virus was isolated from babaco (Vasconcellea x heilbornii), a natural hybrid native to Ecuador. The virus contains a 6,692 nt long genome organized in five open reading frames in an arrangement typical of other potexviruses. Sequence comparisons revealed close relatedness with Papaya mosaic virus (PapMV), Alternathera mosaic virus (AltMV) and Senna mosaic virus (SenMV), exhibiting nucleotide identities up to 67% for the polymerase (Pol) and 68% for the coat protein (CP), with deduced amino acid identities of 70% and 72% for the Pol and CP, respectively. The presence of an AlkB domain, in the polymerase region, was observed. Terminal nucleotide sequences were conserved across potexviruses with characteristic motifs and predicted secondary structures at the 3' UTR. Although serologically undistinguishable from PapMV and AltMV, the new virus showed differences in host range and symptom induction. The name babaco mosaic virus is proposed for this newly characterized Potexvirus. The complete genome sequence of the new virus has been deposited in NCBI GenBank under accession number MF978248.

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.