Amesuviridae: a new family of plant-infecting viruses in the phylum Cressdnaviricota, realm Monodnaviria.

The phylum Cressdnaviricota comprises viruses with single-stranded, circular DNA genomes that encode an HUH-type endonuclease (known as Rep). The phylum includes two classes, eight orders, and 11 families. Here, we report the creation of a twelfth family in the order Mulpavirales, class Arfiviricetes of the phylum Cressdnaviricota. The family Amesuviridae comprises viruses that infect plants and is divided into two genera: Temfrudevirus, including the species Temfrudevirus temperatum (with temperate fruit decay-associated virus as a member), and Yermavirus, including the species Yermavirus ilicis (with yerba mate-associated circular DNA virus as a member). Both viruses encode Rep proteins with HUH endonuclease and SH3 superfamily helicase domains. Phylogenetic analysis indicates that the replicative module of amesuviruses constitutes a well-supported monophyletic clade related to Rep proteins from viruses in the order Mulpavirales. Furthermore, both viruses encode a single capsid protein (CP) related to geminivirus CPs. Phylogenetic incongruence between the replicative and structural modules of amesuviruses suggests a chimeric origin resulting from remote recombination events between ancestral mulpavirales and geminivirids. The creation of the family Amesuviridae has been ratified by the International Committee on Taxonomy of Viruses (ICTV).

An Unwanted Association: The Threat to Papaya Crops by a Novel Potexvirus in Northwest Argentina.

An emerging virus isolated from papaya (Carica papaya) crops in northwestern (NW) Argentina was sequenced and characterized using next-generation sequencing. The resulting genome is 6667-nt long and encodes five open reading frames in an arrangement typical of other potexviruses. This virus appears to be a novel member within the genus Potexvirus. Blast analysis of RNA-dependent RNA polymerase (RdRp) and coat protein (CP) genes showed the highest amino acid sequence identity (67% and 71%, respectively) with pitaya virus X. Based on nucleotide sequence similarity and phylogenetic analysis, the name papaya virus X is proposed for this newly characterized potexvirus that was mechanically transmitted to papaya plants causing chlorotic patches and severe mosaic symptoms. Papaya virus X (PapVX) was found only in the NW region of Argentina. This prevalence could be associated with a recent emergence or adaptation of this virus to papaya in NW Argentina.

A Novel Lineage of Cile-Like Viruses Discloses the Phylogenetic Continuum Across the Family Kitaviridae.

An increasing number of plant species have been recognized or considered likely reservoirs of viruses transmitted by Brevipalpus mites. A tiny fraction of these viruses, primarily those causing severe economic burden to prominent crops, have been fully characterized. In this study, based on high-throughput sequencing, transmission electron microscopy analyses of virions in plant-infected tissues, viral transmission experiments, and the morphoanatomical identification of the involved Brevipalpus mites, we describe molecular and biological features of viruses representing three new tentative species of the family Kitaviridae. The genomes of Solanum violifolium ringspot virus (SvRSV, previously partially characterized), Ligustrum chlorotic spot virus (LigCSV), and Ligustrum leprosis virus (LigLV) have five open reading frames (ORFs) > 500 nts, two distributed in RNA1 and three in RNA2. RNA1 of these three viruses display the same genomic organization found in RNA1 of typical cileviruses, while their RNA2 are shorter, possessing only orthologs of genes p61, p32, and p24. LigCSV and LigLV are more closely related to each other than to SvRSV, but the identities between their genomic RNAs were lower than 70%. In gene-by-gene comparisons, ORFs from LigCSV and LigLV had the highest sequence identity values (nt sequences: 70-76% and deduced amino acid sequences: 74-83%). The next higher identity values were with ORFs from typical cileviruses, with values below 66%. Virions of LigLV (≈ 40 nm × 55 nm) and LigCSV (≈ 54 nm × 66 nm) appear almost spherical, contrasting with the bacilliform shape of SvRSV virions (≈ 47 nm × 101 nm). Mites collected from the virus-infected plants were identified as Brevipalpus papayensis, B. tucuman, and B. obovatus. Viruliferous B. papayensis mites successfully transmitted LigCSV to Arabidopsis thaliana. SvRSV, LigCSV, and LigLV seem to represent novel sub-lineages of kitaviruses that descent on parallel evolutionary branches from a common ancestor shared with the tentative cile-like virus hibiscus yellow blotch virus and typical cileviruses. Biological and molecular data, notably, the phylogenetic reconstruction based on the RdRp proteins in which strong support for monophyly of the family Kitaviridae is observed, mark an advance in the understanding of kitavirids.

Molecular characterization of a novel cytorhabdovirus with a unique genomic organization infecting yerba mate (Ilex paraguariensis) in Argentina.

The genome of a novel rhabdovirus was detected in yerba mate (Ilex paraguariensis St. Hil.). The newly identified virus, tentatively named "yerba mate virus A" (YmVA), has a genome of 14,961 nucleotides. Notably, eight open reading frames were identified in the antigenomic orientation of the negative-sense, single-stranded viral RNA, including two novel accessory genes, in the order 3'-N-P-3-4-M-G-L-8-5'. Sequence comparisons of the encoded proteins as well as phylogenetic analysis suggest that YmVA is a new member of the genus Cytorhabdovirus, family Rhabdoviridae. YmVA's unique genomic organization and phylogenetic relationships indicate that this virus likely represents a distinct evolutionary lineage among the cytorhabdoviruses.

Redefining the medicago sativa alphapartitiviruses genome sequences.

In alfalfa samples analyzed by hightroughput sequencing, four de novo assembled contigs encoding gene products showing identities to alphapartitiviruses proteins were found based on BlastX analysis. The predicted amino acid (aa) sequences of two contigs presented 99-100% identity to the RNA-dependent RNA polymerase (RdRp) and the capsid protein (CP) of the recently reported medicago sativa alphapartitivirus 1 (MsAPV1). In addition, the remaining two contigs shared only 56% (CP) and 70% (RdRp) pairwise aa identity with the proteins of MsAPV1, suggesting that these samples presented also a novel Alphapartitivirus species. Further analyses based on complete genome segments termini and the presence/absence of alphapartitivirus RNA in several samples and public alfalfa RNA datasets corroborated the identification of two different alphapartitivirus members. Our results likely indicate that the reported MsAPV1 genome was previously reconstructed with genome segments of two different alphapartitiviruses. Overall, we not only revisited the MsAPV1 genome sequence but also report a new tentative alphapartitivirus species, which we propose the name medicago sativa alphapartitivirus 2. In addition, the RT-PCR detection of both MsAPV1 and MsAPV2 in several alfalfa cultivars suggests a broad distribution of both viruses.

Identification and molecular characterization of a novel circular single-stranded DNA virus associated with yerba mate in Argentina.

A single-stranded DNA (ssDNA) virus was detected in Yerba mate samples showing chlorotic linear patterns, chlorotic rings and vein yellowing. The full-genome sequences of six different isolates of this ssDNA circular virus were obtained, which share > 99% sequence identity with each other. The newly identified virus has been tentatively named as yerba mate-associated circular DNA virus (YMaCV). The 2707 nt-long viral genome has two and three open reading frame on its complementary and virion-sense strands, respectively. The coat protein is more similar to that of mastreviruses (44% identity), whereas the replication-associated protein of YMaCV is more similar (49% identity) to that encoded by a recently described, unclassified ssDNA virus isolated on trees in Brazil. This is the first report of a circular DNA virus associated with yerba mate. Its unique genome organization and phylogenetic relationships indicates that YMaCV represents a distinct evolutionary lineage within the ssDNA viruses and therefore this virus should be classified as a member of a new species within an unassigned genus or family.

Molecular characterization of yerba mate chlorosis-associated virus, a putative cytorhabdovirus infecting yerba mate (Ilex paraguariensis).

We present the molecular characterization of a new virus infecting yerba mate (Ilex paraguariensis St. Hil.) in Argentina. Deep sequencing of diseased yerba mate plants showing chlorotic linear patterns, chlorotic rings, and vein yellowing resulted in the identification of a new virus resembling plant rhabdoviruses in sequence and genome structure. We have determined the complete genome sequence of this virus, which is 12,876 nt long. Seven open reading frames (ORFs) were identified in the antigenomic orientation of the negative-sense, single-stranded viral RNA, in the order 3'-N-P-P3-P4-M-G-L-5'. Phylogenetic analysis suggested that the described virus is a new member of the genus Cytorhabdovirus, which was supported by the observation of rhabdovirus-like particles within the cytoplasm of infected yerba mate cells. The virus has been tentatively named "yerba mate chlorosis-associated virus" (YmCaV). The availability of the YmCaV genome sequence will contribute to assessing the genetic variability of this virus and determining its role in this yerba mate disease.

Complete genome sequence and integrated protein localization and interaction map for alfalfa dwarf virus, which combines properties of both cytoplasmic and nuclear plant rhabdoviruses.

We have determined the full-length 14,491-nucleotide genome sequence of a new plant rhabdovirus, alfalfa dwarf virus (ADV). Seven open reading frames (ORFs) were identified in the antigenomic orientation of the negative-sense, single-stranded viral RNA, in the order 3'-N-P-P3-M-G-P6-L-5'. The ORFs are separated by conserved intergenic regions and the genome coding region is flanked by complementary 3' leader and 5' trailer sequences. Phylogenetic analysis of the nucleoprotein amino acid sequence indicated that this alfalfa-infecting rhabdovirus is related to viruses in the genus Cytorhabdovirus. When transiently expressed as GFP fusions in Nicotiana benthamiana leaves, most ADV proteins accumulated in the cell periphery, but unexpectedly P protein was localized exclusively in the nucleus. ADV P protein was shown to have a homotypic, and heterotypic nuclear interactions with N, P3 and M proteins by bimolecular fluorescence complementation. ADV appears unique in that it combines properties of both cytoplasmic and nuclear plant rhabdoviruses.

Detection and identification of a novel 16SrXIII subgroup phytoplasma associated with strawberry red leaf disease in Argentina.

Strawberry red leaf phytoplasma was found in strawberry plants from production fields in Lules (Tucumán province) and Bella Vista (Corrientes province), Argentina. Characteristic strawberry red leaf symptoms were stunting, young leaves with yellowing at the edges, mature leaves which curled and were reddish at the abaxial face, flower and fruit deformation and death. The pathogen was detected with phytoplasma-universal primer pairs P1/P7 followed by R16F2n/R16R2 as nested primers in 13 diseased plants. Based on RFLP and sequence analysis of the amplified 16S rRNA gene, the phytoplasma was related to the 16SrXIII group (Mexican periwinkle virescence). In silico the RFLP profile of all the samples analysed revealed the presence of a unique pattern, showing that the novel phytoplasma is different from all the phytoplasmas currently composing the 16SrXIII group. The phylogenetic analysis was consistent with RFLP analysis as the strawberry red leaf phytoplasma was grouped within the 16SrXIII group, but formed a particular cluster. On this basis, the Strawberry red leaf phytoplasma associated with strawberry red leaf disease was assigned to a new subgroup, 16SrXIII-F.

Differences in intracellular localization of corn stunt spiroplasmas in magnesium treated maize.

Maize plants infected with Spiroplasma kunkelii show symptoms similar to that of plants in a magnesium-deficient soil, and it has been shown that the spiroplasma alters the plants' magnesium absorption. In the current study we compared changes associated to either spiroplasma infection, two soil magnesium levels and their combinations. Plant symptoms were recorded and correlated with transmission electron microscopy observations. Plants grown on a high magnesium treatment showed no macroscopical alterations nor organelle ultrastructural alterations, while plants on a low magnesium treatment showed macroscopical vein yellowing and, ultrastructurally, they had most chloroplasts and mitochondrial membranes altered. Infected plants on a low magnesium treatment had an ageing aspect, ultrastructurally showed chloroplasts and mitochondrial alterations similar to those non-infected and grown on a low magnesium treatment, and spiroplasma cells were found in phloem cells, but outside their cytoplasm. Infected plants on a high magnesium treatment showed similar symptoms and ultrastructural alterations as either non-infected plants on the low magnesium treatment or in infected plants on the low magnesium treatment, but differ from them in that the spiroplasma cells were located inside the cytoplasm. Results suggest that magnesium is involved in the plant-pathogen interaction.

Differences in intracellular localization of corn stunt spiroplasmas in magnesium treated maize

Maize plants infected with Spiroplasma kunkelii show symptoms similar to that of plants in a magnesium-deficient soil, and it has been shown that the spiroplasma alters the plants magnesium absorption. In the current study we compared changes associated to either spiroplasma infection, two soil magnesium levels and their combinations. Plant symptoms were recorded and correlated with transmission electron microscopy observations. Plants grown on a high magnesium treatment showed no macroscopical alterations nor organelle ultrastructural alterations, while plants on a low magnesium treatment showed macroscopical vein yellowing and, ultrastructurally, they had most chloroplasts and mitochondrial membranes altered. Infected plants on a low magnesium treatment had an ageing aspect, ultrastructurally showed chloroplast s and mitochondrial alterations similar to those non-infected and grown on a low magnesium treatment, and spiroplasma cells were found in phloem cells, but outside their cytoplasm. Infected plants on a high magnesium treatment showed similar symptoms and ultrastructural alterations as either non-infected plants on the low magnesium treatment or in infected plants on the low magnesium treatment, but differ from them in that the spiroplasma cells were located inside the cytoplasm. Results suggest that magnesium is involved in the plant-pathogen interaction.(AU)

Differences in intracellular localization of corn stunt spiroplasmas in magnesium treated maize

Maize plants infected with Spiroplasma kunkelii show symptoms similar to that of plants in a magnesium-deficient soil, and it has been shown that the spiroplasma alters the plants' magnesium absorption. In the current study we compared changes associated to either spiroplasma infection, two soil magnesium levels and their combinations. Plant symptoms were recorded and correlated with transmission electron microscopy observations. Plants grown on a high magnesium treatment showed no macroscopical alterations nor organelle ultrastructural alterations, while plants on a low magnesium treatment showed macroscopical vein yellowing and, ultrastructurally, they had most chloroplasts and mitochondrial membranes altered. Infected plants on a low magnesium treatment had an ageing aspect, ultrastructurally showed chloroplast s and mitochondrial alterations similar to those non-infected and grown on a low magnesium treatment, and spiroplasma cells were found in phloem cells, but outside their cytoplasm. Infected plants on a high magnesium treatment showed similar symptoms and ultrastructural alterations as either non-infected plants on the low magnesium treatment or in infected plants on the low magnesium treatment, but differ from them in that the spiroplasma cells were located inside the cytoplasm. Results suggest that magnesium is involved in the plant-pathogen interaction.

Localization of sweet potato chlorotic stunt virus (SPCSV) in synergic infection with potyviruses in sweet potato.

Among diseases reported worldwidely for sweet potato (Ipomoea batatas (L) Lam) crop, one of the most frequent is the Sweet potato virus disease (SPVD), caused by sweet potato chlorotic stunt virus (SPCSV) and sweet potato feathery mottle virus (SPFMV) co-infection. In Argentina, there exists the sweet potato chlorotic dwarf (SPCD), a sweet potato disease caused by triple co-infection with SPCSV, SPFMV and sweet potato mild speckling virus (SPMSV). Both diseases cause a synergism between the potyviruses (SPFMV and SPMSV) and the crinivirus (SPCSV). Up to date, studies carried out on the interaction among these three viruses have not described their localization in the infected tissues. In single infections, virions of the crinivirus genus are limited to the phloem while potyviral virions are found in most tissues of the infected plant. The purpose of this work was to localize the heat shock protein 70 homolog (HSP70h), a movement protein for genus crinivirus, of an Argentinean SPCSV isolate in its single infection and in its double and triple co-infection with SPFMV and SPMSV. The localization was made by in situ hybridization (ISH) for electron microscopy (EM) on ultrathin sections of sweet potato cv. Morada INTA infected tissues. The results demonstrated that viral RNA coding HSP70h is restricted to phloem cells during crinivirus single infection, while it was detected outside the phloem in infections combined with the potyviruses involved in chlorotic dwarf disease.