Geographically distant isolates of the persistent southern tomato virus (STV) show very low genetic diversity in the putative coat protein gene.

Southern tomato virus (STV) from genus Amalgavirus (Family Amalgaviridae) is a persistent virus infecting tomato crops worldwide. Information on genetic diversity and evolutionary mechanisms for plant persistent viruses are very scarce in comparison with plant acute viruses. In this work, the putative coat protein gene of worldwide STV isolates was analyzed showing very low nucleotide diversity (< 0.0100). Phylogenetic analysis separated STV isolates into two clades, but no correlation was found between genetic and geographic distances. Also, no recombination events among STV isolates were detected. Comparison of synonymous and nonsynonymous substitutions indicated negative selection at the amino acid level.

Localization of southern tomato virus (STV) in tomato tissues.

Southern tomato virus (STV) is a dsRNA virus, which belongs to the newly formed Amalgavirus genus of the Amalgaviridae family. Currently there is no report regarding the presence of STV in tomato tissues. In this study, we performed in situ hybridization to examine the distribution of STV in host tissues. STV was found in the leaves, stems, seeds, shoot apexes and root tips of tomato and localized in the cortex tissue, vascular tissues, pith, seed coat, endosperm, cotyledon (including inner cotyledons and outer cotyledons), hypocotyls and radicles of infected tomato tissues. In addition, STV was detected in the apical part of the stems and roots for the first time. This indicates that STV is a systemic infectious virus.

Molecular characterization of three novel mycoviruses in the plant pathogenic fungus Exobasidium.

The plant pathogen Exobasidium gracile, which belongs to the basidiomycetous genus Exobasidium, can lead to swollen and thicker leaves of C. oleifera. To our knowledge, there have been no reports of mycoviruses infecting Exobasidium gracile. This study characterized three mycoviruses coinfecting the plant pathogen Exobasidium gracile strain Z-1. Based on phylogenetic and genomic analyses, E. gracile strain Z-1 was infected two putative Totiviruses designated Exobasidium gracile Totivirus 1 (EgTV1) and Exobasidium gracile Totivirus 2 (EgTV2) and a putative Zybavirus of the family Amalgaviridae defined Exobasidium gracile Zybavirus 1 (EgZV1). Similar to the genomic organization of other Totiviruses, the EgTV1 and EgTV2 genomes are composed of one dsRNA segment that exhibits two large ORFs encoding a CP (capsid protein) and an RdRp (RNA-dependent RNA polymerase), respectively. Moreover, EgTV1 and EgTV2 genomes with a candidate -1 slippery heptamer sequence were discovered between CP and RdRp, respectively. Similar to other Zybaviruses of the family Amalgaviridae, the EgZV1 genome is composed of one dsRNA segment that contains two large ORFs encoding an unknown protein and an RdRp. In addition, the EgZV1 genome has a candidate +1 slippery heptamer sequence between an unknown protein and RdRp.

Persistent Southern Tomato Virus (STV) Interacts with Cucumber Mosaic and/or Pepino Mosaic Virus in Mixed- Infections Modifying Plant Symptoms, Viral Titer and Small RNA Accumulation.

Southern tomato virus (STV) is a persistent virus that was, at the beginning, associated with some tomato fruit disorders. Subsequent studies showed that the virus did not induce apparent symptoms in single infections. Accordingly, the reported symptoms could be induced by the interaction of STV with other viruses, which frequently infect tomato. Here, we studied the effect of STV in co- and triple-infections with Cucumber mosaic virus (CMV) and Pepino mosaic virus (PepMV). Our results showed complex interactions among these viruses. Co-infections leaded to a synergism between STV and CMV or PepMV: STV increased CMV titer and plant symptoms at early infection stages, whereas PepMV only exacerbated the plant symptoms. CMV and PepMV co-infection showed an antagonistic interaction with a strong decrease of CMV titer and a modification of the plant symptoms with respect to the single infections. However, the presence of STV in a triple-infection abolished this antagonism, restoring the CMV titer and plant symptoms. The siRNAs analysis showed a total of 78 miRNAs, with 47 corresponding to novel miRNAs in tomato, which were expressed differentially in the plants that were infected with these viruses with respect to the control mock-inoculated plants. These miRNAs were involved in the regulation of important functions and their number and expression level varied, depending on the virus combination. The number of vsiRNAs in STV single-infected tomato plants was very small, but STV vsiRNAs increased with the presence of CMV and PepMV. Additionally, the rates of CMV and PepMV vsiRNAs varied depending on the virus combination. The frequencies of vsiRNAs in the viral genomes were not uniform, but they were not influenced by other viruses.

Amalga-like virus infecting Antonospora locustae, a microsporidian pathogen of grasshoppers, plus related viruses associated with other arthropods.

A previously reported Expressed Sequence Tag (EST) library from spores of microsporidian Antonospora locustae includes a number of clones with sequence similarities to plant amalgaviruses. Reexamining the sequence accessions from that library, we found additional such clones, contributing to a 3247-nt contig that approximates the length of an amalga-like virus genome. Using A. locustae spores stored from that previous study, and new ones obtained from the same source, we newly visualized the putative dsRNA genome of this virus and obtained amplicons yielding a 3387-nt complete genome sequence. Phylogenetic analyses suggested it as prototype strain of a new genus in family Amalgaviridae. The genome contains two partially overlapping long ORFs, with downstream ORF2 in the +1 frame relative to ORF1 and a proposed motif for +1 ribosomal frameshifting in the region of overlap. Subsequent database searches using the predicted fusion protein sequence of this new amalga-like virus identified related sequences in the transcriptome of a basal hexapod, the springtail species Tetrodontophora bielanensis. We speculate that this second new amalga-like virus (contig length, 3475 nt) likely also derived from a microsporidian, or related organism, which was associated with the springtail specimens at the time of sampling for transcriptome analysis. Other findings of interest include evidence that the ORF1 translation products of these two new amalga-like viruses contain a central region of predicted α-helical coiled coil, as recently reported for plant amalgaviruses, and transcriptome-based evidence for another new amalga-like virus in the transcriptome of another basal hexapod, the two-pronged bristletail species Campodea augens.

Deep Sequencing Data and Infectivity Assays Indicate that Chickpea Chlorotic Dwarf Virus is the Etiological Agent of the "Hard Fruit Syndrome" of Watermelon.

Chickpea chlorotic dwarf virus (CpCDV), a polyphagous mastrevirus, family Geminiviridae, has been recently linked to the onset of the "hard fruit syndrome" of watermelon, first described in Tunisia, that makes fruits unmarketable due to the presence of white hard portions in the flesh, chlorotic mottling on the rind, and an unpleasant taste. To investigate the etiological agent of this disease, total RNA extracted from symptomatic watermelon fruits was subjected to small RNA sequencing through next generation sequencing (NGS) techniques. Data obtained showed the presence of CpCDV and two other viral species. However, following validation through polymerase chain reaction (PCR), CpCDV was the only viral species consistently detected in all samples. Watermelon seedlings were then challenged by an agroinfectious CpCDV clone; several plants proved to be CpCDV-infected, and were able to produce fruits. CpCDV infected and replicated in watermelon fruits and leaves, leading to abnormality in fruits and in seed production, similar to those described in field. These results indicate that CpCDV is the etiological agent of the "hard fruit syndrome" of watermelon.

Nucleotide sequence of Zygosaccharomyces bailii virus Z: Evidence for +1 programmed ribosomal frameshifting and for assignment to family Amalgaviridae.

Zygosaccharomyces bailii virus Z (ZbV-Z) is a monosegmented dsRNA virus that infects the yeast Zygosaccharomyces bailii and remains unclassified to date despite its discovery >20years ago. The previously reported nucleotide sequence of ZbV-Z (GenBank AF224490) encompasses two nonoverlapping long ORFs: upstream ORF1 encoding the putative coat protein and downstream ORF2 encoding the RNA-dependent RNA polymerase (RdRp). The lack of overlap between these ORFs raises the question of how the downstream ORF is translated. After examining the previous sequence of ZbV-Z, we predicted that it contains at least one sequencing error to explain the nonoverlapping ORFs, and hence we redetermined the nucleotide sequence of ZbV-Z, derived from the same isolate of Z. bailii as previously studied, to address this prediction. The key finding from our new sequence, which includes several insertions, deletions, and substitutions relative to the previous one, is that ORF2 in fact overlaps ORF1 in the +1 frame. Moreover, a proposed sequence motif for +1 programmed ribosomal frameshifting, previously noted in influenza A viruses, plant amalgaviruses, and others, is also present in the newly identified ORF1-ORF2 overlap region of ZbV-Z. Phylogenetic analyses provided evidence that ZbV-Z represents a distinct taxon most closely related to plant amalgaviruses (genus Amalgavirus, family Amalgaviridae). We conclude that ZbV-Z is the prototype of a new species, which we propose to assign as type species of a new genus of monosegmented dsRNA mycoviruses in family Amalgaviridae. Comparisons involving other unclassified mycoviruses with RdRps apparently related to those of plant amalgaviruses, and having either mono- or bisegmented dsRNA genomes, are also discussed.

A +1 ribosomal frameshifting motif prevalent among plant amalgaviruses.

Sequence accessions attributable to novel plant amalgaviruses have been found in the Transcriptome Shotgun Assembly database. Sixteen accessions, derived from 12 different plant species, appear to encompass the complete protein-coding regions of the proposed amalgaviruses, which would substantially expand the size of genus Amalgavirus from 4 current species. Other findings include evidence for UUU_CGN as a +1 ribosomal frameshifting motif prevalent among plant amalgaviruses; for a variant version of this motif found thus far in only two amalgaviruses from solanaceous plants; for a region of α-helical coiled coil propensity conserved in a central region of the ORF1 translation product of plant amalgaviruses; and for conserved sequences in a C-terminal region of the ORF2 translation product (RNA-dependent RNA polymerase) of plant amalgaviruses, seemingly beyond the region of conserved polymerase motifs. These results additionally illustrate the value of mining the TSA database and others for novel viral sequences for comparative analyses.