Viromes in Xylariaceae fungi infecting avocado in Spain.

Four isolates of Entoleuca sp., family Xylariaceae, Ascomycota, recovered from avocado rhizosphere in Spain were analyzed for mycoviruses presence. For that, the dsRNAs from the mycelia were extracted and subjected to metagenomics analysis that revealed the presence of eleven viruses putatively belonging to families Partitiviridae, Hypoviridae, Megabirnaviridae, and orders Tymovirales and Bunyavirales, in addition to one ourmia-like virus plus other two unclassified virus species. Moreover, a sequence with 98% nucleotide identity to plant endornavirus Phaseolus vulgaris alphaendornavirus 1 has been identified in the Entoleuca sp. isolates. Concerning the virome composition, the four isolates only differed in the presence of the bunyavirus and the ourmia-like virus, while all other viruses showed common patterns. Specific primers allowed the detection by RT-PCR of these viruses in a collection of Entoleuca sp. and Rosellinia necatrix isolates obtained from roots of avocado trees. Results indicate that intra- and interspecies horizontal virus transmission occur frequently in this pathosystem.

The complete nucleotide sequence and genome organization of a novel virus of the order Tymovirales isolated from Prunus davidiana (Carr.) Franch. in Liaoning, China.

In September 2017, a yellow spot leaf disease was noted on the leaves of Prunus davidiana (Carr.) Franch. plants in Liaoning, China, and spherical virions (approx. 30 nm in diameter) were later observed in preparations of symptomatic leaves. Subsequent deep sequencing of small RNA revealed the presence of a virus in these symptomatic leaves The complete genome of this viral isolate consists of 6,072 nucleotides, excluding the poly(A) tail. The virus showed the closest genetic relationship to grapevine-associated tymo-like virus, reported in Colmar, France (GaTLV, MH383239), which is the sole member of the newly proposed genus "Gratylivirus" within the order Tymovirales, which is currently unassigned to a particular family. The virus clustered closely with GaTLV in a phylogenetic tree constructed based on complete genomic sequences. On the basis of the nucleotide and amino acid sequences of the replicase and coat protein genes, this virus shares the highest (although still relatively low) sequence similarity with those of GaTLV (41.6%-60.8% identity), indicating that the virus is a distinct member of the order Tymovirales, for which the name "prunus yellow spot-associated virus" (PYSaV) is proposed. To our knowledge, this is the first report of a virus naturally infecting P. davidiana.

Discovery of a novel Tymoviridae-like virus in mosquitoes from Mexico.

A novel Tymoviridae-like virus, designated Ek Balam virus, was isolated from male Culex quinquefasciatus mosquitoes collected in Yucatan, Mexico. The genome was fully sequenced and shown to have no more than 69% nt sequence identity to its closest known relative. Mosquito cells were permissive to Ek Balam virus replication, but mammalian and avian cells were refractory, suggesting that vertebrates are not involved in the maintenance of the virus in nature.

Characterization of alfalfa virus F, a new member of the genus Marafivirus.

Viral infections of alfalfa are widespread in major cultivation areas and their impact on alfalfa production may be underestimated. A new viral species, provisionally named alfalfa virus F (AVF), was identified using a virion-associated nucleic acid (VANA) metagenomics-based approach in alfalfa (Medicago sativa L.) samples collected in Southern France. The nucleotide sequence of the viral genome was determined by de-novo assembly of VANA reads and by 5'/3' RACE with viral RNA extracted from enriched viral particles or with total RNA, respectively. The virus shares the greatest degree of overall sequence identity (~78%) with Medicago sativa marafivirus 1 (MsMV1) recently deduced from alfalfa transcriptomic data. The tentative nucleotide sequence of the AVF coat protein shares ~83% identity with the corresponding region of MsMV1. A sequence search of the predicted single large ORF encoding a polyprotein of 235kDa in the Pfam database resulted in identification of five domains, characteristic of the genus Marafivirus, family Tymoviridae. The AVF genome also contains a conserved "marafibox", a 16-nt consensus sequence present in all known marafiviruses. Phylogenetic analysis of the complete nucleotide sequences of AVF and other viruses of the family Tymoviridae grouped AVF in the same cluster with MsMV1. In addition to 5' and 3' terminal extensions, the identity of the virus was confirmed by RT-PCRs with primers derived from VANA-contigs, transmission electron microscopy with virus-infected tissues and transient expression of the viral coat protein gene using a heterologous virus-based vector. Based on the criteria demarcating species in the genus Marafivirus that include overall sequence identity less than 80% and coat protein identity less than 90%, we propose that AVF represents a distinct viral species in the genus Marafivirus, family Tymoviridae.

Complete nucleotide sequence and genome organization of peach virus D, a putative new member of the genus Marafivirus.

The complete nucleotide sequence of peach virus D (PeVD) from Prunus persica was determined. The PeVD genome consists of 6,612 nucleotides excluding the 3' poly(A) tail and contains a single open reading frame coding for a polyprotein of 227 kDa. Sequence comparisons and phylogenetic analysis revealed that PeVD is most closely related to viruses in the genus Marafivirus, family Tymoviridae. The complete nucleotide and CP amino acid sequences of PeVD were most similar (51.1-57.8% and 32.2-48.0%, respectively) to members of the genus Marafivirus, suggesting that PeVD is a new member of this genus.

Complete nucleotide sequence of a novel strain of fig fleck-associated virus from China.

The complete nucleotide sequence of fig fleck-associated virus from Xinjiang Uygur Autonomous Region of China (FFkaV-CN) was determined. The 6,723-nucleotide-long viral genome, excluding a terminal poly(A) tail, contains three open reading frames (ORFs). Pairwise comparisons showed that FFkaV-CN shares 83% and 92% sequence identity with FFkaV-Italy based on the complete genomic sequence and CP aa sequence, respectively, slightly higher than the species demarcation criterion for the genus Maculavirus. Phylogenetic analysis showed that FFkaV-CN and FFkaV-Italy clustered into one group. These results indicate that FFkaV-CN is a novel strain of FFkaV with a genome organization somewhat different from what was reported for FFkaV-Italy.

Identification of a novel mycovirus isolated from Rhizoctonia solani (AG 2-2 IV) provides further information about genome plasticity within the order Tymovirales.

The complete genome of a novel mycovirus, named Rhizoctonia solani flexivirus 1 (RsFV-1), which infects an avirulent strain of Rhizoctonia solani AG 2-2 IV, was sequenced and analyzed. Its RNA genome consists of 10,621 nucleotides, excluding the poly-A tail, and encodes a single protein of 3477 amino acids. The identification of conserved motifs of methyltransferase, helicase and RNA-dependent RNA polymerase revealed its relatedness to members of the alphavirus-like superfamily of positive-strand RNA viruses. Phylogenetic analysis of these fused domains suggested that this virus should be assigned to the order Tymovirales. The recently described Fusarium graminearum deltaflexivirus 1 was found to be its closest relative. However, the whole genome, as well as the encoded protein of RsFV-1, is larger than that of other known members of the order Tymovirales, and unlike all other viruses belonging to this order, its methyltransferase domain is not located at the N-terminus of the replicase. Although genome diversity, as a result of recombination and gene loss, is a well-documented trait in members of the order Tymovirales, no related virus with a comparable genome alteration has been reported before. For these reasons, RsFV-1 broadens our perception about genome plasticity and diversity within the order Tymovirales.

The complete nucleotide sequence and genomic characterization of grapevine asteroid mosaic associated virus.

In analyzing grapevine clones infected with grapevine red blotch associated virus, we identified a small number of isometric particles of approximately 30nm in diameter from an enriched fraction of leaf extract. A dominant protein of 25kDa was isolated from this fraction using SDS-PAGE and was identified by mass spectrometry as belonging to grapevine asteroid mosaic associated virus (GAMaV). Using a combination of three methods RNA-Seq, sRNA-Seq, and Sanger sequencing of RT- and RACE-PCR products, we obtained a full-length genome sequence consisting of 6719 nucleotides without the poly(A) tail. The virus possesses all of the typical conserved functional domains concordant with the genus Marafivirus and lies evolutionarily between citrus sudden death associated virus and oat blue dwarf virus. A large shift in RNA-Seq coverage coincided with the predicted location of the subgenomic RNA involved in coat protein (CP) expression. Genus wide sequence alignments confirmed the cleavage motif LxG(G/A) to be dominant between the helicase and RNA dependent RNA polymerase (RdRp), and the RdRp and CP domains. A putative overlapping protein (OP) ORF lacking a canonical translational start codon was identified with a reading frame context more consistent with the putative OPs of tymoviruses and fig fleck associated virus than with those of marafiviruses. BLAST analysis of the predicted GAMaV OP showed a unique relatedness to the OPs of members of the genus Tymovirus.

Genetic Structure and Molecular Variability Analysis of Citrus sudden death-associated virus Isolates from Infected Plants Grown in Brazil.

Citrus sudden death-associated virus (CSDaV) is a monopartite positive-sense single-stranded RNA virus that was suggested to be associated with citrus sudden death (CSD) disease in Brazil. Here, we report the first study of the genetic structure and molecular variability among 31 CSDaV isolates collected from both symptomatic and asymptomatic trees in CSD-affected areas. Analyses of partial nucleotide sequences of five domains of the CSDaV genomic RNA, including those encoding for the methyltransferase, the multi-domain region (MDR), the helicase, the RNA-dependent RNA polymerase and the coat protein, showed that the MDR coding region was the most diverse region assessed here, and a possible association between this region and virus adaption to different host or plant tissues is considered. Overall, the nucleotide diversity (π) was low for CSDaV isolates, but the phylogenetic analyses revealed the predominance of two main groups, one of which showed a higher association with CSD-symptomatic plants. Isolates obtained from CSD-symptomatic plants, compared to those obtained from asymptomatic plants, showed higher nucleotide diversity, nonsynonymous and synonymous substitution rates and number of amino acid changes on the coding regions located closer to the 5' end region of the genomic RNA. This work provides new insights into the genetic diversity of the CSDaV, giving support for further epidemiological studies.

Molecular characterization of a novel mycovirus of the family Tymoviridae isolated from the plant pathogenic fungus Fusarium graminearum.

We isolated a novel mycovirus, Fusarium graminearum mycotymovirus 1 (FgMTV1/SX64), which is related to members of the family Tymoviridae, from the plant pathogenic fungus F. graminearum strain SX64. The complete 7863 nucleotide sequence of FgMTV1/SX64, excluding the poly (A) tail, was determined. The genome of FgMTV1/SX64 is predicted to contain four open reading frames (ORFs). The largest ORF1 is 6723 nucleotides (nt) in length and encodes a putative polyprotein of 2242 amino acids (aa), which contains four conserved domains, a methyltransferase (Mtr), tymovirus endopeptidase (Pro), viral RNA helicase (Hel), and RNA-dependent RNA polymerase (RdRp), of the replication-associated proteins (RPs) of the positive-strand RNA viruses. ORFs 2-4 putatively encode three putative small hypothetical proteins, but their functions are still unknown. Sequence alignments and phylogenetic analyses based on the putative RP protein and the three conserved domains (Mtr, Hel and RdRp) showed that FgMTV1/SX64 is most closely related to, but distinctly branched from, the viruses from the family Tymoviridae. Although FgMTV1/SX64 infection caused mild or no effect on conidia production, biomass and virulence of its host F. graminearum strain SX64, its infection had significant effects on the growth rate, colony diameter and deoxynivalenol (DON) production. This is the first molecular characterization of a tymo-like mycovirus isolated from a plant pathogenic fungus. It is proposed that the mycovirus FgMTV1/SX64 is a representative member of new proposed lineage Mycotymovirus in the family Tymoviridae.

Detection and molecular characterisation of Grapevine Syrah virus-1 isolates from Central Europe.

Grapevine Syrah virus-1 (GSyV-1) was identified by small-RNA deep sequencing in Slovak grapevine co-infected by several other viruses. The RT-PCR assays developed in this work substantially improved the virus detection and allowed the identification of GSyV-1 in tested grapevine samples from Slovakia and the Czech Republic at an unexpectedly high rate (ca. 30 %). Subsequently, complete genome sequences of 3 GSyV-1 isolates (2 Slovak and 1 Czech) were determined by Sanger sequencing, showing a typical marafivirus genome organization. Analyses of complete genome sequences showed a higher intra-group diversity among these 3 central European GSyV-1 isolates (differences reaching 7.1 % at the nucleotide level) in comparison to 3 previously characterized North American isolates (only 1.2 % intra-group divergence). A substantially higher divergence among central European isolates and their clustering into two major phylogenetic groups was further confirmed by the partial genome analysis of additional 26 isolates. The CP-centered study did not support the geography-based clustering among central European and American isolates. Nevertheless, the sequence data of the highly variable 5'-proximal portion of the genome obtained for few additional isolates from Slovakia and Czech Republic showed the presence of both, "European-" and "north American-like", GSyV-1 isolates in the analyzed grapevine samples.

Genomic characterization of Ambrosia asymptomatic virus 1 and evidence of other Tymovirales members in the Oklahoma tallgrass prairie revealed by sequence analysis.

The Plant Virus Biodiversity and Ecology project was undertaken to better understand the nature of plant-viral interactions and the occurrence of non-pathogenic viruses. Plants from the Tallgrass Prairie Preserve (TPP), Osage County, Oklahoma, were surveyed from 2005 to 2008 for the presence of viruses, resulting in the detection, using a virus-like particle enrichment method, of the genome a novel virus, Ambrosia asymptomatic virus 1 (AAV1), from Ambrosia psilostachya DC (western ragweed). Here, we present the genomic organization and genetic variability of AAV1. The virus has a single-stranded RNA genome of about 7408 nt, which has six open reading frames (ORFs). Phylogenetic analysis of the replicase and coat protein ORFs of the virus indicates strongly that the virus should be placed in the genus Mandarivirus. No evidence of recombination was detected. We also report the detection in the TPP of two known viruses and seven other putative viruses, members of the order Tymovirales.

Unraveling complex viral infections in cassava (Manihot esculenta Crantz) from Colombia.

In the Americas, different disease symptoms have been reported in cassava including leaf mosaics, vein clearings, mottles, ring spots, leaf distortions and undeveloped and deformed storage roots. Some viruses have been identified and associated with these symptoms while others have been reported in symptomless plants or latent infections. We observed that reoviruses associated with severe root symptoms (RS) of Cassava Frogskin Disease (CFSD) are not associated with leaf symptoms (LS) observed in the cassava indicator plant 'Secundina'. Neither were these LS associated with the previously characterized Cassava common mosaic virus, Cassava virus X, Cassava vein mosaic virus or phytoplasma, suggesting the presence of additional pathogens. In order to explain LS observed in cassava we used a combination of biological, serological and molecular tests. Here, we report three newly described viruses belonging to the families Secoviridae, Alphaflexiviridae and Luteoviridae found in cassava plants showing severe RS associated with CFSD. All tested plants were infected by a mix of viruses that induced distinct LS in 'Secundina'. Out of the three newly described viruses, a member of family Secoviridae could experimentally induce LS in single infection. Our results confirm the common occurrence of complex viral infections in cassava field-collected since the 1980s.

Complete nucleotide sequences of two isolates of cherry green ring mottle virus from peach (Prunus persica) in China.

Two complete nucleotide sequences of cherry green ring mottle virus (CGRMV) isolated from peach in Hebei (Hs10) and Fujian (F9) Provinces, China, were determined. Five open reading frames (ORFs) were found in the genomes of both isolates. The F9 and Hs10 isolates shared 82.2 % and 83.4-94.4 % nucleotide sequence identity, respectively, with two CGRMV isolates from cherry. Analysis of the nucleotide and amino acid sequences from the five ORFs of both isolates showed that Hs10 shares the greatest sequence identity with P1A (GenBank AJ291761) from cherry. Phylogenetic analysis indicated that CGRMV isolates from peach and cherry are closely related to members of the genus Foveavirus.

Real-time multiplex RT-PCR for the simultaneous detection of the five main grapevine viruses.

A real-time multiplex RT-PCR has been developed for the simultaneous detection and identification of the major RNA viruses that infect grapevines (Grapevine fanleaf virus, Arabis mosaic virus, Grapevine leafroll-associated virus 1, Grapevine leafroll-associated virus 3 and Grapevine fleck virus). Serial dilutions of infected plant extracts were tested using the new method, and the results were compared with those obtained using a commercially available ELISA and real-time singleplex RT-PCR. The two real-time RT-PCR versions detected up to the same level of dilution and were at least 10,000 times more sensitive than the ELISA. In addition, 158 grapevine plants collected in a survey of the Protected Designation of Origin in Alicante, Spain were compared using the three methods. The results of the molecular methods were very similar, with only four discordant results, and both were able to detect many more infected plants than the ELISA. The high prevalence of Grapevine fleck virus, Grapevine leafroll-associated virus 3 and Grapevine fanleaf virus suggests that the main pathways of viral introduction are infected plant material that has escaped controls and/or uncontrolled traffic of propagating plant material. Real-time multiplex RT-PCR could be used to facilitate a better control of grapevine viruses.

Genomic characterization of a novel virus of the family Tymoviridae isolated from mosquitoes.

BACKGROUND: The family Tymoviridae comprises three plant virus genera, including Tymovirus, Marafivirus, and Maculavirus, which are found in most parts of the world and cause severe agricultural losses. We describe a putatively novel member of the family Tymoviridae, which is isolated from mosquitoes (Culex spp.), referred to as CuTLV. METHODS AND RESULTS: The CuTLV was isolated by cell culture, which replicates and causes cytopathic effects in Aedes albopictus C6/36 cells, but not in mammalian BHK-21 or Vero cells. The complete 6471 nucleotide sequence of CuTLV was determined. The genome of CuTLV is predicted to contain three open reading frames (ORFs). The largest ORF1 is 5307 nucleotides (nt) in length and encodes a putative polypeptide of 1769 amino acids (aa), which contains the conserved motifs for the methyltransferase (MTR), Tymovirus endopeptidase (PRO), helicase (HEL), and RNA-dependent RNA polymerase (RdRp) of the replication-associated proteins (RPs) of positive-stranded RNA viruses. In contrast, the ORF1 sequence does not contain the so-called "tymobox" or "marafibox", the conserved subgenomic RNA promoter present in all tymoviruses or marafiviruses, respectively. ORF2 and ORF3 putatively encode a 248-aa coat protein (CP) and a proline-rich 149-aa polypeptide. The whole genomic nucleotide identity of CuTLV with other members of family Tymoviridae ranged from 46.2% (ChiYMV) to 52.4% (GFkV). Phylogenetic analysis based on the putative RP and CP genes of CuTLV demonstrated that the virus is most closely related to viruses in the genus Maculavirus. CONCLUSIONS: The CuTLV is a novel virus related to members of the family Tymoviridae, with molecular characters that are distinct from those of tymoviruses, marafiviruses, and other maculaviruses or macula-like viruses. This is the first report of the isolation of a Tymoviridae-like virus from mosquitoes. Further investigations are required to clarify the origin, replication strategy, and the public health or agricultural importance of the CuTLV.

Complete sequence of Fig fleck-associated virus, a novel member of the family Tymoviridae.

The complete nucleotide sequence and the genome organization were determined of a novel virus, tentatively named Fig fleck-associated virus (FFkaV). The viral genome is a positive-sense, single-stranded RNA 7046 nucleotides in size excluding the 3'-terminal poly(A) tract, and comprising two open reading frames. ORF1 encodes a polypeptide of 2161 amino acids (p240), which contains the signatures of replication-associated proteins and the coat protein cistron (p24) at its 3' end. ORF2 codes for a 461 amino acid protein (p50) identified as a putative movement proteins (MP). In phylogenetic trees constructed with sequences of the putative polymerase and CP proteins FFkaV consistently groups with members of the genus Maculavirus, family Tymoviridae. However, the genome organization diverges from that of the two completely sequenced maculaviruses, Grapevine fleck virus (GFkV) and Bombix mori Macula-like virus (BmMLV), as it exhibits a structure resembling that of Maize rayado fino virus (MRFV), the type species of the genus Marafivirus and of Olive latent virus 3 (OLV-3), an unclassified virus in the family Tymoviridae. FFkaV was found in field-grown figs from six Mediterranean countries with an incidence ranging from 15% to 25%.

Presence of a polyA tail at the 3' end of maize rayado fino virus RNA.

Maize rayado fino virus (MRFV) is distinct from other marafiviruses in that its genome reportedly lacks a poly(A) tail at the 3' terminus. We now show that the MRFV genome is indeed polyadenylated.

Selective constraints, molecular recombination structure and phylogenetic reconstruction of isometric plant RNA viruses of the families Luteoviridae and Tymoviridae.

In an effort to enhance the knowledge on molecular evolution of currently the known members of the families Luteoviridae and Tymoviridae, in-depth molecular investigations in the entire genome of 147 accessions retrieved from the international databases, were carried out. Two algorithms (RECCO and RDP version 3.31ß) adapted to the mosaic structure of viruses were utilized. The recombination frequency along the sequences was dissected and demonstrated that the three virus genera of the family Luteoviridae comprise numerous members subjected to recombination. It has pointed out that the major viruses swapped a few but long genomic segments. In addition, in Barley yellow dwarf virus, heredity material might be exchanged between two different serotypes. Even more, putative recombination events occurred between two different genera. Based on Fisher's Exact Test of Neutrality, positive selection acting on protein expression was detected only in the poleroviruses Cereal yellow dwarf virus, Potato leafroll virus and Wheat yellow dwarf virus. In contrast, several components of the family Tymoviridae were highly recombinant. Genomic portion exchange arose in many positions consisting of short fragments. Furthermore, no positive selection was detected. The evolutionary history showed, in the Luteoviridae, that all screened isolates split into three clusters corresponding to the three virus genera forming this family. Moreover, in the serotype PAV of Barley yellow dwarf virus, two major clades corresponding to PAV-USA and PAV-China, were delineated. Similarly, in the Tymoviridae, all analyzed isolates fell into four groups corresponding to the three virus genera composing this family along with the unclassified Tymoviridae. Inferred phylogenies reshuffled the existing classification and showed that Wheat yellow dwarf virus-RPV was genetically closely related to Cereal yellow dwarf virus and the unclassified Tymoviridae Grapevine syrah virus-1 constituted an integral part of the genus Marafivirus.

Genetic heterogeneity found in the replicase gene of poinsettia mosaic virus isolates.

The complete nucleotide sequences of five isolates of poinsettia mosaic virus (PnMV) from Japan (JN, JO1, JO2, JO4, and JO5) were determined. These isolates contained a single large open reading frame in their genomes and shared 96.6-97.8% identity at the nucleotide level and 91.3-98.1% identity at the amino acid level with two previously reported European isolates. Interestingly, the JO isolates were found to possess eight common translational frameshift sites in the interdomain region between the methyltransferase and protease domains, resulting in considerable variation in the interdomain region compared to the other isolates. This suggests that PnMV might have evolved by creating variations in its genome by such translational frameshifts.