Complete genome sequence of mimosa mosaic virus, a new sobemovirus infecting Mimosa sensitiva L.

A new sobemovirus, which we have named "mimosa mosaic virus" (MimMV), was found by high-throughput sequencing and isolated from a mimosa (Mimosa sensitiva L.) plant. The genome sequence was confirmed by Sanger sequencing and comprises 4595 nucleotides. Phylogenetic analysis based on the predicted amino acid (aa) sequences of the P2b protein (encoded by ORF2b) and the coat protein showed 52.7% and 31.8% aa sequence identity, respectively, to those of blueberry shoestring virus. The complete genome sequence of MimMV was less than 47% identical to those of other sobemoviruses. These data suggest that MimMV is a member of a new species in the genus Sobemovirus, for which the binomial name "Sobemovirus mimosae" is proposed.

A novel deltacryptic virus identified in Allium cepa from Brazil.

 This work describes a novel partitivirus genome assembled from RNA-seq data generated from onion tissue from fields in Brazil. A new partitivirus genome composed of three dsRNAs, which was closely related to arhar cryptic virus 1, was assembled from Allium cepa samples from Brazil. The genomic sequences were also identified from available transcriptomic datasets of onion samples from China, Czech Republic, India, South Korea and USA. According to the species demarcation in the Partitiviridae family, the new virus was classified into the genus Deltapartitivirus with the suggested name of allium deltapartitivirus. This is the first report of the occurrence of a cryptic virus in plants of the genus Allium, and therefore, this work contributes to the understanding of the genetic diversity of partitiviruses  that infect the genus Allium. Keywords: Allium sp.; high-throughput sequencing; partitiviruses.

Complete genome sequence of patchouli chlorosis-associated cytorhabdovirus, a new cytorhabdovirus infecting patchouli plants in Brazil.

A cytorhabdovirus, tentatively named "patchouli chlorosis-associated cytorhabdovirus" (PCaCV), was identified in a patchouli plant, using high-throughput sequencing, and its genome sequence was confirmed by Sanger sequencing. The PCaCV genome consists of 12,913 nucleotides and contains six open reading frames in the order 3'-N-P'-P-P3-M-(G)-L-5'. The glycoprotein gene was found to contain stop codons in the coding frame; hence, this gene is considered defective. PCaCV is most closely related to tomato yellow mottle-associated virus, sharing 61.1% nucleotide sequence identity in the complete genome and 73.9% amino acid sequence identity in the L protein. These data suggest that PCaCV should be considered a new member of the genus Cytorhabdovirus, and the binomial species name "Cytorhabdovirus patchoulii" is proposed.

Characterization of yam mosaic viruses from Brazil reveals a new phylogenetic group and possible incursion from the African continent.

Yam (Dioscorea spp.) is an important crop for smallholder farmers in the Northeast region of Brazil. Wherever yam is grown, diseases caused by yam mosaic virus (YMV) are prevalent. In the present study, the diversity of YMV infecting Dioscorea cayennensis-rotundata was analyzed. In addition, five species of Dioscorea (D. alata, D. altissima, D. bulbifera, D. subhastata, and D. trifida) commonly found in Brazil were analyzed using ELISA and high-throughput sequencing (HTS). YMV was detected only in D. cayennensis-rotundata, of which 66.7% of the samples tested positive in ELISA. Three YMV genome sequences were assembled from HTS and one by Sanger sequencing to group the sequences in a clade phylogenetically distinct from YMV from other origins. Temporal phylogenetic analyses estimated the mean evolutionary rate for the CP gene of YMV as 1.76 × 10-3 substitutions per site per year, and the time to the most recent common ancestor as 168.68 years (95% Highest Posterior Density, HPD: 48.56-363.28 years), with a most likely geographic origin in the African continent. The data presented in this study contribute to reveal key aspects of the probable epidemiological history of YMV in Brazil.

Transcriptomic analyses reveal highly conserved plant amalgavirus genomes in different species of Allium.

This work aims to study amalgavirus diversity in different species of allium collected around the world. Transcriptomic data of 19 Sequence Read Archive runs available at GenBank, as well as RNA-seq data generated from onion tissue from fields in Brazil were used to assemble nine allium cepa amalgavirus 1 (AcAV-1) and nine allium cepa amalgavirus 2 (AcAV-2) genomes from different species of allium worldwide. Sequence demarcation tool analyses of RdRp amino acid sequences revealed identities above 99% within each species, except for an isolate of AcAV-1 from Allium escalonicum from China. This work contributes to the understanding of the genetic diversity of amalgaviruses that infect the genus Allium. Keywords: amalgaviruses; Allium transcriptomic datasets; Allium sp.

Characterization of an infectious clone of pepper ringspot virus and its use as a viral vector.

The genus Tobravirus comprises three species: Tobacco rattle virus, Pea early-browning virus and Pepper ringspot virus. The genomes of tobraviruses consist of two positive-sense single-stranded RNA segments (RNA1 and RNA2). Infectious clones of TRV are extensively used as virus-induced gene-silencing (VIGS) vectors for studies of virus-host interactions and functions of plant genes. Complete infectious clones of pepper ringspot virus (PepRSV), the only tobravirus present in Brazil, however, have not yet been reported. Infectious clones will help to identify unique features of PepRSV RNA2 and provide another option for development of VIGS vectors. We constructed infectious clones based on two PepRSV isolates, CAM (RNA1 and RNA2) and LAV (RNA2). The cDNA constructs for both homologous (RNA1 and RNA2 of the CAM isolate) and heterologous (RNA1/CAM and RNA2/LAV) combinations were infectious in Nicotiana benthamiana plants. VIGS vector constructs with green fluorescent protein or phytoene desaturase genes inserted in RNA2 silenced the target genes. The systemic translocation of the PepRSV RNA1 construct alone (nonmultiple infection) was also confirmed in an N. benthamiana plant. These results are similar to those reported for tobacco rattle virus.

Arachis virus Y, a new potyvirid from Brazilian forage peanut (Arachis pintoi).

The complete nucleotide sequence of a new member of the family Potyviridae, which we propose to name "Arachis virus Y" (ArVY), is reported from forage peanut plants (Arachis pintoi) exhibiting virus-like symptoms. The ArVY positive-sense RNA genome is 9,213 nucleotides long and encodes a polyprotein with 2,947 amino acids that is predicted to be cleaved into 10 mature proteins. The complete single open reading frame (ORF) of ArVY shares 47% and 34% nucleotide and amino acid sequence identity, respectively, with the closest related virus, soybean yellow shoot virus. Electron microscopic analysis revealed elongated viral particles typical of those found in plant cells infected with potyviruses.

Complete genome sequence of a new bipartite begomovirus infecting tomato in Brazil.

A novel bipartite begomovirus infecting begomovirus-resistant tomato plants was detected via Illumina sequencing analysis, and its genome sequence was confirmed by Sanger sequencing. The DNA-A (2627 nt) and DNA-B (2587 nt) have a genome organization that is typical of New World bipartite begomoviruses, sharing 82.5% identity with tomato golden leaf distortion virus and 75.1% identity with sida chlorotic vein virus. Based on the current classification criteria for begomoviruses, this isolate should be considered a member of a new species, and the name "tomato interveinal chlorosis virus-2" (ToICV2) is proposed for this virus.

Cucurbit aphid-borne yellows virus from melon plants in Brazil is an interspecific recombinant.

Melon plants with severe yellowing symptoms from in Brazil were analyzed by high-throughput sequencing. Sequences homologous to the genome of the polerovirus cucurbit aphid-borne yellows virus (CABYV) were frequently retrieved. Two draft CABYV genomes were assembled from two pooled melon samples that contained an identical putative recombinant fragment in the 3' region with an unknown polerovirus. The complete genomes of these isolates revealed by Sanger sequencing share 96.8% nucleotide identity, while both sequences share 73.7% nucleotide identity with a CABYV-N isolate from France. A molecular-clock analysis suggested that CABYV was introduced into Brazil ~ 68 years ago.

Assembly of tomato blistering mosaic virus-like particles using a baculovirus expression vector system.

The expression of several structural proteins from a wide variety of viruses in heterologous cell culture systems results in the formation of virus-like particles (VLPs). These VLPs structurally resemble the wild-type virus particles and have been used to study viral assembly process and as antigens for diagnosis and/or vaccine development. Tomato blistering mosaic virus (ToBMV) is a tymovirus that has a 6.3-kb positive-sense ssRNA genome. We have employed the baculovirus expression vector system (BEVS) for the production of tymovirus-like particles (tVLPs) in insect cells. Two recombinant baculoviruses containing the ToBMV wild-type coat protein (CP) gene or a modified short amino-terminal deletion (Δ2-24CP) variant were constructed and used to infect insect cells. Both recombinant viruses were able to express ToBMV CP and Δ2-24CP from infected insect cells that self-assembled into tVLPs. Therefore, the N-terminal residues (2-24) of the native ToBMV CP were shown not to be essential for self-assembly of tVLPs. We also constructed a third recombinant baculovirus containing a small sequence coding for the major epitope of the chikungunya virus (CHIKV) envelope protein 2 (E2) replacing the native CP N-terminal 2-24 amino acids. This recombinant virus also produced tVLPs. In summary, ToBMV VLPs can be produced in a baculovirus/insect cell heterologous expression system, and the N-terminal residues 2-24 of the CP are not essential for this assembly, allowing its potential use as a protein carrier that facilitates antigen purification and might be used for diagnosis.

Human virome in nasopharynx and tracheal secretion samples.

BACKGROUND: In Brazil the implementation of the Sentinel Surveillance System of Influenza began in 2000. Central public health laboratories use reverse transcription-quantitative polymerase chain reaction (RT-qPCR) for diagnosis of respiratory viruses, but this protocol identifies only specific targets, resulted in inconclusive diagnosis for many samples. Thus, high-throughput sequencing (HTS) would be complementary method in the identification of pathogens in inconclusive samples for RT-qPCR or other specific detection protocols. OBJECTIVES: This study aimed to detect unidentified viruses using HTS approach in negative samples of nasopharynx/tracheal secretions by the standard RT-qPCR collected in the Federal District, Brazil. METHODS: Nucleic acids were extracted from samples collected in winter period of 2016 and subjected to HTS. The results were confirmed by the multiplex PR21 RT-qPCR, which identifies 21 respiratory pathogens. FINDINGS: The main viruses identified by HTS were of families Herpesviridae, Coronaviridae, Parvoviridae and Picornaviridae, with the emphasis on rhinoviruses. The presence of respiratory viruses in the samples was confirmed by the PR21 multiplex RT-qPCR. Coronavirus, enterovirus, bocavirus and rhinovirus were found by multiplex RT-qPCR as well as by HTS analyses. MAIN CONCLUSIONS: Wide virus diversity was found by different methodologies and high frequency of rhinovirus occurrence was confirmed in population in winter, showing its relevance for public health.

High-throughput sequencing reveals a novel closterovirus in arracacha (Arracacia xanthorrhiza).

High-throughput sequencing analysis detected a clostero-like virus from arracacha plants (Arracacia xanthorrhiza) in Brazil. The complete genome sequence, confirmed by RACE and Sanger sequencing, consists of 15,763 nucleotides with nine predicted open reading frames (ORFs) in a typical closterovirus genome organisation. The putative RNA-dependent RNA polymerase (RdRp), heat shock protein 70 homologue (Hsp70h), and coat protein showed 55-65, 38-44, and 20-36% amino acid sequence identity, respectively, to the homologous proteins of known closteroviruses. Phylogenetic analysis of Hsp70h showed that this putative novel arracacha plant virus was related to members of the genus Closterovirus in the family Closteroviridae. These results suggest that this virus, tentatively named "arracacha virus 1" (AV-1), is a novel member of the genus Closterovirus. This is the first closterovirus identified in arracacha plants.

A novel vitivirus-like sequence found in Arracacia xanthorrhiza plants by high throughput sequencing.

High throughput sequencing (HTS) is a very powerful tool for detecting and discovering novel viral-like sequences without prior knowledge of the sequence. Here we describe the complete genome of a new vitivirus-like sequence that was found in arracacha (Arracacia xanthorrhiza) plants using HTS technology. The complete genome sequence was validated by Sanger sequencing. The genomic organization of the new putative vitivirus resembles that of grapevine virus B (GVB) and grapevine virus D (GVD). The putative coat protein showed 41 to 49% identity with similar proteins of known vitiviruses, while the RNA-dependent RNA polymerase shared 52 to 55% identity with those encoded by grapevine vitiviruses. Based on the demarcation criteria for the genus Vitivirus, the virus described in this work, provisionally named as "Arracacha virus V", represents a novel species in this taxon.

Complete genome sequence of a putative new secovirus infecting yam (Dioscorea) plants.

The complete genome sequence of a new virus infecting yam plants exhibiting mosaic symptom in Brazil was determined. The genome of this virus is composed of two molecules of positive-sense RNAs of 5979 and 3809 nucleotides in length, excluding the poly(A) tails. One large open reading frame (ORF) in each genomic segment (RNA1-ORF1 and RNA2-ORF2) was predicted. The highest amino acid sequence similarity in the Pro-Pol core region of RNA1 and the CP region of RNA2 was observed with chocolate lily virus A (a putative member of the family Secoviridae), with 54.6 and 27.7 % identity, respectively. This virus is thus likely to be a new member of the family Secoviridae, and we propose the tentative name "dioscorea mosaic-associated virus" (DMaV) for this virus.

Complete genome sequence of melon yellowing-associated virus from melon plants with the severe yellowing disease in Brazil.

Here, we describe the complete genome sequence of melon yellowing-associated virus (MYaV), found in melon plants with severe yellowing disease, determined by high-throughput and Sanger sequencing. MYaV has an RNA genome of 9073 nucleotides plus a poly(A) tail. At least six open reading frames were predicted, with a typical carlavirus genomic organisation. Phylogenetic analysis of the complete genome sequence and the amino acid sequences of the RNA-dependent RNA polymerase confirmed that MYaV belongs to the genus Carlavirus, with the highest genome-wide nucleotide sequence identity of 59.8% to sweet potato yellow mottle virus.

Discovery and molecular characterization of a novel enamovirus, Grapevine enamovirus-1.

In this study, we describe a novel putative Enamovirus member, Grapevine enamovirus-1 (GEV-1), discovered by high-throughput sequencing (HTS). A limited survey using HTS of 17 grapevines (Vitis spp.) from the south, southeast, and northeast regions of Brazil led to the detection of GEV-1 exclusively on southern plants, infecting four grapevine cultivars (Cabernet Sauvignon, Semillon, CG 90450, and Cabernet franc) with a remarkable identity of around 99% at the nucleotide level. This novel virus was only detected in multiple-virus infected plants exhibiting viral-like symptoms. GEV-1 was also detected on a cv. Malvasia Longa by RT-PCR. We performed graft-transmissibility assays on GEV-1. The organization, products, and cis-acting regulatory elements of GEV-1 genome are also discussed here. The near complete genome sequence of GEV-1 was obtained during the course of this study, lacking only part of the 3' untranslated terminal region. This is the first report of a virus in the family Luteoviridae infecting grapevines. Based on its genomic properties and phylogenetic analyses, GEV-1 should be classified as a new member of the genus Enamovirus.

Construction of an agroinfectious clone of bean rugose mosaic virus using Gibson Assembly.

Construction of agroinfectious viral clones usually requires many steps of cloning and sub-cloning and also a binary vector, which makes the process laborious, time-consuming, and frequently susceptible to some degree of plasmid instability. Nowadays, novel methods have been applied to the assembly of infectious viral clones, and here we have applied isothermal, single-step Gibson Assembly (GA) to construct an agroinfectious clone of Bean rugose mosaic virus (BRMV) using a small binary vector. The procedure has drastically reduced the cloning steps, and BRMV could be recovered from agroinfiltrated common bean twenty days after inoculation, indicating that the infectious clone could spread in the plant tissues and efficiently generate a systemic infection. The virus was also recovered from leaves of common bean and soybean cultivars mechanically inoculated with infectious clone two weeks after inoculation, confirming the efficiency of GA cloning procedure to produce the first BRMV agroinfectious clone to bean and soybean.

Complete genome sequence of tobacco mosqueado virus.

We describe the genomic characteristics of a new potyvirus isolated from tobacco plants showing mottling ("mosqueado" in Portuguese) in southern Brazil. The complete genomic sequence consists of 9896 nucleotides, without the poly(A) tail, and shares the highest pairwise nucleotide sequence identities of 68.5 % with pepper yellow mosaic virus and 68.2 % with Brugmansia mosaic virus isolate D437. These identity values are below the level of 76.0 % used as a criterion for species demarcation in the genus Potyvirus based on the complete genome sequence. The viral genomic organization and sequence comparison thus suggest that this virus, tentatively named "tobacco mosqueado virus" (TMosqV), represents a new potyvirus species.

A highly divergent isolate of tomato blistering mosaic virus from Solanum violaefolium.

The complete genome of a tymovirus infecting Solanum violaefolium was sequenced. The genome comprised 6284 nt, with a 5'-UTR of 137 nt and a comparatively longer 3'-UTR of 121 nt. Sequence analysis confirmed three ORFs encoding a movement protein, a polyprotein, and a coat protein (CP). The isolate was considered to be the Tomato blistering mosaic virus (ToBMV) based on a CP amino acid sequence identity of 95.3 %. The nucleotide sequence of the complete genome of the S. violaefolium isolate, however, differed markedly from the other two reported ToBMV isolates, with identities of 76.6 and 76.3 %, below one of the demarcation criteria of the genus Tymovirus (overall genome identity of 80 %). No recombination signals were detected in the genome of this isolate. The high identity of the CP amino acid sequence and similar host responses suggest that the S. violaefolium isolate belongs to the same species as the Tomato blistering mosaic virus. The sequence analysis of this ToBMV isolate thus suggests that the demarcation criterion of 80 % overall genome sequence identity in the genus Tymovirus may require revision.

Subcellular localization of p29, a putative movement protein of pepper ringspot virus.

Pepper ringspot virus (PepRSV) is a member of the genus Tobravirus. It possesses a bipartite single-strand RNA genome in a positive-sense polarity. The p29 protein is encoded by RNA 1 and is presumed to be the movement protein (MP) of this virus. In this study, the intracellular distribution of the p29 protein was analyzed by confocal microscopy. Transient expression of the PepRSV p29 protein fused to green fluorescent protein was observed as punctate spots localized next to the cell wall. This protein partially co-localized with the eCFP-tagged tobacco mosaic virus 30K MP, which is known to associate with plasmodesmata. This result suggests that the p29 protein is most probably the movement protein for PepRSV.