High-Throughput Sequencing Reveals New Viroid Species in Opuntia in Mexico.

In the main cactus pear (Opuntia ficus-indica)-producing region in the State of Mexico, fruit production occupies the largest cultivated area with 15,800 ha, while 900 ha are cultivated for edible young Opuntia pads ("nopalitos") which are consumed as vegetables. Two composite samples consisting of cladodes of plants for fruit production (n = 6) and another of "nopalitos" (n = 6) showing virus-like symptoms were collected. Both sample sets were subjected to high-throughput sequencing (HTS) to identify the viruses and viroids. The HTS results were verified using RT-PCR and Sanger sequencing. Subsequently, 86 samples including cladodes from "nopalitos", plants for fruit production, xoconostles, and some wild Opuntia were analyzed via RT-PCR with specific primers for the viruses and viroids previously detected via HTS. Three viruses were discovered [Opuntia virus 2 (OV2), cactus carlavirus 1 (CCV-1), and Opuntia potexvirus A (OPV-A)], along with a previously reported viroid [Opuntia viroid 1 (OVd-1)]. Additionally, two new viroids were identified, provisionally named the Mexican opuntia viroid (MOVd, genus Pospiviroid) and Opuntia viroid 2 (OVd-2, genus Apscaviroid). A phylogenetic analysis, pairwise identity comparison, and conserved structural elements analysis confirmed the classification of these two viroids as new species within the Pospiviroidae family. This is the first report of a pospiviroid and two apscaviroids infecting cactus pears in the world. Overall, this study enhances our understanding of the virome associated with cactus pears in Mexico.

Complete genome sequence of a new isolate of caper latent virus in caper.

The genome of a new carlavirus isolate from asymptomatic wild Capparis spinosa L. plants in Sicily was sequenced via high-throughput sequencing (HTS) and 5'/3' RACE experiments. The complete genomic sequence was found to be 8,280 nt in length, excluding the poly(A) tail, and contained five putative open reading frames (ORFs). Molecular characterization revealed a close relationship to caper latent virus (CapLV), with 87% and 90% nucleotide sequence identity to available partial sequences of the ORFs encoding the replicase and coat protein of that virus. According to the molecular criteria for species demarcation, which is based on the ORF-1- and ORF-5-encoded proteins, the virus characterized in this study could be considered a variant of CapLV, and we have thus designated it as CapLV-W.

First complete genome sequence of carnation latent virus, the type member of the genus Carlavirus.

The genus Carlavirus (family Betaflexiviridae, order Tymovirales) currently includes 53 species recognized by the ICTV. The NCBI GenBank database has 43 full-length carlavirus genome sequences (7,890 to 9,073 nt). Surprisingly, the type species Carnation latent virus is not associated with a complete genome sequence of a carnation latent virus (CLV) isolate; GenBank only has accessions with 1313 or fewer nucleotides. The goal of this study was to determine the full-length genome sequence of CLV. Naturally infected greenhouse-grown 'Kiwi Lace' carnation plants that tested positive for CLV by ELISA and RT-PCR were used as source plants for high-throughput sequencing, completed by 5' and 3' RACE and validated by Sanger sequencing of CLV-specific RT-PCR-generated amplicons. The complete CLV-KL sequence (MN450069) was determined to be 8,513 nt in length. In pairwise analysis, the genome shares 40-46% identity with recognized carlaviruses and the six in silico-translated proteins have 15-62% amino acid sequence identity to their respective carlavirus orthologs. The CLV-KL coat protein shares 98.4% identity with the NCBI reference sequence CLV-UK. In phylogenetic analysis, CLV clusters with butterbur mosaic virus, coleus vein necrosis virus, and garlic common latent virus. This is the first report of the full genome sequence of an isolate of carnation latent virus, the type member of the genus Carlavirus.

Complete nucleotide sequence of a new carlavirus infecting Aconitum carmichaelii in China.

A novel virus was identified in aconite (Aconitum carmichaelii Debx.) in China by high-throughput sequencing (HTS) and tentatively named "aconite virus A" (AcVA). The genomic RNA of AcVA consists of 8,844 nucleotides, excluding the poly(A) at the 3' end. Analysis of the genomic organization of AcVA indicated that it possesses a genomic structure that is typical of carlaviruses and contains six putative open reading frames (ORFs). Pairwise analysis revealed that the replicase and coat protein of AcVA share the highest amino acid sequence identity (43.78% and 57.01%) with those of coleus vein necrosis virus (CVNV) and butterbur mosaic virus (ButMV), respectively. Based on the current classification criteria for carlaviruses, AcVA should be considered a distinct member of the genus Carlavirus.

Complete genome sequence of rose virus A, the first carlavirus identified in rose.

A novel virus was discovered in a Rosa wichuraiana Crep. by high-throughput sequencing and tentatively named "rose virus A" (RVA). Based on sequence identity and phylogenetic analysis, RVA represents a new member of the genus Carlavirus (family Betaflexiviridae). The genome of RVA is 8,849 nucleotides long excluding the poly(A) tail and contains six open reading frames (ORFs). The predicted ORFs code for a replicase, triple gene block (TGB), coat protein, and nucleic acid binding protein, as in a typical carlavirus. RVA is the first carlavirus identified in rose and has the highest nucleotide sequence similarity to poplar mosaic virus. Reverse transcription-PCR-based assays were developed to confirm the presence of RVA in the original source and to screen additional rose plants.

Complete genome sequence of a divergent strain of potato virus P isolated from Solanum tuberosum in Russia.

Contigs with sequence similarity to potato virus P (PVP), which belongs to the genus Carlavirus, were identified by high-throughput sequencing analysis in potato tubers collected from a farmer's potato production field in Surazhevka, Artyom, Primorskiy Krai (Russia) in 2018. The complete genome sequence of this virus consisted of 8,394 nucleotides, excluding the poly(A) tail. This is the first report of PVP being detected outside South America. The isolate had high sequence similarity to PVP isolates from Argentina and Brazil, but low sequence similarity was observed in the genes encoding the RNA-dependent RNA polymerase (69% nucleotide sequence identity and 80% amino acid sequence identity) and coat protein (78% nucleotide sequence identity and 89% amino acid sequence identity). Phylogenetic analysis revealed that this PVP-like virus clustered with known PVP isolates but was distinct from them. Comparison of the sequences using the classification criteria of the ICTV indicated that this PVP-like virus is a strain of PVP.

Molecular characterization and detection of two carlaviruses infecting cactus.

Two large contigs with sequence similarities to different carlaviruses were identified by high-throughput sequencing in samples from a cactus plant. The complete genomes of the two viruses, tentatively named "cactus carlavirus 1" (CCV-1) and "cactus carlavirus 2" (CCV-2), were determined to be 8,441 and 8,396 nucleotides long, respectively, excluding the poly(A) tail. These viruses have the typical genomic organization of members of the genus Carlavirus. CCV-1 appears to be a cactus isolate of the carlavirus HSO-2016a, with 90.1% nucleotide sequence identity between the two virus genomes, whereas CCV-2 may be classified as a member of a new species. The sequences of CCV-2 and other carlaviruses are 48.9-60.0% identical at the whole-genome level.

Phylogenetic relationships of two Ukrainian tomato isolates of potato virus M and genetic variability analysis of its population.

The aim of this study was to investigate biological and molecular properties of two Ukrainian tomato isolates of potato virus M (PVM), K-16 and Pol-14, to determine their phylogenetic relationships and the genetic variability of PVM isolates. Study of phylogenetic relationships of two Ukrainian tomato PVM isolates with 35 isolates represented in GenBank was conducted. It was found that the coat protein (CP) gene sequence identity between two Ukrainian PVM isolates is 94.3% at the nucleotide level and 100% at the amino acid level. The highest level of the sequence identity (97.0% and 96.5% nt and 100% aa) have the isolates K-16 and Pol-14 with the German potato isolate DSMZ PV0273, Indian potato isolates Del 123, Del 134, Del 147, M 34 and Chinese isolate from pepino GS-6-2 (isolate K-16), which testifies about their common origin. Ukrainian tomato isolates K-16 and Pol-14 belong together with all European, Chinese, Iranian, Indian isolates to PVM-o clade or group I. It was found that the nucleotide substitutions in the capsid protein gene of all tomato PVM isolates (except the Italian) are synonymous. Analysis showed that the global dN/dS ratio for the entire CP gene sequences used in the study was 0.041 (p Keywords: potato virus M; Solanum lycopersicum; phylogenetic analysis; genetic variability; selection pressure.

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.

Mixed Infections of Four Viruses, the Incidence and Phylogenetic Relationships of Sweet Potato Chlorotic Fleck Virus (Betaflexiviridae) Isolates in Wild Species and Sweetpotatoes in Uganda and Evidence of Distinct Isolates in East Africa.

Viruses infecting wild flora may have a significant negative impact on nearby crops, and vice-versa. Only limited information is available on wild species able to host economically important viruses that infect sweetpotatoes (Ipomoea batatas). In this study, Sweet potato chlorotic fleck virus (SPCFV; Carlavirus, Betaflexiviridae) and Sweet potato chlorotic stunt virus (SPCSV; Crinivirus, Closteroviridae) were surveyed in wild plants of family Convolvulaceae (genera Astripomoea, Ipomoea, Hewittia and Lepistemon) in Uganda. Plants belonging to 26 wild species, including annuals, biannuals and perennials from four agro-ecological zones, were observed for virus-like symptoms in 2004 and 2007 and sampled for virus testing. SPCFV was detected in 84 (2.9%) of 2864 plants tested from 17 species. SPCSV was detected in 66 (5.4%) of the 1224 plants from 12 species sampled in 2007. Some SPCSV-infected plants were also infected with Sweet potato feathery mottle virus (SPFMV; Potyvirus, Potyviridae; 1.3%), Sweet potato mild mottle virus (SPMMV; Ipomovirus, Potyviridae; 0.5%) or both (0.4%), but none of these three viruses were detected in SPCFV-infected plants. Co-infection of SPFMV with SPMMV was detected in 1.2% of plants sampled. Virus-like symptoms were observed in 367 wild plants (12.8%), of which 42 plants (11.4%) were negative for the viruses tested. Almost all (92.4%) the 419 sweetpotato plants sampled from fields close to the tested wild plants displayed virus-like symptoms, and 87.1% were infected with one or more of the four viruses. Phylogenetic and evolutionary analyses of the 3'-proximal genomic region of SPCFV, including the silencing suppressor (NaBP)- and coat protein (CP)-coding regions implicated strong purifying selection on the CP and NaBP, and that the SPCFV strains from East Africa are distinguishable from those from other continents. However, the strains from wild species and sweetpotato were indistinguishable, suggesting reciprocal movement of SPCFV between wild and cultivated Convolvulaceae plants in the field.

The complete sequence and genome organization of ligustrum virus A, a novel carlavirus.

The complete genome sequence of ligustrum virus A (LVA) from a Ligustrum obtusifolium Sieb. & Zucc. plant was determined. The genomic RNA has 8,525 nucleotides, excluding the poly(A) tail, and consists of six open reading frames typical of members of the genus Carlavirus, family Betaflexiviridae. Phylogenetic analysis of the viral replicase and coat protein (CP) indicated that LVA is closely related to daphne virus S and helenium virus. The replicase and CP of LVA shared 44.73-52.35 % and 25.39-62.46 % amino acid identity, respectively, with those of other carlaviruses. These results suggest that LVA is a member of a distinct carlavirus species.

Evidence of sympatric speciation of elderberry carlaviruses.

Five new carlaviruses infecting elderberry were characterized and tentatively named as elderberry virus A-E (ElVA-ElVE). Their genome organization is similar to that of other carlaviruses with size ranging from 8540 to 8628 nucleotides, excluding the polyadenylated tails. ElVA, ElVB and ElVD share a common ancestor as do ElVC and ElVE, indicating that speciation may be sympatric with all viruses having emerged in elderberry. Analyses of the carlavirus conserved domains indicate that the 2-oxoglutarate and Fe(II)-dependent oxygenase motifs are reliable indicators of virus phylogenetic classification with recombination playing a significant role in the evolution of the genus. A universal RT-PCR assay that detects all the elderberry carlaviruses and potentially other members of the genus has been developed. This tool can be used for research and regulatory purposes as elderberry cultivation is rapidly expanding to new areas where the viruses may be absent.

Nucleotide sequence and genome organization of atractylodes mottle virus, a new member of the genus Carlavirus.

The complete genome sequence of a member of a distinct species of the genus Carlavirus in the family Betaflexiviridae, tentatively named atractylodes mottle virus (AtrMoV), has been determined. Analysis of its genomic organization indicates that it has a single-stranded, positive-sense genomic RNA of 8866 nucleotides, excluding the poly(A) tail, and consists of six open reading frames typical of members of the genus Carlavirus. The individual open reading frames of AtrMoV show moderately low sequence similarity to those of other carlaviruses at the nucleotide and amino acid sequence levels. Pairwise comparison and phylogenetic analysis suggest that AtrMoV is most closely related to chrysanthemum virus B.

The complete nucleotide sequence and genome organization of pea streak virus (genus Carlavirus).

Pea streak virus (PeSV) is a member of the genus Carlavirus in the family Betaflexiviridae. Here, the first complete genome sequence of PeSV was determined by deep sequencing of a cDNA library constructed from dsRNA extracted from a PeSV-infected sample and Rapid Amplification of cDNA Ends (RACE) PCR. The PeSV genome consists of 8041 nucleotides excluding the poly(A) tail and contains six open reading frames (ORFs). The putative peptide encoded by the PeSV ORF6 has an estimated molecular mass of 6.6 kDa and shows no similarity to any known proteins. This differs from typical carlaviruses, whose ORF6 encodes a 12- to 18-kDa cysteine-rich nucleic-acid-binding protein.

Genetic variation in potato virus M isolates infecting pepino (Solanum muricatum) in China.

Potato virus M (PVM, genus Carlavirus, family Betaflexviridae) is considered to be one of most economically important pathogens of pepino in China. However, the details and the mechanisms underlying PVM evolution are unknown. In this study, we determined and analyzed 40 TGB 1 gene sequences, 67 TGB 2 and TGB 3 gene sequences, and 88 CP and NABP gene sequences from viruses isolated from 19 samples of pepino (Solanum muricatum) and one sample of tomato (S. lycopersicum) collected from different areas of China. Recombination analysis identified only one clear recombinant in the TGB2-TGB3-CP region, but no recombinants were detected for each of the five individual genes. Phylogenetic analysis showed that all PVM isolates could be divided into at least two lineages in trees derived from the TGB 2, CP, and NABP gene sequences, and the lineages seemed to reflect geographical origin. The five PVM genes in this study were found to be under strong negative selection pressure. The PVM isolates examined showed frequent gene flow between the Chinese and European populations, and also within the Chinese population. Clear star phylogenies and the neutral equilibrium model test showed that pepino isolates of PVM appear to be experiencing a new expansion after a recent introduction into China, and these isolates display low levels of genetic diversity. To our knowledge, this study is the first report describing genetic structure, recombination, and gene flow in PVM populations, and it provides strong evolutionary evidence for the virus populations from different geographic regions of China.

Genetic structure and molecular variability of potato virus M populations.

To investigate the genetic diversity of potato virus M (PVM; genus Carlavirus, family Betaflexiviridae), the complete nucleotide sequence of the coat protein gene of 30 PVM isolates from a major potato-growing region in Iran were determined. Phylogenetic analysis of these Iranian PVM isolates together with those available in the GenBank database suggested two divergent evolutionary lineages that did not reflect the origin of the isolates, and these were designated as PVM-o and PVM-d. Examination of the genetic variability of the coat protein of Iranian isolates and their counterparts whose sequences are available in the Genbank database revealed 16 genotype groups in the PVM population. Analysis of the synonymous-tononsynonymous ratio showed strong purifying selection in the CP gene in the genotype groups of divergent clades.

Molecular characterization of domestic and exotic potato virus S isolates and a global analysis of genomic sequences.

Five potato virus S (PVS) isolates from the USA and three isolates from Chile were characterized based on biological and molecular properties to delineate these PVS isolates into either ordinary (PVS(O)) or Andean (PVS(A)) strains. Five isolates - 41956, Cosimar, Galaxy, ND2492-2R, and Q1 - were considered ordinary strains, as they induced local lesions on the inoculated leaves of Chenopodium quinoa, whereas the remaining three (FL206-1D, Q3, and Q5) failed to induce symptoms. Considerable variability of symptom expression and severity was observed among these isolates when tested on additional indicator plants and potato cv. Defender. Additionally, all eight isolates were characterized by determining the nucleotide sequences of their coat protein (CP) genes. Based on their biological and genetic properties, the 41956, Cosimar, Galaxy, ND2492-2R, and Q1 isolates were identified as PVS(O). PVS-FL206-1D and the two Chilean isolates (PVS-Q3 and PVS-Q5) could not be identified based on phenotype alone; however, based on sequence comparisons, PVS-FL206-1D was identified as PVS(O), while Q3 and Q5 clustered with known PVS(A) strains. C. quinoa may not be a reliable indicator for distinguishing PVS strains. Sequences of the CP gene should be used as an additional criterion for delineating PVS strains. A global genetic analysis of known PVS sequences from GenBank was carried out to investigate nucleotide substitution, population selection, and genetic recombination and to assess the genetic diversity and evolution of PVS. A higher degree of nucleotide diversity (π value) of the CP gene compared to that of the 11K gene suggested greater variation in the CP gene. When comparing PVS(A) and PVS(O) strains, a higher π value was found for PVS(A). Statistical tests of the neutrality hypothesis indicated a negative selection pressure on both the CP and 11K proteins of PVS(O), whereas a balancing selection pressure was found on PVS(A).

Molecular variability of cowpea mild mottle virus infecting soybean in Brazil.

Molecular variability was assessed for 18 isolates of cowpea mild mottle virus (CPMMV, genus Carlavirus, family Betaflexiviridae) found infecting soybean in various Brazilian states (Bahia, Goiás, Maranhão, Mato Grosso, Minas Gerais, Pará) in 2001 and 2010. A variety of symptoms was expressed in soybean cv. CD206, ranging from mild (crinkle/blistering leaves, mosaic and vein clearing) to severe (bud blight, dwarfing, leaf and stem necrosis). Recombination analysis revealed only one CPMMV isolate to be recombinant. Pairwise comparisons and phylogenetic analysis were performed for partial genomes (ORF 2 to the 3' terminus) and for each ORF individually (ORFs 2 to 6), showing the isolates to be distinct. The topology of the phylogenetic tree could be related to symptoms, but not to the year of collection or geographical origin. Additionally, the phylogenetic analysis supported the existence of two distinct strains of the virus, designated CPMMV-BR1 and CPMMV-BR2, with molecular variations between them.

Genome-wide scan for analysis of simple and imperfect microsatellites in diverse carlaviruses.

An exhaustive compilation and analysis of incidence, distribution and variation of simple sequence repeats (SSRs) in viruses are required to understand the evolution and functional aspects of repetitive sequences. Present study focuses on the analysis of SSRs in 32 species of carlaviruses. The full length genome sequences were assessed from NCBI (http://www.ncbi.nlm.nih.-gov/) and analyzed using IMEx software. Variance in incidence of SSRs was observed, independent of genome size. Though the conversion of SSRs to imperfect microsatellite or compound SSR is low; compound microsatellites constituted by variant motifs accounted for up to 12.5% of the SSRs. Mononucleotide A/T is most prevalent followed by dinucleotide GT/TG and trinucleotide AAG/GAA in these genomes. The SSR and cSSR are predominantly localized to the coding region RDRP (RNA dependent RNA polymerase) and ORF-6 (open reading frame). The relative frequency of different classes of simple and compound microsatellites has been highlighted in accordance with the biology of carlavirus. Characterization of such variations would be pivotal for deciphering the enigma of these widely used, but incompletely understood sequences.

[Detection and molecular characterization of Potato virus S (PVS, Carlavirus) from Colombia]. / Detección serológica y caracterización molecular de Potato virus S (PVS, Carlavirus) en cultivos de papa de Colombia.

In Colombia, potato crops are affected by a wide variety of viruses such as PVY, PLRV, PVX, PMTV and PVS. Unfortunately, there are very few studies on the biology, distribution and pathogenicity of these viruses; this situation is even worse for the latent virus PVS. In this work, we evaluated the presence ofPVS in four Colombian provinces (Antioquia, Boyacá, Cundinamarca, Nariño) by the use of ELISA. We also studied the degree of molecular variation by sequence comparison of a segment of the gene encoding for the viral coat protein. In average, PVS was detected in 40% of 320 analyzed samples of potato leaves; the highest levels were observed in the East ofAntioquia (49%) and Pasto (Nariño) (47%), while in the other regions ranged between 35% and 42%. Analysis of sequence revealed the presence of two PVS strains in Colombia: three isolates were associated to PVSo (Ordinary) and twelve belonged to PVSA (Andean). A high diversity was observed among PVSA strains with percent identities in the range of 88-99%. These findings highlight the importance of strengthening seed certification programs and quarantine measures in Colombia for viruses like PVS, which can cause losses of up to 20% in potato crops and even higher in mixed virus infection.