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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.

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., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

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.

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.

To elucidate the etiology of a new disease of shallot in France, double-stranded RNAs from asymptomatic and symptomatic shallot plants were analyzed using high-throughput sequencing (HTS). Annotation of contigs, molecular characterization and phylogenetic analyses revealed the presence in symptomatic plants of a virus complex consisting of shallot virus X (ShVX, Allexivirus), shallot latent virus (SLV, Carlavirus) and two novel viruses belonging to the genera Carlavirus and Potyvirus, for which the names of shallot virus S (ShVS) and shallot mild yellow stripe associated virus (SMYSaV), are proposed. Complete or near complete genomic sequences were obtained for all these agents, revealing divergent isolates of ShVX and SLV. Trials to fulfill Koch's postulates were pursued but failed to reproduce the symptoms on inoculated shallots, even though the plants were proved to be infected by the four viruses detected by HTS. Replanting of bulbs from SMYSaV-inoculated shallot plants resulted in infected plants, showing that the virus can perpetuate the infection over seasons. A survey analyzing 351 shallot samples over a four years period strongly suggests an association of SMYSaV with the disease symptoms. An analysis of SMYSaV diversity indicates the existence of two clusters of isolates, one of which is largely predominant in the field over years., Competing Interests: The authors have declared that no competing interests exist.

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.

A new virus causing a serious stunt disease of chrysanthemum was identified in China by high-throughput sequencing (HTS) and named chrysanthemum virus R (CVR). The complete sequence of CVR was determined by reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). The genomic RNA of CVR consists of 8,874 nucleotides (nt), excluding the poly(A) tail, contains six putative open reading frames (ORFs), and has a genomic organization typical of members of the genus Carlavirus. BLAST analysis of the full genome sequence showed low similarity (38%-56% sequence identity) to other members of the genus Carlavirus. BLAST analysis and phylogenetic analysis based on the amino acid (aa) sequences of the CVR replicase and coat protein (CP) confirmed that CVR is a distinct member of the genus Carlavirus.

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.

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.

Potato virus S (PVS) often causes significant losses in potato production in potato-growing countries. In this study, the ordinary strain of PVS (PVS O ) was purified from PVS-infected potato plants and used as the immunogen to produce hybridomas secreting monoclonal antibodies (MAbs). Five highly specific and sensitive murine MAbs (1A3, 16C10, 18A9, 20B12, and 22H4) against PVS were prepared using conventional hybridoma technology. Using these MAbs, tissue print-enzyme-linked immunosorbent assay (ELISA), dot-ELISA, and double-antibody sandwich (DAS)-ELISA were developed for sensitive and specific detection of PVS infection in potato plants. The results of sensitivity assays revealed that PVS could be reliably detected in PVS-infected leaf crude extracts diluted at 1:10 240 and 1:163 840 (w/v, g/ml) in phosphate buffer saline (PBS) by dot-ELISA and DAS-ELISA, respectively. Twenty-two samples collected from potato fields in Yunnan Province, China were tested for PVS infection using the serological assays we had developed, and 14 of them were found to be positive. This indicates that PVS is now prevalent in potato fields in Yunnan Province.

Viruses commonly infecting lily (Lilium spp.) include: Lily symptomless virus (LSV), Cucumber mosaic virus (CMV) and Lily mottle virus (LMoV). These viruses usually co-infect lilies causing severe economic losses in terms of quantity and quality of flower and bulb production around the world. Reliable and precise detection systems need to be developed for virus identification. We describe the development of a triplex immunocapture (IC) reverse transcription (RT) polymerase chain reaction (PCR) assay for the simultaneous detection of LSV, CMV and LMoV. The triplex IC-RT-PCR was compared with a quadruplex RT-PCR assay. Relative to the quadruplex RT-PCR, the specificity of the triplex IC-RT-PCR system for LSV, CMV and LMoV was 100% for field samples. The sensitivity of the triplex IC-RT-PCR system was 99.4%, 81.4% and 98.7% for LSV, CMV and LMoV, respectively. Agreement (κ) between the results obtained from the two tests was 0.968, 0.844 and 0.984 for LSV, CMV and LMoV, respectively. This is the first report of the simultaneous detection of LSV, CMV and LMoV in a triplex IC-RT-PCR assay. In particular we believe this convenient and reliable triplex IC-RT-PCR method could be used routinely for large-scale field surveys or crop health monitoring of lily., (Copyright © 2017. Published by Elsevier B.V.)

A dot-immunobinding assay (DIBA) was optimized and used successfully for the rapid detection of 15 known viruses [Alfalfa mosaic virus (AMV), Bean pod mottle virus (BPMV), Bean yellow mosaic virus (BYMV), Cowpea mild mottle virus (CPMMV), Cowpea severe mosaic virus (CPSMV), Cucumber mosaic virus (CMV), Peanut mottle virus (PeMoV), Peanut stunt virus (PSV), Southern bean mosaic virus (SBMV), Soybean dwarf virus (SbDV), Soybean mosaic virus (SMV), Soybean vein necrosis virus (SVNV), Tobacco ringspot virus (TRSV), Tomato ringspot virus (ToRSV), and Tobacco streak virus (TSV)] infecting soybean plants in Oklahoma. More than 1000 leaf samples were collected in approximately 100 commercial soybean fields in 24 counties of Oklahoma, during the 2012-2013 growing seasons. All samples were tested by DIBA using polyclonal antibodies of the above 15 plant viruses. Thirteen viruses were detected, and 8 of them were reported for the first time in soybean crops of Oklahoma. The highest average incidence was recorded for PeMoV (13.5%) followed by SVNV (6.9%), TSV (6.4%), BYMV, (4.5%), and TRSV (3.9%), while the remaining seven viruses were detected in less than 2% of the samples tested. The DIBA was quick, and economical to screen more than 1000 samples against 15 known plant viruses in a very short time., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The complete genome sequence of a new potato virus M (PVM) isolate (PVM-YN), collected from potato (Solanum tuberosum) in Yunnan, China, was determined. It was 8,530 nucleotides (nt) in length, excluding the poly(A) tail at the 3' end, and shared 71.4-72.0% nucleotide sequence identity with available PVM isolates in the NCBI database. The coat proteins (CP) of PVM-YN shared 79.0-97.4% amino acid sequence identity with that of other isolates. It is the first report of the complete genomic sequence of a new PVM isolate infecting S. tuberosum in China.

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., Competing Interests: The authors have declared that no competing interests exist.

Lily symptomless virus (LSV) is one of the most prevalent viruses that infect lily plants worldwide. A rapid and sensitive reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for detection of LSV, using two primer pairs that specifically amplified the conserved sequence of LSV coat protein. The optimum reaction conditions were as follows: 4mM MgCl 2 and 0.8M betaine with incubation at 64°C for 30min. The limit of detection of LSV from infected lily leaves was 10-fold higher for RT-LAMP than for conventional RT-PCR. Moreover, RT-LAMP detected LSV in not only symptomatic, but also in symptomless tissues of infected plants. These findings indicate that our RT-LAMP method for LSV can serve as a low-cost, simple, and rapid alternative to conventional detection assays., (Copyright © 2016 Elsevier B.V. All rights reserved.)

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.

Virus-like particles (VLPs) are multisubunit self-assembly competent protein structures with identical or highly related overall structure to their corresponding native viruses. To construct a new filamentous VLP carrier, the coat protein (CP) gene from potato virus M (PVM) was amplified from infected potato plants, cloned, and expressed in Escherichia coli cells. As demonstrated by electron microscopy analysis, the PVM CP self-assembles into filamentous PVM-like particles, which are mostly 100-300 nm in length. Adding short Gly-Ser peptide at the C-terminus of the PVM, CP formed short VLPs, whereas peptide and protein A Z-domain fusions at the CP N-terminus retained its ability to form typical PVM VLPs. The PVM-derived VLP carrier accommodates up to 78 amino acid-long foreign sequences on its surface and can be produced in technologically significant amounts. PVM-like particles are stable at physiological conditions and also, apparently do not become disassembled in high salt and high pH solutions as well as in the presence of EDTA or reducing agents. Despite partial proteolytic processing of doubled Z-domain fused to PVM VLPs, the rabbit IgGs specifically bind to the particles, which demonstrates the functional activity and surface location of the Z-domain in the PVM VLP structure. Therefore, PVM VLPs may be recognized as powerful structural blocks for new human-made nanomaterials.

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.

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.

A rapid immunochromatographic strip (ICS) test for specific detection of lily symptomless virus (LSV) was developed. The test is based on a double-antibody sandwich format and employs two distinct anti-LSV polyclonal antibodies (IgG1 and IgG2). The first antibody, IgG1 was used as the detection antibody conjugated to colloidal gold and the second antibody, IgG2 was used to as the capture antibody at the test line. The performance of the ICS test was evaluated and the results obtained were compared with a quadruplex RT-PCR assay. When serial dilutions of purified LSV were tested, the LSV detection limit of the ICS test was 6.0 × 10(-8)mg/mL, which was the same as the quadruplex RT-PCR assay. Relative to quadruplex RT-PCR, the specificity and sensitivity of the ICS were 98.6% and 100%, respectively for field leaf samples. There was significant agreement between the results of the ICS and quadruplex RT-PCR tests (κ = 0.983). Compared with conventional lily virus detection methods, our ICS test has many advantages: simple, fast, low cost, high sensitivity and specificity, and has applications in the laboratory and in the field to detect and control LSV., (Copyright © 2015 Elsevier B.V. All rights reserved.)

A multiplex reverse transcription loop-mediated isothermal amplification (mRT-LAMP) assay was developed for the simultaneous detection of Chrysanthemum Virus B (CVB) and Chrysanthemum stunt viroid (CSVd), which are the major viral pathogens of chrysanthemum worldwide. Two sets of mRT-LAMP primers were designed for the coat protein gene of CVB and the complete nucleotide sequence of CSVd, and a restriction enzyme cleavage site was inserted into two pairs of species-specific primers. The mRT-LAMP assay was designed by combining these two sets for a total of eight primers. The mRT-LAMP method distinguished between CVB and CSVd due to the subsequent restriction enzyme analysis. The sensitivity of the mRT-LAMP method was 10(3) times higher than classical PCR regarding the detection limits for CVB and CSVd. No positive results were observed when RNA from other chrysanthemum pathogens were used as mRT-LAMP templates. The method was verified by testing chrysanthemum samples collected from Beijing and Henan Province and showed high reliability and sensitivity. The developed mRT-LAMP assay also offers an efficient, convenient, and rapid tool for screening chrysanthemum virus and viroid, especially CVB and CSVd, and can be diagnosed in a single reaction. These results suggest that the new mRT-LAMP method may be used routinely for virus and viroid surveys., (Copyright © 2014 Elsevier B.V. All rights reserved.)

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.

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.

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).

Indian garlic is infected with Onion yellow dwarf virus (OYDV), Shallot latent virus (SLV), Garlic common latent virus (GarCLV) and allexiviruses. Identity and distribution of garlic viruses in various garlic accessions from different geographical regions of India were investigated. OYDV and allexiviruses were observed in all the garlic accessions, while SLV and GarCLV were observed only in a few accessions. A multiplex reverse transcription (RT)-PCR method was developed for the simultaneous detection and identification of OYDV, SLV, GarCLV and Allexivirus infecting garlic accessions in India. This multiplex protocol standardized in this study will be useful in indexing of garlic viruses and production of virus free seed material., (Copyright © 2014 Elsevier B.V. All rights reserved.)

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.

The coat protein (CP) gene of five Indian Garlic common latent virus (GarCLV) isolates was sequenced and it was 960 bp long in all the five isolates, encoding a protein of 319 amino acids. Comparative nucleotide sequence analysis revealed diversity of 4.3% among the Indian isolates and of 11.9% among all isolates worldwide. Amino acid sequence comparison showed a significant variability in the N-terminal of CP of GarCLV. Various protein analysis tools identified thirteen conserved domains and motifs including Carlavirus and Potexvirus-specific Flexi CP and Flexi N CP. Phylogenetic analysis clustered GarCLV isolates in the subgroup II with isolates from Australia, Brazil, Japan, and South Korea. Intraspecies recombination study revealed that only one of the Indian isolates was a recombinant. Interspecies recombination study suggested the absence of genetic exchange from Carlavirus species to GarCLV; conversely, GarCLV was identified as a putative donor for at least two other Carlavirus species. This is the first report of molecular variability and recombination in GarCLV isolates.

A new carlavirus, tentatively named Potato virus H (PVH), was found on potato plants with mild symptoms in Hohhot, Inner Mongolia Autonomous Region, China. PVH was confirmed by genome sequencing, serological reactions, electron microscopy, and host index assays. The PVH particles were filamentous and slightly curved, with a modal length of 570 nm. Complete RNA genomic sequences of two isolates of PVH were determined using reverse transcription-PCR (RT-PCR) and the 5' rapid amplification of cDNA ends (5' RACE) method. Sequence analysis revealed that PVH had the typical genomic organization of members of the genus Carlavirus, with a positive-sense single-stranded genome of 8410 nt. It shared coat protein (CP) and replicase amino acid sequence identities of 17.9-56.7% with those of reported carlaviruses. Phylogenetic analyses based on the protein-coding sequences of replicase and CP showed that PVH formed a distinct branch, which was related only distantly to other carlaviruses. Western blotting assays showed that PVH was not related serologically to other potato carlaviruses (Potato virus S, Potato virus M, and Potato latent virus). PVH systemically infected Nicotianaglutinosa but not Nicotiana tabacum, Nicotianabenthamiana, or Chenopodiumquinoa, which is in contrast with the other potato carlaviruses. These results support the classification of PVH as a novel species in the genus Carlavirus. Preliminary results also indicated that a cysteine-rich protein encoded by the smallest ORF located in the 3' proximal region of the genome suppressed local RNA silencing and enhanced the pathogenicity of the recombinant PVX.

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.

Yardlong bean (Vigna unguiculata subsp. sesquipedalis) plants with virus-like systemic mottling and leaf distortion were observed in both experimental and commercial fields in Aragua State, Venezuela. Symptomatic leaves were shown to contain carlavirus-like particles. RT-PCR analysis with carlavirus-specific primers was positive in all tested samples. Nucleotide sequences of the obtained amplicons showed 84%-74% similarity to corresponding sequences of Cowpea mild mottle virus (CPMMV) isolates deposited in the GenBank database. This is the first report of CPMMV in Venezuela and is thought to be the first report of CPMMV infecting yardlong bean.

Multiple viruses such as Lily symptomless virus (LSV), Lily mottle virus (LMoV), and cucumber mosaic virus (CMV) are the most prevalent viruses infecting lilies in Korea. Leaf samples and bulbs showing characteristic symptoms of virus infection were collected from Gangwon, Chungnam, and Jeju provinces of Korea in 2008-2011. Coat protein (CP) genes of LSV and LMoV were amplified from collected samples by reverse transcription-polymerase chain reaction (RT-PCR) and cloned into a pET21d(+) expression vector to generate recombinant CPs. The resulting carboxy-terminal His-tagged CPs were expressed in Escherichia coli strain BL21(DE3) by isopropyl-1-thio- beta-D-galactoside induction. The recombinant proteins were purified using Ni-NTA agarose beads, and the purified proteins were used as an immunogen to produce polyclonal antibodies in rabbits. The resulting polyclonal antisera recognized specifically LSV and LMoV from infected plant tissues in Western blotting assays. Indirect enzyme-linked immunosorbent assay and immunocapture RTPCR using these polyclonal antisera were developed for the sensitive, efficient, economic, and rapid detection of Lily viruses. These results suggest that large-scale bulb tests and economic detection of Lily viruses in epidemiological studies can be performed routinely using these polyclonal antisera.

Complete genome sequences were obtained from two isolates of the carlavirus nerine latent virus from hippeastrum and narcissus plants, two isolates of the potyvirus hippeastrum mosaic virus from a hippeastrum plant, and one isolate each of the potyviruses narcissus degeneration virus, narcissus yellow stripe virus and Vallota speciosa virus from narcissus plants. Proposals are made to clarify the current confusion surrounding the naming of some of these viruses.

Chrysanthemums worldwide suffer from a high incidence of infection with chrysanthemum virus B (CVB), a member of the genus Carlavirus, family Betaflexiviridae. Three major lineages or strains of this virus have been found in India, but none have been characterized beyond the genetic variation they display in their coat protein genes. Here, we describe the analysis of four near-complete genome sequences (from the three lineages) representing the genetic diversity of these strains. Ranging in size from 8815 to 8855 nucleotides (excluding the polyA tail), these four isolates have a genome organization very similar to that of the recently reported Japanese isolate of CVB, with which they share between 70 and 73% genome-wide sequence identity. We present further evidence that recombination may feature quite prominently in the evolution of CVB.

Hemp (Cannabis sativa) was found to be a useful propagation host for hop latent virus, a carlavirus. However, when virus preparations were analysed by electron microscopy, along with the expected filamentous particles, spherical particles with a diameter of around 34 nm were found. RNA from virus preparations was purified, and cDNA was prepared and cloned. Sequence information was used to search databases, and the greatest similarity was found with Primula malacoides virus 1, a putative new member of the genus Partitivirus. The full sequences of RNA 1 and RNA 2 of this new hemp cryptic virus were obtained.

Two hundred forty potato samples with one or more symptoms of leaf mosaic, distortion, mottling and yellowing were collected between 2005 and 2008 from seven Iranian provinces. Forty-four of these samples tested positive with double-antibody sandwich enzyme-linked immunosorbent assays (DAS-ELISA) using a Potato virus S (PVS) polyclonal antibody. Of these 12 isolates of PVS were selected based on the geographical location for biological and molecular characterization. The full coat protein (CP) and 11K genes from 12 PVS isolates were PCR amplified, cloned and sequenced. All 12 PVS isolates showed mosaic symptoms on Nicotiana debneyii and N. tabacum cv. Whiteburly and local lesion on Chenopodium amaranticolor, C. quinoa and C. album. The Iranian isolates share between 93 and 100% pairwise nucleotide identity with other PVS(O) isolates. Based on maximum likelihood phylogenetic analysis coupled with pairwise identity analysis, we propose 15 genotypes for the PVS(O) strain and 3 genotypes for the PVS(A) strain.

During blueberry scorch virus (BlScV) surveys of highbush blueberries in Italy between 2005 and 2010, we initially discovered infected orchards only in Piedmont. Since 2009, however, three infected orchards have also been found in Trentino, where a new host species, Vaccinium ashei, was found to be infected by BlScV. Molecular characterization of isolates during the study period suggests that two very distinct epidemics are now present in Italy: the Piedmont isolates belong to a new BlScV strain, whereas the Trentino isolates are almost identical to the Washington State strain.

Lily symptomless virus (LSV) is the most common lily virus, being detected in many species and hybrids. We established a microsphere-based fluorescent immunoassay for the determination of LSV, using a polyclonal antibody against LSV covalently bound to carboxy-modified microspheres able to capture LSV antigen. A monoclonal antibody against LSV conjugated to quantum dots (QDs) was used as a fluorescent probe, enabling LSV to be fluorescently detected by a combination of encoded beads and QDs. This method was 16 times more sensitive than ELISA in the detection of LSV, and could potentially be applied to the simultaneous detection of inhomogeneous matter.

Hop mosaic virus (HpMV), a member of the genus Carlavirus, is importance to hop production worldwide. We identified variation in nucleic and amino acid sequences among 23 HpMV isolates from Australia, the USA, the Czech Republic, South Africa and Japan using a 1,455-bp fragment covering the 3' end of the virus genome including ORFs 4, 5 and 6. Three clusters of two or more isolates were identified in phylogenies of the total nucleotide sequence and the coat protein (ORF5) amino acid sequence. Two of these clusters combined in analyses of ORF4 and ORF6 amino acid sequences. Isolates from within and outside of Australia were found in each cluster, indicating that sequence variation was not associated with geographic source. Monitoring of HpMV variants in the field and evaluation of the impact of variants on vector association, rate of spread, and hop yield and quality can now be undertaken.

Potato virus M (PVM, Carlavirus) is considered to be one of the most common potato viruses distributed worldwide. Sequences of the coat protein (CP) gene of several Canadian PVM isolates were determined. Phylogenetic analysis indicated that all known PVM isolates fell into two distinct groups and the isolates from Canada and the US clustered in the same group. The Canadian PVM isolates could be further divided into two sub-groups. Two molecular procedures, reverse transcription - polymerase chain reaction (RT-PCR) and restriction fragment length polymorphism (RFLP) were developed in this study for the detection and identification of PVM in potato tubers. RT-PCR was highly specific and only amplified PVM RNA from potato samples. PVM RNAs were easily detected in composite samples of 400 to 800 potato leaves or 200 to 400 dormant tubers. Restriction analysis of PCR amplicons with MscI was a simple method for the confirmation of PCR tests. Thus, RT-PCR followed by RFLP analysis may be a useful approach for screening potato samples on a large scale for the presence of PVM.

An isolate of the tentative carlavirus species Hydrangea chlorotic mottle virus (HdCMV) was found in New Zealand (NZ) in 2007. The host range, serological properties and complete genome sequence of this isolate were determined in this study. While the NZ isolate shared 98% nucleotide sequence identity with the US isolate of HdCMV, differences in titre and host range were found. The HdCMV-NZ genome sequence of 8,433 nt possessed a typical carlavirus organisation with six open reading frames. HdCMV is most closely related (60.4% nt identity) to blueberry scorch virus, a relationship also suggested by serology. These data suggest that HdCMV is a new carlavirus species.

A high throughput, real-time multiplex, single tube RT-PCR assay was developed for simultaneous detection of Potato leafroll virus (PLRV), Potato virus X (PVX) and Potato virus S (PVS) in potato leaves and tubers, and Tomato spotted wilt virus (TSWV) in potato tubers and tomato leaves. The test uses four different fluorescently labelled TaqMan probes. Limits of detection sensitivity were established using a range of virus transcript copy numbers (8 x 10(1) to 8 x 10(9) copies of PVX and PVS, 1 x 10(2) to 1 x 10(10) copies of PLRV and 1 x 10(3) to 1 x 10(10) copies of TSWV). For each individual assay, the inter-assay reproducibility was high, with a coefficient of variation of the combined assays of <2%. Total RNA was extracted rapidly and efficiently from bulked samples equivalent to 300 dormant tubers to detect single infections of PLRV, PVX, PVS and TSWV simultaneously in a single assay. The multiplexed assay was validated in blind studies with leaves and tubers. This high-throughput test is accurate and sensitive, and provides seed potato industries with a cost-effective diagnostic tool to detect viruses reliably in bulked samples of dormant potato tubers.

During 2006-2007 growing seasons, survey were carried to identify a virus disease causing mosaic of soybean in the field in Southern region (Khozestan Province) of Iran. To detect the viral infection, diseased leaf samples showing mild mosaic and leaf malformation were collected from soybean fields in Dezful, located in Khozestan Province. Infected samples were carried to the lab in a proper condition on ice packages. TPIA and DAS-ELISA serological tests were applied to identify the viral agent. To investigate the host-range, several indicator plants were mechanically inoculated under green-house condition. Seed transmission of CPMMV was examined using the seeds obtained from infected plants. The virus isolate was not found to be seed-borne in Clark variety of soybean. Different steps of ultracentrifugation including sucrose density gradient (10-40%) were carried out in order to obtain partial purified virus. On the basis of biological, serological and EM results, CPMMV-Carla virus was identified in the infected soybean samples. This is the first report of CPMMV infection of soybean in Iran.

To facilitate efficient and accurate detection of potato-infecting carlaviruses, degenerated universal primers were designed based on conserved amino acid and nucleotide sequences. Two sense primers, Car-F1 and Car-F2, were based on the amino acid sequences "SNNMA" and "GLGVPTE", respectively, in the coat protein. The reverse primer, Car-R, which was located at the border of the nucleic acid binding protein gene and the 3' untranslated region, and dT-B, which was derived from the oligo-dT targeting the poly(A) tail, were selected. Successful application of fragments within the predicted size range of carlaviruses was obtained using Car-F1 paired with either Car-R or dT-B from tested carlaviruses (Potato virus S, M and latent) by RT-PCR. The Car-F2 failed to yield clear-cut fragments within the predicted size range when paired with either Car-R or dT-B in RT-PCR. However, a less degenerated version of the primer, Car-F2b, resulted in amplicons within the predicted size range when paired with either Car-R or dT-B. Sequencing of the tentative carlavirus-fragments resulting from Car-F1/Car-R and Car-F2b/dT-B proved their carlavirus-origin, thus indicating the high specificity of these primers. The sensitivity of Car-F1/Car-R or Car-F2b/Car-R mediated RT-PCR for the detection of carlavirus-infected potato tubers were assessed using composite samples containing one carlavirus-infected-potato-tuber RNA sample with up to 49 virus-free-potato-tuber RNA samples under the optimal annealing temperature. The target carlaviruses were detected readily from all composites, demonstrating a high sensitivity. The method was further evaluated using presumed virus-free or carlavirus-infected potatoes of several cultivars, and reliable results were obtained.