First detection and complete genome sequence of a new potexvirus naturally infecting Adenium obesum.

Here, we report the detection and complete genome sequence of a novel potexvirus, tentatively named "Adenium obesum virus X" (AobVX), isolated from Adenium obesum, that was sent for virus screening at Australian Government post-entry quarantine (PEQ) facilities after being imported into Australia from China. The AobVX genome is 6781 nucleotides in length excluding the poly(A) tail and is predicted to encode conserved potexvirus proteins and sequence motifs across five open reading frames. The RNA-dependent RNA polymerase of this virus shares the highest amino acid sequence similarity with that of nerine potexvirus 1 (58.7% identity) and nerine virus X (58.58% identity). This is the first report of a positive-sense single-stranded RNA virus in A. obesum related to members of the genus Potexvirus in the family Alphaflexiviridae.

Complete genome sequence of viola mottle virus, revealing its synonymous relationship to tulip virus X.

Viola mottle virus (VMoV) was discovered in Viola odorata showing symptoms of reduced growth, leaf mottling, and whitish stripes on flowers in northern Italy in 1977. This virus has been provisionally classified as a member of the genus Potexvirus based on its morphological, serological, and biological characteristics. However, since genetic information of VMoV has never been reported, the taxonomic status of this virus is unclear. Here, we report the first complete genome sequence of VMoV to clarify its taxonomic position. Its genomic RNA is 6,052 nucleotides long, excluding the 3'-terminal poly(A) tail, and has five open reading frames (ORFs) typical of potexviruses. Among potexviruses, VMoV showed the most similarity to tulip virus X (TVX) with 81.1-81.2% nucleotide and 90.4-90.7% amino acid sequence identity in ORF1 and 82.9-83.5% nucleotide and 93.2-95.2% amino acid sequence identity in ORF5. These values are much higher than the species demarcation threshold for the genus. Phylogenetic analysis also indicated that VMoV is nested within the clade of TVX isolates. These data demonstrate that VMoV and TVX are members of the same species.

The Phylogeography of Potato Virus X Shows the Fingerprints of Its Human Vector.

Potato virus X (PVX) occurs worldwide and causes an important potato disease. Complete PVX genomes were obtained from 326 new isolates from Peru, which is within the potato crop's main domestication center, 10 from historical PVX isolates from the Andes (Bolivia, Peru) or Europe (UK), and three from Africa (Burundi). Concatenated open reading frames (ORFs) from these genomes plus 49 published genomic sequences were analyzed. Only 18 of them were recombinants, 17 of them Peruvian. A phylogeny of the non-recombinant sequences found two major (I, II) and five minor (I-1, I-2, II-1, II-2, II-3) phylogroups, which included 12 statistically supported clusters. Analysis of 488 coat protein (CP) gene sequences, including 128 published previously, gave a completely congruent phylogeny. Among the minor phylogroups, I-2 and II-3 only contained Andean isolates, I-1 and II-2 were of both Andean and other isolates, but all of the three II-1 isolates were European. I-1, I-2, II-1 and II-2 all contained biologically typed isolates. Population genetic and dating analyses indicated that PVX emerged after potato's domestication 9000 years ago and was transported to Europe after the 15th century. Major clusters A-D probably resulted from expansions that occurred soon after the potato late-blight pandemic of the mid-19th century. Genetic comparisons of the PVX populations of different Peruvian Departments found similarities between those linked by local transport of seed potato tubers for summer rain-watered highland crops, and those linked to winter-irrigated crops in nearby coastal Departments. Comparisons also showed that, although the Andean PVX population was diverse and evolving neutrally, its spread to Europe and then elsewhere involved population expansion. PVX forms a basal Potexvirus genus lineage but its immediate progenitor is unknown. Establishing whether PVX's entirely Andean phylogroups I-2 and II-3 and its Andean recombinants threaten potato production elsewhere requires future biological studies.

Construction of an infectious full-length cDNA clone of potato aucuba mosaic virus.

Potato aucuba mosaic virus (PAMV), a positive single-strand RNA virus, has one of the longest genomes of the viruses in the genus Potexvirus. In 2019, potato samples with mottle and crinkling symptoms from Huzhou, Zhejiang province, China, were identified to be infected with PAMV, potato virus X (PVX), and potato virus Y (PVY) by transcriptome sequencing. To study the effects of single infection by PAMV, the full-length sequence of PAMV from Huzhou (MT193476) was determined and an infectious full-length cDNA clone was constructed. This cDNA clone was infectious by agro-infiltration, leading to systemic symptoms in Nicotiana benthamiana, tomato, pepper, and potato.

Genetic diversity of strawberry mild yellow edge virus from eastern Canada.

Strawberry mild yellow edge virus (SMYEV) is a member of the genus Potexvirus, family Alphaflexiviridae. It is one of the most common pathogenic viruses infecting cultivated strawberries worldwide. In this study, we investigated the genetic diversity of SMYEV in strawberry fields that were severely affected by strawberry decline disease in the eastern Canadian provinces of New Brunswick, Nova Scotia, Prince Edward Island and Quebec. A total of 134 SMYEV coat protein (CP) gene sequences, representing 85 nucleic acid haplotypes, were identified in 56 field samples. A highly divergent SMYEV population was found in all four provinces, but there was little genetic differentiation among the populations, and moreover, the Canadian SMYEV isolates formed a unique dissimilar, genetically divergent population group when compared to those reported in other countries. Phylogenetic analysis revealed three new SMYEV subclades that consisted mainly of Canadian variants and were composed of 76 sequence haplotypes (76/85, 88%). Mixed infections by different SMYEV variants were observed in 38 samples (38/56, 68%). Evolutionary analysis suggested that the SMYEV strains in eastern Canada possibly originated outside of Canada but adapted to conditions in the region through genetic mutations.

Complete nucleotide sequence of a new potexvirus, 'Cnidium virus X', isolated from Cnidium officinale in Japan.

A flexuous virus was detected in a Cnidium officinale plant in Japan showing mosaic symptoms. The virus was assigned to the genus Potexvirus based on analysis of its complete nucleotide sequence. The genomic RNA of the virus was 5,964 nucleotides in length, excluding the 3'-terminal poly(A) tail. It contained five open reading frames (ORFs), consistent with other members of Potexvirus. The ORF sequences differ from those of previously reported potexviruses. Phylogenetic analysis indicated that the polymerase of the virus is closely related to that of strawberry mild yellow edge virus; and the CP, to those of both yam virus X and vanilla virus X. We propose that this virus be designated as "cnidium virus X" (CnVX).

Characterization of the complete genome of euonymus yellow vein associated virus, a distinct member of the genus Potexvirus, family Alphaflexiviridae, isolated from Euonymus bungeanus Maxim in Liaoning, Northern China.

In August 2016, a yellow vein disease was observed on leaves of Euonymus bungeanus Maxim (Euonymus, Celastraceae) in Liaoning, China. Virions measuring 750 × 13 nm were observed in a sample from the diseased plant. A potexvirus was detected in the sample by small-RNA deep sequencing analysis and recovered by traditional cloning. The genome of this potexvirus consists of 7,279 nucleotides, excluding the poly(A) tail at the 3' end, and contains five open reading frames (ORFs). Based on the nucleotide and amino acid sequences of the coat protein gene, the virus shared the highest sequence similarity with white clover mosaic virus (WCMV, X16636) (40.1%) and clover yellow mosaic virus (ClYMV, D00485) (37.1%). Phylogenetic analysis showed that the virus clustered with potexviruses and is most closely related to strawberry mild yellow edge virus. These results indicate that this virus is a distinct member of the genus Potexvirus, for which the name euonymus yellow vein associated virus (EuYVAV) is proposed. To our knowledge, this is the first report of a potexvirus on E. bungeanus.

First report of Hosta virus X infecting hosta plants in Ukraine.

In September 2011, the leaf samples of hosta cultivar 'Sum and substance' were collected from the collection of Gryshko' National Botanical Garden in Kyiv. The leaves showed dark green streaking and puckering along the leaf veins. Transmission electron microscopy revealed the presence of filamentous viral particles 13 nm in diameter and 470-580 nm in length. Reverse transcription PCR (RT-PCR) analysis confirmed the presence of Hosta virus X (HVX). The sequencing of the complete genome revealed 99% identity to HVX-37 and 97.5% identity to HVX-Kr. Notably, ORF4 initiation codon presented a non-conventional start codon (UUG) like it was previously identified in HVX-37.

Two novel Alphaflexiviridae members revealed by deep sequencing of the Vanilla (Orchidaceae) virome.

The genomes of two novel viruses were assembled from 454 pyrosequencing data obtained from vanilla leaves from La Réunion. Based on genome organization and homologies, one agent was unambiguously classified as a member of the genus Potexvirus and named vanilla virus X (VVX). The second one, vanilla latent virus (VLV), is phylogenetically close to three unclassified members of the family Alphaflexiviridae with similarity to allexiviruses, and despite the presence of an additional 8-kDa open reading frame, we propose to include VLV as a new member of the genus Allexivirus. Both VVX and VLV were mechanically transmitted to vanilla plants, resulting in asymptomatic infections.

Resolution of cassava-infecting alphaflexiviruses: Molecular and biological characterization of a novel group of potexviruses lacking the TGB3 gene.

Several potexviruses (Family Alphaflexiviridae) have been reported infecting cassava (Manihot esculenta Crantz) in the Americas. They were isolated from severely diseased plants during the last 30-40 years and include: Cassava common mosaic virus (CsCMV), Cassava Caribbean mosaic virus (CsCaMV), Cassava Colombian symptomless virus (CsCSV) and Cassava virus X (CsVX). However, their definitive classification as distinct species remains unresolved for several reasons, including the lack of sequence data and unavailability of samples from original isolates. This complicates disease diagnostics, cassava germplasm exchange certification, evaluation of virus cleaning protocols and epidemiological studies. Furthermore, a recently detected novel alphaflexivirus, indicates that cassava-infecting potexviruses may be more diverse. To solve the identity of these viruses, we started indexing samples from different parts of Colombia using different sets of PCR primers, antisera available and inoculation to indicator plants. Results show that there are three major phylogenetic groups of potexviruses infecting cassava, and they correspond to CsCMV, CsVX and the newly identified Cassava new alphaflexivirus (CsNAV). Bioassays and sequence analysis established that isolates of CsNAV and CsVX cause latent infections in different cassava landraces, they are not efficiently transmitted to the indicator plant Nicotiana benthamiana and they lack the gene 3 of the conserved potexviral 'triple gene block' (TGB). In contrast, all isolates of CsCMV (which have a characteristic potexvirus genome arrangement) caused Cassava Common Mosaic Disease (CCMD) in single infections and were efficiently transmitted to N. benthamiana. Although phylogenetic analysis of the replicase sequence placed CsNAV and CsVX as members of the Potexvirus genus, their distinct genome arrangement and biological characteristics suggest they can be considered as members of a separate taxonomic group.

Strain-dependent mutational effects for Pepino mosaic virus in a natural host.

BACKGROUND: Pepino mosaic virus (PepMV) is an emerging plant pathogen that infects tomatoes worldwide. Understanding the factors that influence its evolutionary success is essential for developing new control strategies that may be more robust against the evolution of new viral strains. One of these evolutionary factors is the distribution of mutational fitness effect (DMFE), that is, the fraction of mutations that are lethal, deleterious, neutral, and beneficial on a given viral strain and host species. The goal of this study was to characterize the DMFE of introduced nonsynonymous mutations on a mild isolate of PepMV from the Chilean 2 strain (PepMV-P22). Additionally, we also explored whether the fitness effect of a given mutation depends on the gene where it appears or on epistatic interactions with the genetic background. To address this latter possibility, a subset of mutations were also introduced in a mild isolate of the European strain (PepMV-P11) and the fitness of the resulting clones measured. RESULTS: A collection of 25 PepMV clones each containing a single nucleotide nonsynonymous substitution was created by site-directed mutagenesis and the fitness of each mutant was determined. PepMV-P22 genome showed a high degree of robustness against point mutations, with 80% of mutations being either neutral or even beneficial and only 20% being deleterious or lethal. We found that the effect of mutations strongly depended on the gene in which they were introduced. Mutations with the largest average beneficial effects were those affecting the RdRp gene, in contrast to mutations affecting TGB1 and CP genes, for which the average effects were deleterious. Moreover, significant epistatic interactions were observed between nonsynonymous mutations and the genetic background, meaning that the effect of a given nucleotide substitution on a particular genomic context cannot be predicted by knowing its effect in a different one. CONCLUSIONS: Our results indicated that PepMV genome has a surprisingly high robustness against mutations. We also found that fitness consequences of a given mutation differ between the two strains analyzed. This discovery suggests that the strength of selection, and thus the rates of evolution, vary among PepMV strains.

Identification and characterization of Bamboo mosaic virus isolates from a naturally occurring coinfection in Bambusa xiashanensis.

Bamboo mosaic virus (BaMV) is a well-characterized virus and a model of virus-host interaction in plants. Here, we identified naturally occurring BaMV isolates from Fujian Province, China and furthermore describe a naturally occurring BaMV coinfection in bamboo (Bambusa xiashanensis) plants. Two different types of BaMV were identified, represented by isolates BaMV-XSNZHA7 (X7) and BaMV-XSNZHA10 (X10). The phylogenetic relationships between X7- and X10-like isolates and published BaMV isolates were determined based on genomic RNA and amino acid sequences. Three clusters were identified, indicating that BaMV is highly diverse. The in planta viral replication kinetics were determined for X7 and X10 in single infections and in an X7/X10 coinfection. The peak viral load during coinfection was significantly greater than that during single infection with either virus and contained a slightly higher proportion of X10 virus than X7, suggesting that X10-like viruses may have a fitness advantage when compared to X7-like viruses.

Complete genome sequences of two highly divergent Japanese isolates of Plantago asiatica mosaic virus.

Plantago asiatica mosaic virus (PlAMV) is a member of the genus Potexvirus and has an exceptionally wide host range. It causes severe damage to lilies. Here we report on the complete nucleotide sequences of two new Japanese PlAMV isolates, one from the eudicot weed Viola grypoceras (PlAMV-Vi), and the other from the eudicot shrub Nandina domestica Thunb. (PlAMV-NJ). Their genomes contain five open reading frames (ORFs), which is characteristic of potexviruses. Surprisingly, the isolates showed only 76.0-78.0 % sequence identity with each other and with other PlAMV isolates, including isolates from Japanese lily and American nandina. Amino acid alignments of the replicase coding region encoded by ORF1 showed that the regions between the methyltransferase and helicase domains were less conserved than other regions, with several insertions and/or deletions. Phylogenetic analyses of the full-length nucleotide sequences revealed a moderate correlation between phylogenetic clustering and the original host plants of the PlAMV isolates. This study revealed the presence of two highly divergent PlAMV isolates in Japan.

Characterisation of siRNAs derived from new isolates of bamboo mosaic virus and their associated satellites in infected ma bamboo (Dendrocalamus latiflorus).

We characterised the virus-derived small interfering RNAs (vsiRNA) of bamboo mosaic virus (Ba-vsiRNAs) and its associated satellite RNA (satRNA)-derived siRNAs (satsiRNAs) in a bamboo plant (Dendrocalamus latiflorus) by deep sequencing. Ba-vsiRNAs and satsiRNAs of 21-22 nt in length, with both (+) and (-) polarity, predominated. The 5'-terminal base of Ba-vsiRNA was biased towards A, whereas a bias towards C/U was observed in sense satsiRNAs, and towards A in antisense satsiRNAs. A large set of bamboo genes were identified as potential targets of Ba-vsiRNAs and satsiRNAs, revealing RNA silencing-based virus-host interactions in plants. Moreover, we isolated and characterised new isolates of bamboo mosaic virus (BaMV; 6,350 nt) and BaMV-associated satRNA (satBaMV; 834 nt), designated BaMV-MAZSL1 and satBaMV-MAZSL1, respectively.

Biological and molecular characterization of a putative new potexvirus infecting Senna occidentalis.

In this work, we report the complete genome sequence of, production of polyclonal antibodies against, and development of biological assays for a putative new potexvirus, named senna mosaic virus (SenMV), found infecting Senna occidentalis in the state of São Paulo, Brazil. The complete genome sequence of SenMV comprises 6775 nucleotides excluding the poly(A) tail. The genome organization is similar to those of other potexviruses, with five open reading frames coding for RNA-dependent RNA polymerase (RdRp), the triple gene block (TGB 1, 2, and 3) proteins, and coat protein (CP). The virus was transmitted to S. occidentalis by mechanical inoculation and trimming scissors, but not by seeds.

Complete genome sequence of a strain of Actinidia virus X detected in Ribes nigrum cv. Baldwin showing unusual symptoms.

A Ribes-infecting strain of the potexvirus Actinidia virus X (AVX-RV3124) was isolated from black currant plants (Ribes nigrum cv. Baldwin, accession 3124-03D1) showing symptoms of leaf chlorosis and deformity. This is the first description of the complete genome sequence of an isolate of this virus and the first detection of a potexvirus in Ribes. The genome of AVX-RV3124 consists of 6,888 nucleotides (nt) excluding the poly(A) tail at the 3' terminus. When AVX-RV3124 was compared to the available sequence of the AVX isolate in GenBank (accession no. KC568202), two large indel events (72 nt and 33 nt) were identified in the replicase coding region of RV3124. Evidence of recombination was detected upstream of the 3' terminus of the replicase gene of both virus isolates, providing further evidence of a common origin.

Simultaneous detection of papaya ringspot virus, papaya leaf distortion mosaic virus, and papaya mosaic virus by multiplex real-time reverse transcription PCR.

Both the single infection of papaya ringspot virus (PRSV), papaya leaf distortion mosaic virus (PLDMV) or papaya mosaic virus (PapMV) and double infection of PRSV and PLDMV or PapMV which cause indistinguishable symptoms, threaten the papaya industry in Hainan Island, China. In this study, a multiplex real-time reverse transcription PCR (RT-PCR) was developed to detect simultaneously the three viruses based on their distinctive melting temperatures (Tms): 81.0±0.8°C for PRSV, 84.7±0.6°C for PLDMV, and 88.7±0.4°C for PapMV. The multiplex real-time RT-PCR method was specific and sensitive in detecting the three viruses, with a detection limit of 1.0×10(1), 1.0×10(2), and 1.0×10(2) copies for PRSV, PLDMV, and PapMV, respectively. Indeed, the reaction was 100 times more sensitive than the multiplex RT-PCR for PRSV, and 10 times more sensitive than multiplex RT-PCR for PLDMV. Field application of the multiplex real-time RT-PCR demonstrated that some non-symptomatic samples were positive for PLDMV by multiplex real-time RT-PCR but negative by multiplex RT-PCR, whereas some samples were positive for both PRSV and PLDMV by multiplex real-time RT-PCR assay but only positive for PLDMV by multiplex RT-PCR. Therefore, this multiplex real-time RT-PCR assay provides a more rapid, sensitive and reliable method for simultaneous detection of PRSV, PLDMV, PapMV and their mixed infections in papaya.

Simultaneous detection of Cymbidium mosaic virus and Odontoglossum ringspot virus in orchids using multiplex RT-PCR.

A system for simultaneous detection of two orchid-infecting viruses was developed and applied to several orchid species. The detection system involved multiplex reverse transcription-polymerase chain reaction (RT-PCR) and could simultaneously identify Cymbidium mosaic virus (CymMV) and Odontoglossum ringspot virus (ORSV) from the orchid species studied. Multiplex RT-PCR was conducted using two virus-specific primer pairs and an internal control pair of primers to amplify the CymMV and ORSV coat protein regions, and orchid 18S rDNA, respectively. For optimization of multiplex RT-PCR conditions, serial dilutions of total RNA and cDNA were performed and the detection limit of the system was evaluated. The optimized multiplex detection system for CymMV and ORSV was applied to various orchid species, including several cultivars of Doritaenopsis, Cymbidium, Dendrobium, and Phalaenopsis to test the efficacy of this method. Our results indicate that the multiplex RT-PCR detection system will be a rapid, simple, and precise diagnosis tool in a range of orchid species.

Molecular characterization of yam virus X, a new potexvirus infecting yams (Dioscorea spp) and evidence for the existence of at least three distinct potexviruses infecting yams.

The genome of yam virus X (YVX), a new member of the genus Potexvirus from yam (Dioscorea trifida), was completely sequenced. Structural and phylogenetic analysis showed that the closest relative of YVX is nerine virus X. A prevalence study found YVX only in plants maintained in Guadeloupe and showed that it also infects members of the complex D. cayenensis rotundata. This study provides evidence for the existence of two additional potexviruses, one of which infects D. nummularia in Vanuatu and the other, D. bulbifera and D. rotundata in Haiti and D. trifida and D. rotundata in Guadeloupe. This work also shows that existing potexvirus-specific degenerate primers targeting the ORF1-encoded polymerase domain are well suited for the identification of the three potexviruses reported here.

Complete genome sequences of new divergent potato virus X isolates and discrimination between strains in a mixed infection using small RNAs sequencing approach.

Potato virus X (PVX; genus Potexvirus, family Alphaflexiviridae, order Tymovirales) is one of the most widespread and intensively studied viruses of potato. However, little is known about its diversity in its likely center of radiation, the Andean region of South America. To fill this gap, the strategy of Illumina deep sequencing of small RNAs was used to obtain complete or near complete genome sequence of PVX from 5 symptomatically infected greenhouse and 3 field samples (Solanum tuberosum) from Peru. PVX sequences determined in this study were assigned into three different phylogenetic groups of isolates. Notably, a complete genome sequence of a representative of a new PVX phylogenetic lineage was obtained, which shows a high level of sequence dissimilarity to other completely sequenced isolates (∼17%). The new PVX genotype was detected in greenhouse and field samples. One of the field samples was infected with the mixture of two PVX strains, which were efficiently discriminated using small RNA sequencing approach. The study confirms the utility of small RNAs deep sequencing for successful viral strain differentiation and discovery of new viral strains and indicates a high diversity of PVX in the Andean region of South America, a pattern which may be expected also for other potato pathogens.