Complete genome sequence of almond luteovirus 1, a novel luteovirus infecting almond.

In this study, we report the complete genome sequence of a novel luteovirus detected in almond using high-throughput sequencing. The genome of the new luteovirus comprises 5,047 nucleotides, and its genomic organization is similar to that of the recently described nectarine stem pitting associated virus (NSPaV), with only four open reading frames, encoding replication-related proteins, the coat protein (CP), and a CP readthrough protein involved in the aphid transmission of luteovirids. Phylogenic and pairwise distance analyses showed that this virus shares 79% and 57.8% amino acid identity in the P1-P2 fusion protein and the P3-P5 protein, respectively, with the most closely related luteovirus, NSPaV, suggesting that it represents a novel species, for which the name "Almond associated luteovirus 1" is proposed. To our knowledge, this is the first report of an almond-infecting luteovirus.

Wheat Virus Identification Within Infected Tissue Using Nanopore Sequencing Technology.

Viral diseases are a limiting factor to wheat production. Viruses are difficult to diagnose in the early stages of disease development and are often confused with nutrient deficiencies or other abiotic problems. Immunological methods are useful to identify viruses, but specific antibodies may not be available or require high virus titer for detection. In 2015 and 2017, wheat plants containing Wheat streak mosaic virus (WSMV) resistance gene, Wsm2, were found to have symptoms characteristic of WSMV. Serologically, WSMV was detected in all four samples. Additionally, High Plains wheat mosaic virus (HPWMoV) was also detected in one of the samples. Barley yellow dwarf virus (BYDV) was not detected, and a detection kit was not readily available for Triticum mosaic virus (TriMV). Initially, cDNA cloning and Sanger sequencing were used to determine the presence of WSMV; however, the process was time-consuming and expensive. Subsequently, cDNA from infected wheat tissue was sequenced with single-strand, Oxford Nanopore sequencing technology (ONT). ONT was able to confirm the presence of WSMV. Additionally, TriMV was found in all of the samples and BYDV in three of the samples. Deep coverage sequencing of full-length, single-strand WSMV revealed variation compared with the WSMV Sidney-81 reference strain and may represent new variants which overcome Wsm2. These results demonstrate that ONT can more accurately identify causal virus agents and has sufficient resolution to provide evidence of causal variants.

Red clover-associated luteovirus - a newly classifiable member of the genus Luteovirus with an enamo-like P5 protein.

This study reports the complete genomic sequence of a novel virus isolated from red clover. According to its genomic organization, its similarity to luteoviruses, and a greater than 10% difference in all genes, this virus isolate likely represents a new luteovirus species. As seen in nectarine stem pitting-associated virus (NSPaV) and NSPaV-South Korea (SK) luteoviruses, it differs from typical luteoviruses through the absence of ORF3a and ORF4 encoding movement proteins. Furthermore, its P5 protein (responsible for aphid transmission) is more similar to the P5 of enamoviruses than that of luteoviruses. The virus isolate has been named red clover-associated luteovirus (RCaV).

Characterization of a new apple luteovirus identified by high-throughput sequencing.

BACKGROUND: 'Rapid Apple Decline' (RAD) is a newly emerging problem of young, dwarf apple trees in the Northeastern USA. The affected trees show trunk necrosis, cracking and canker before collapse in summer. In this study, we discovered and characterized a new luteovirus from apple trees in RAD-affected orchards using high-throughput sequencing (HTS) technology and subsequent Sanger sequencing. METHODS: Illumina NextSeq sequencing was applied to total RNAs prepared from three diseased apple trees. Sequence reads were de novo assembled, and contigs were annotated by BLASTx. RT-PCR and 5'/3' RACE sequencing were used to obtain the complete genome of a new virus. RT-PCR was used to detect the virus. RESULTS: Three common apple viruses and a new luteovirus were identified from the diseased trees by HTS and RT-PCR. Sequence analyses of the complete genome of the new virus show that it is a new species of the genus Luteovirus in the family Luteoviridae. The virus is graft transmissible and detected by RT-PCR in apple trees in a couple of orchards. CONCLUSIONS: A new luteovirus and/or three known viruses were found to be associated with RAD. Molecular characterization of the new luteovirus provides important information for further investigation of its distribution and etiological role.

Molecular characterization of a novel luteovirus infecting apple by next-generation sequencing.

A new single-stranded positive-sense RNA virus, which shares the highest nucleotide (nt) sequence identity of 53.4% with the genome sequence of cherry-associated luteovirus South Korean isolate (ChALV-SK, genus Luteovirus), was discovered in this work. It is provisionally named apple-associated luteovirus (AaLV). The complete genome sequence of AaLV comprises 5,890 nt and contains eight open reading frames (ORFs), in a very similar arrangement that is typical of members of the genus Luteovirus. When compared with other members of the family Luteoviridae, ORF1 of AaLV was found to encompass another ORF, ORF1a, which encodes a putative 32.9-kDa protein. The ORF1-ORF2 region (RNA-dependent RNA polymerase, RdRP) showed the greatest amino acid (aa) sequence identity (59.7%) to that of cherry-associated luteovirus Czech Republic isolate (ChALV-CZ, genus Luteovirus). The results of genome sequence comparisons and phylogenetic analysis, suggest that AaLV should be a member of a novel species in the genus Luteovirus. To our knowledge, it is the sixth member of the genus Luteovirus reported to naturally infect rosaceous plants.

Identification and characterization of a new member of the genus Luteovirus from cherry.

The complete genomic sequence of a new virus from cherry trees was determined. Its genome is 5857 nt long and resembles that of members of the genus Luteovirus in its genomic organization and nucleotide sequence. Based on the species demarcation criteria for luteoviruses, the virus represents a new luteovirus species. Furthermore, a 47-nt-long inverted repeat was found at the 3' end of its genome. The virus has been provisionally named cherry-associated luteovirus (ChALV) and is the fourth member of the family Luteoviridae reported to naturally infect woody plants.

Molecular characterization of barley yellow dwarf virus in Tunisia.

Barley yellow dwarf disease is a worldwide ubiquitous virus disease of cereal crops. In order to characterize the B/CYDV isolates occurring in Tunisia, 240 barley leaves were randomly sampled from 6 fields following a North-South trend and analyzed by serological and molecular tests. DAS-ELISA results showed 40 positive samples with a prevalence of barley yellow dwarf virus (BYDV)-PAV (77.5%), followed by cereal yellow dwarf virus (CYDV)-RPV (25%) and BYDV-MAV (15%). Studies of the geographic distribution showed a high incidence of B/CYDV in the Tunisian Southern provinces. RT-PCR assays were performed to amplify the viral coat protein gene (CP) and sequence analyses revealed six BYDV-PAV haplotypes named PAV-TN1 to PAV-TN6. Phylogenetic analysis showed that the six Tunisian haplotypes were close to BYDV-PAV-II subspecies and had a strong similarity with Moroccan, Czech, French and German haplotypes. Although PAV-TN2 and PAV-TN5 showed up to 10% divergence from BYDV-PAV-II at the amino acid level, it seems to belong to the same subspecies but in a separated cluster. Our results will be important in developing appropriate control measures against BYDV disease in Tunisia.

Coat protein based molecular characterization of Barley yellow dwarf virus isolates identified on oat plants in Pakistan.

Barley yellow dwarf virus (BYDV) is a potential threat to the agriculture production. The amplified complete coat protein sequences of the isolate M07 and M12 were determined to be 597 bp and 603bp, respectively. M07 showed maximum nucleotide sequence identity of 87.6% (84.3% amino acid sequence identity) to a Chinese isolate of BYDV-PAV. Whereas, the isolate M12 showed maximum nucleotide sequence identity of 94.5% (94.0% amino acid sequence identity) to French isolate BYDV-PAV. Since more than 10 o/o differences, among the amino acid level of any gene product, is the sole criterion to discriminate between species within the family Luteoviridae, the isolate M07 that shows maximum of 84.3% (less than 90%) amino acid sequence identity with previously known Luteovirus species, is thus, recommended to be a distinct PAV species within the genus Luteovirus.

Distribution of Barley yellow dwarf virus-PAV in the Sub-Antarctic Kerguelen Islands and Characterization of Two New Luteovirus Species.

A systematic search for viral infection was performed in the isolated Kerguelen Islands, using a range of polyvalent genus-specific PCR assays. Barley yellow dwarf virus (BYDV) was detected in both introduced and native grasses such as Poa cookii. The geographical distribution of BYDV and its prevalence in P. cookii were analyzed using samples collected from various sites of the archipelago. We estimate the average prevalence of BYDV to be 24.9% in P. cookii, with significant variability between sites. BYDV genetic diversity was assessed using sequence information from two genomic regions: the P3 open reading frame (ORF) (encoding the coat protein) and the hypervariable P6 ORF region. The phylogenetic analysis in the P3 region showed that BYDV sequences segregate into three major lineages, the most frequent of which (Ker-I cluster) showed close homology with BYDV-PAV-I isolates and had very low intra-lineage diversity (0.6%). A similarly low diversity was also recorded in the hypervariable P6 region, suggesting that Ker-I isolates derive from the recent introduction of BYDV-PAV-I. Divergence time estimation suggests that BYDV-PAV-I was likely introduced in the Kerguelen environment at the same time frame as its aphid vector, Rhopalosiphum padi, whose distribution shows good overlap with that of BYDV-Ker-I. The two other lineages show more than 22% amino acid divergence in the P3 region with other known species in the BYDV species complex, indicating that they represent distinct BYDV species. Using species-specific amplification primers, the distribution of these novel species was analyzed. The high prevalence of BYDV on native Poaceae and the presence of the vector R. padi, raises the question of its impact on the vulnerable plant communities of this remote ecosystem.

[Codon bias and nucleotide substitutions in soybean dwarf virus].

Computational analysis of codon usage bias and spontaneous nucleotide substitutions in six strains of soybean dwarf virus was performed. It was shown that synonymous codon usage in the virus genes varies widely depending on the gene, gene overlapping, codon, codon's two first nucleotides, mononucleotide context located upstream and downstream of the codon, GC-content in virus-encoded genes and in the third codon position. Overlapping of genes causes a 2.5-fold decrease of the total number of nucleotide substitutions, 2.8-4.3-fold decrease of the number of synonymous substitutions in the third codon position, 1.4-1.6-fold decrease of the dicodon content in genes. At the same time there is a significant increase of nonsynonymous substitutions in the second codon position as well as a high codon bias (Fop = 0.94-1.0) and correlation between the nucleotide content in genes and in the third codon positions (r = 0.73-0.74). The results obtained evidence for selection of dicodons in the viral genes.

Molecular analysis of soybean dwarf virus isolates in the eastern United States confirms the presence of both D and Y strains and provides evidence of mixed infections and recombination.

Soybean dwarf virus (SbDV), first identified as an agricultural problem in Japan, has emerged as a growing problem in the Midwestern United States. The majority of research on SbDV had been limited to four lab maintained strains from Japan. SbDV had been found in clover in the eastern United States, but these isolates rarely emerged into soybeans. These isolates were analyzed by multiplex PCR and sequencing, revealing that some were infections of both Y and D components, including a recombinant subisolate. Phylogenetic analyses for the US isolates revealed a broad diversity of SbDV, with selection pressure greater on the movement protein than the coat protein. The field isolates from the Eastern United States showed differences in symptoms, aphid transmission and host range, demonstrating that a study of field isolates is an important complement to laboratory maintained strains in understanding the biology and evolution of plant viruses.

A novel strain of Beet western yellows virus infecting sugar beet with two distinct genotypes differing in the 5'-terminal half of genome.

The complete genomic sequences of two distinct Beet western yellows virus (BWYV) genotypes infecting sugar beet in Beijing, named as BWYV-BJ(A) and BWYV-BJ(B) (GenBank accession number HM804471, HM804472, respectively), were determined by RT-PCR sub-cloning approach. BWYV-BJ(A) and BWYV-BJ(B) were 5674 and 5626nt in length, respectively. BWYV-BJ(B) was 48nt shorter than BWYV-BJ(A) in the regions 1589-1615 and 1629-1649nt. Sequence alignment analysis showed that the full length of BWYV-BJ(A) and BWYV-BJ(B) shared 93% nucleotide sequence identity, with relatively high variability within ORFs 0, 1, 2 (at the nucleotide level was 86.3-88.8%) and high conservation within ORFs 3, 4, 5 (at the nucleotide level was 99.3-99.5%). The complete nucleotide sequences of BWYV-BJ(A) and BWYV-BJ(B) were most related to BWYV-US (80.6 and 79.0%, respectively). ORFs 1, 2 of BWYV-BJ(A) and BWYV-BJ(B) shared the highest homology with BWYV-US (nucleotide identity 91.2-93.3, 86.7-89.5%, respectively) and their ORFs 3, 4 were more closely related to BWYV-IM. However, their ORF5 were more closely related to that of Cucurbit aphid-borne yellows virus China strain (CABYV-CHN), with 68.1 and 68.5% nucleotide identity, respectively. Based on the sequence and phylogenetic analysis, we proposed that BWYV-BJ was at least a novel strain of BWYV, and BWYV-BJ(A), BWYV-BJ(B) were two distinct genotypes of BWYV-BJ. In addition, phylogenetic analysis and recombination analysis suggested that BWYV-BJ(A) and BWYV-BJ(B) might be recombinant viruses.

Modeling the competition between viruses in a complex plant-pathogen system.

In this article, we propose a mathematical model that describes the competition between two plant virus strains (MAV and PAV) for both the host plant (oat) and their aphid vectors. We found that although PAV is transmitted by two aphids and MAV by only one, this fact, by itself, does not explain the complete replacement of MAV by PAV in New York State during the period from 1961 through 1976; an interpretation that is in agreement with the theories of A. G. Power. Also, although MAV wins the competition within aphids, we assumed that, in 1961, PAV mutated into a new variant such that this new variant was able to overcome MAV within the plants during a latent period. As shown below, this is sufficient to explain the swap of strains; that is, the dominant MAV was replaced by PAV, also in agreement with Power's expectations.

The complete nucleotide sequence of the barley yellow dwarf GPV isolate from China shows that it is a new member of the genus Polerovirus.

The complete nucleotide sequence of the ssRNA genome of a Chinese GPV isolate of barley yellow dwarf virus (BYDV) was determined. It comprised 5673 nucleotides, and the deduced genome organization resembled that of members of the genus Polerovirus. It was most closely related to cereal yellow dwarf virus-RPV (77% nt identity over the entire genome; coat protein amino acid identity 79%). The GPV isolate also differs in vector specificity from other BYDV strains. Biological properties, phylogenetic analyses and detailed sequence comparisons suggest that GPV should be considered a member of a new species within the genus, and the name Wheat yellow dwarf virus-GPV is proposed.

Sequence diversity of readthrough proteins of Soybean dwarf virus isolates from the Midwestern United States.

The amino acid sequence diversity of readthrough proteins (RTPs) of 24 dwarfing isolates of Soybean dwarf virus (SbDV) from Wisconsin and Illinois was analyzed. The RTP, a minor component of viral capsids, has a significant role in specificity of aphid transmission of luteovirids. Among the isolates, nucleotide sequence identities ranged from 95 to 100%. The predicted amino acid sequences differed at 56 amino acid positions in the 54 kDa RTD compared to only five positions in the 22 kDa CP. Phylogenetic analysis of both amino acid and nucleotide sequences showed three distinct clusters of SbDV isolates.

A Chinese isolate of barley yellow dwarf virus-PAV represents a third distinct species within the PAV serotype.

The complete nucleotide sequence of barley yellow dwarf virus (BYDV) PAV-CN genomic RNA was determined. This represents the seventh complete genome sequence of a BYDV-PAV serotype. The genome organization of PAV-CN was comparable to that of other BYDV-PAV serotypes, but the nucleotide sequence of full genome was only 76.9-80.3% similar. Sequence similarity of individual open reading frames and untranslated regions (UTR) between PAV-CN and other PAV isolates ranged from 37.9 to 98.2%. Overall, PAV-CN was most similar to BYDV-PAS, which belongs to one of two distinct species within the PAV serotype of BYDV, although the 5' UTR and ORF1 of PAV-CN was most similar to BYDV-GAV, another member of the genus Luteovirus that is not serologically related to BYDV-PAV. These data suggest that PAV-CN may have undergone a recombination event with GAV and that PAV-CN represents a third distinct species within the PAV serotype of BYDV.

Genetic analysis of a novel Alaska barley yellow dwarf virus in the family Luteoviridae.

A new plant virus belonging to the family Luteoviridae and isolated from diseased oat (Avena sativa L.) plants was discovered in Alaska in 2003. Even though plants with red/orange leaves were indicative of barley yellow dwarf disease, they were not reactive to specific antibodies corresponding to barley yellow dwarf virus (BYDV)-MAV, -PAV, -SGV, and cereal yellow dwarf virus-RPV from enzyme-linked immunosorbent assays (ELISA). An alternative RT-PCR assay that incorporated Shu-F/Yan-R primers for detection of BYDV-MAV, -PAS, -PAV, and SGV was effective in producing approximately 830-nt fragments that contained genomic sequences to the 3'-terminus of the polymerase gene (ORF 2), the intergenic region ( approximately 113 nt), the coat protein gene (ORF 3), and the putative movement gene (ORF 4). The Alaskan isolates were most similar to BYDV-MAV with only about 77 and 80% amino acid identity in the CP and ORF 4, respectively. The Alaska isolates coat protein gene sequences differed in several regions that otherwise are conserved among BYDV-MAV isolates, and may be important in serological variations, accounting for the negative ELISA results. Based upon sequence and serological differences, we concluded that the Alaskan BYDV-MAV-like isolates formed a novel species tentatively in the genus Luteovirus, and propose the name BYDV-ORV (oat red-leaf virus).

Discrimination of four soybean dwarf virus strains by dot-blot hybridization with specific probes.

Soybean dwarf virus (SbDV) is divided into four strains (YS, YP, DS, and DP) on the basis of host symptoms in infected soybean plants and on aphid vector specificity. To detect and discriminate each strain of SbDV by dot-blot hybridization, probes Y, D, S, and P were prepared. Probes Y and D, covering most of the 3'-noncoding region of the viral genome containing the sequence of small subgenomic RNA, could discriminate strains in accord with the host symptoms. Probes S and P were derived from the 5'-half of open reading frame 5 encoding the N-terminal half of the readthrough domain which is closely related to the aphid vector specificity of each strain. Thus, the four SbDV strains could be discriminated by the combination of these probes. This method, based on a procedure specific to the SbDV sequence, is a good alternative for routine examination of infected plants in soybean breeding programs for evaluation of resistance to SbDV and for assessment of the distribution of each strain in epidemiological studies.

Subcellular localization of the Triple Gene Block movement proteins of Beet necrotic yellow vein virus by electron microscopy.

The Triple Gene Block proteins TGBp1, TGBp2, and TGBp3 of Beet necrotic yellow vein virus (BNYVV) are required for efficient cell-to-cell spread of the infection. The TGB proteins can drive cell-to-cell movement of BNYVV in trans when expressed from a co-inoculated BNYVV RNA 3-based 'replicon'. TGBp2 and TGBp3 expressed from the replicon were nonfunctional in this assay if they were fused to the green fluorescent protein (GFP), but addition of a hemagglutinin (HA) tag to their C-termini did not incapacitate movement. Immunogold labeling of ultrathin sections treated with HA-specific antibodies localized TGBp2-HA and TGBp3-HA to what are probably structurally modified plasmodesmata (Pd) in infected cells. A similar subcellular localization was observed for TGBp1. Large gold-decorated membrane-rich bodies containing what appear to be short fragments of endoplasmic reticulum were observed near the cell periphery. The modified gold-decorated Pd and the membrane-rich bodies were not observed when the TGB proteins were produced individually in infections using the Tobacco mosaic virus P30 protein to drive cell-to-cell movement, indicating that these modifications are specific for TGB-mediated movement.

Molecular characterization of a virus from the family Luteoviridae associated with cotton blue disease.

Cotton blue disease is an aphid-transmitted cotton disease described in Brazil in 1962 as Vein Mosaic "var. Ribeirão Bonito". At present it causes economically important losses in cotton crops if control measures are not implemented. The observed symptoms and mode of transmission have prompted researchers to speculate that cotton blue disease could be attributed to a member of the family Luteoviridae, but there was no molecular evidence supporting this hypothesis. We have amplified part of the genome of a virus associated with this disease using degenerate primers for members of the family Luteoviridae. Sequence analysis of the entire capsid and a partial RdRp revealed a virus probably belonging to the genus Polerovirus. Based on our results we propose that cotton blue disease is associated with a virus with the putative name Cotton leafroll dwarf virus (CLRDV).