Metatranscriptome analysis of symptomatic bitter apple plants revealed mixed viral infections with a putative novel polerovirus.

BACKGROUND: Next-generation Sequencing (NGS) combined with bioinformatic analyses constitutes a powerful approach for identifying and characterizing previously unknown viral genomes. In this study, leaf samples from bitter apple plants (Citrullus colocynthis (L.) Schrad) exhibiting symptoms such as dwarfing, leaf crinkling, and chlorosis were collected from the southern part of Kerman province, Iran. RESULTS: Putative infecting viruses were identified through de novo assembly of sequencing reads using various tools, followed by BLAST analysis. Complete genomes for Squash vein yellowing virus (SqVYV), Citrus-associated rhabdovirus (CiaRV), and a novel polerovirus-related strain termed Bitter apple aphid-borne yellows virus (BaABYV) were assembled and characterized. Additionally, a partial genome for Watermelon mosaic virus (WMV) was assembled. The genomic organization of the BaABYV was determined to be 5'-ORF0-ORF1-ORF1,2-ORF3a-ORF3-ORF3,5-ORF4-3'. Amino acid sequence identities for inferred proteins (P0 and P1, P1,2) with known poleroviruses were found to be the 90% species delineation limit, implying that BaABYV should be considered a new member of the genus Polerovirus. Recombination events were observed in the BaABYV and WMV strains; such events were not found in the CiaRV strain. CONCLUSIONS: Molecular evidence from this study suggests that C. colocynthis is a reservoir host of several plant viruses. Among them, BaABYV is proposed as a new member of the genus Polerovirus. Furthermore, the CiaRV strain has been reported for the first time from Iran.

Recessive resistance against beet chlorosis virus is conferred by the eukaryotic translation initiation factor (iso)4E in Beta vulgaris.

Eukaryotic translation initiation factors (eIFs) are important for mRNA translation but also pivotal for plant-virus interaction. Most of these plant-virus interactions were found between plant eIFs and the viral protein genome-linked (VPg) of potyviruses. In case of lost interaction due to mutation or deletion of eIFs, the viral translation and subsequent replication within its host is negatively affected, resulting in a recessive resistance. Here we report the identification of the Beta vulgaris Bv-eIF(iso)4E as a susceptibility factor towards the VPg-carrying beet chlorosis virus (genus Polerovirus). Using yeast two-hybrid and bimolecular fluorescence complementation assays, the physical interaction between Bv-eIF(iso)4E and the putative BChV-VPg was detected, while the VPg of the closely related beet mild yellowing virus (BMYV) was found to interact with the two isoforms Bv-eIF4E and Bv-eIF(iso)4E. These VPg-eIF interactions within the polerovirus-beet pathosystem were demonstrated to be highly specific, as single mutations within the predicted cap-binding pocket of Bv-eIF(iso)4E resulted in a loss of interaction. To investigate the suitability of eIFs as a resistance resource against beet infecting poleroviruses, B. vulgaris plants were genome edited by CRISPR/Cas9 resulting in knockouts of different eIFs. A simultaneous knockout of the identified BMYV-interaction partners Bv-eIF4E and Bv-eIF(iso)4E was not achieved, but Bv-eIF(iso)4EKO plants showed a significantly lowered BChV accumulation and decrease in infection rate from 100% to 28.86%, while no influence on BMYV accumulation was observed. Still, these observations support that eIFs are promising candidate genes for polerovirus resistance breeding in sugar beet.

Genomic variation in pepper vein yellows viruses in Australia, including a new putative variant, PeVYV-10.

Since the first identification and full sequence of the polerovirus pepper vein yellows virus in Australia in 2016, virus surveys of crops and weeds have sporadically identified PeVYV in different hosts and locations. Genomic comparisons of 14 PeVYV-like isolates using RT-PCR products spanning the 3' end of the RdRp region (ORF 2), the intergenic region, ORF 3a, ORF 4, and ORF 3 (1388 nt) showed that four of the PeVYV isolates might be a new variant or PeVYV-like virus. From six PeVYV-positive plants, eight PeVYV-like sequences were obtained by high-throughput sequencing, as two hosts, 5352 and 5634, contained two slightly different PeVYV-like isolates. Three of the PeVYV-like isolates were most closely related to PeVYV-6 and PeVYV-5, and two isolates were closely related to PeVYV-9 and PeVYV-2. The other three isolates shared only 69-74% nucleotide sequence identity across the whole genome with any of the other PeVYVs, despite sharing 73-98%, 87-91%, and 84-87% amino acid sequence identity in ORF 3a, ORF 3, and the RdRp (ORF 2), respectively, suggesting that this virus is a new PeVYV-like virus, which we have tentatively called PeVYV-10. This is also the first report of a PeVYV-like virus infecting garlic.

Complete nucleotide sequence of chrysanthemum virus D, a polero-like virus.

A putative new polerovirus, named "chrysanthemum virus D" (ChVD), was detected in a Chrysanthemum morifolium plant in South Korea. The virus was identified by high-throughput sequencing and confirmed by reverse transcription polymerase chain reaction. The entire ChVD genome is composed of 5,963 nucleotides and contains seven open reading frames (ORF0-5 and ORF3a), which are arranged similarly to those of other poleroviruses. These ORFs encode the putative proteins P0-5 and P3a, respectively. Pairwise amino acid sequence comparisons showed that the ChVD P0-5 and P3a proteins have 30.45-75% sequence identity to the corresponding proteins of other members of the genus Polerovirus. Since one of the species demarcation criteria for the genus Polerovirus is > 10% difference in the amino acid sequence of any gene product, the sequence comparisons indicate that ChVD represents a new species in this genus. Phylogenetic analysis of the P1-P2 and P3 amino acid sequences further indicate that ChVD is a novel polerovirus.

Genetic Variability Among U.S.-Sentinel Cotton Plot Cotton Leafroll Dwarf Virus and Globally Available Reference Isolates Based on ORF0 Diversity.

The aphid-transmitted polerovirus, cotton leafroll dwarf virus (CLRDV), first characterized from symptomatic cotton plants in South America, has been identified in commercial cotton plantings in the United States. Here, the CLRDV intraspecific diversity was investigated by comparative sequence analysis of the most divergent CLRDV coding region, ORF0/P0. Bayesian analysis of ORF0 sequences for U.S. and reference populations resolved three well-supported sister clades comprising one U.S. and two South American lineages. Principal component analysis (PCA) identified seven statistically supported intraspecific populations. The Bayesian phylogeny and PCA dendrogram-inferred relationships were congruent. Population analysis of ORF0 sequences indicated most lineages have evolved under negative selection, albeit certain sites/isolates evolved under positive selection. Both U.S. and South American isolates exhibited extensive ORF0 diversity. At least two U.S. invasion foci were associated with their founder populations in Alabama-Georgia and eastern Texas. The Alabama-Georgia founder is implicated as the source of recent widespread expansion and establishment of secondary disease foci throughout the southeastern-central United States. Based on the geographically restricted distribution, spread of another extant Texas population appeared impeded by a population bottleneck. Extant CLRDV isolates represent several putative introductions potentially associated with catastrophic weather events dispersing viruliferous cotton aphids of unknown origin(s).

Advances in Understanding the Mechanism of Cap-Independent Cucurbit Aphid-Borne Yellows Virus Protein Synthesis.

Non-canonical translation mechanisms have been described for many viral RNAs. In the case of several plant viruses, their protein synthesis is controlled by RNA elements in their genomic 3'-ends that are able to enhance cap-independent translation (3'-CITE). The proposed general mechanism of 3'-CITEs includes their binding to eukaryotic translation initiation factors (eIFs) that reach the 5'-end and AUG start codon through 5'-3'-UTR-interactions. It was previously shown that cucurbit aphid-borne yellows virus (CABYV) has a 3'-CITE, which varies in sequence and structure depending on the phylogenetic group to which the isolate belongs, possibly as a result of adaptation to the different geographical regions. In this work, the cap-independent translation mechanisms of two CABYV 3'-CITEs belonging to the Mediterranean (CMTE) and Asian (CXTE) groups, respectively, were studied. In vivo cap-independent translation assays show that these 3'-CITEs require the presence of the CABYV short genomic 5'-UTR with at least 40% adenines in cis and an accessible 5'-end for its activity. Additionally, they suggest that the eIF4E-independent CABYV 3'-CITE activities may not require either eIF4A or the eIF4F complex, but may depend on eIF4G and PABP. By pulling down host proteins using RNA baits containing both 5'- and 3'-CABYV-UTRs, 80 RNA binding proteins were identified. These interacted preferentially with either CMTE, CXTE, or both. One of these proteins, specifically interacting with the RNA containing CMTE, was HSP70.2. Preliminary results suggested that HSP70.2 may be involved in CMTE- but not CXTE-mediated cap-independent translation activity.

Meta-transcriptomic identification of groundnut RNA viruses in western Kenya and the novel detection of groundnut as a host for Cauliflower mosaic virus.

BACKGROUND: Groundnut (Arachis hypogaea L.) is the 13th most important global crop grown throughout the tropical and subtropical regions of the world. One of the major constraints to groundnut production is viruses, which are also the most economically important and most abundant pathogens among cultivated legumes. Only a few studies have reported the characterization of RNA viruses in cultivated groundnuts in western Kenya, most of which deployed classical methods of detecting known viruses. METHODS: We sampled twenty-one symptomatic and three asymptomatic groundnut leaf samples from farmers' fields in western Kenya. Total RNA was extracted from the samples followed by First-strand cDNA synthesis and sequencing on the Illumina HiSeq 2500 platform. After removing host and rRNA sequences, high-quality viral RNA sequences were de novo assembled and viral genomes annotated using the publicly available NCBI virus database. Multiple sequence alignment and phylogenetic analysis were done using MEGA X. RESULTS: Bioinformatics analyses using as low as ∼3.5 million reads yielded complete and partial genomes for Cauliflower mosaic virus (CaMV), Cowpea polerovirus 2 (CPPV2), Groundnut rosette assistor virus (GRAV), Groundnut rosette virus (GRV), Groundnut rosette virus satellite RNA (satRNA) and Peanut mottle virus (PeMoV) falling within the species demarcation criteria. This is the first report of CaMV and the second report of CPPV2 on groundnut hosts in the world. Confirmation of the detected viruses was further verified through phylogenetic analyses alongside reported publicly available highly similar viruses. PeMoV was the only seed-borne virus reported. CONCLUSION: Our findings demonstrate the power of Next Generation Sequencing in the discovery and identification of novel viruses in groundnuts. The detection of the new viruses indicates the complexity of virus diseases in groundnuts and would require more focus in future studies to establish the effect of the viruses as sole or mixed infections on the crop. The detection of PeMoV with potential origin from Malawi indicates the importance of seed certification and cross-boundary seed health testing.

Molecular Characterization of a Novel Enamovirus Infecting Raspberry.

Raspberry plants, valued for their fruits, are vulnerable to a range of viruses that adversely affect their yield and quality. Utilizing high-throughput sequencing (HTS), we identified a novel virus, tentatively named raspberry enamovirus 1 (RaEV1), in three distinct raspberry plants. This study provides a comprehensive characterization of RaEV1, focusing on its genomic structure, phylogeny, and possible transmission routes. Analysis of nearly complete genomes from 14 RaEV1 isolates highlighted regions of variance, particularly marked by indel events. The evidence from phylogenetic and sequence analyses supports the classification of RaEV1 as a distinct species within the Enamovirus genus. Among the 289 plant and 168 invertebrate samples analyzed, RaEV1 was detected in 10.4% and 0.4%, respectively. Most detections occurred in plants that were also infected with other common raspberry viruses. The virus was present in both commercial and wild raspberries, indicating the potential of wild plants to act as viral reservoirs. Experiments involving aphids as potential vectors demonstrated their ability to acquire RaEV1 but not to successfully transmit it to plants.