Metagenomic identification of novel viruses of maize and teosinte in North America.

BACKGROUND: Maize-infecting viruses are known to inflict significant agronomic yield loss throughout the world annually. Identification of known or novel causal agents of disease prior to outbreak is imperative to preserve food security via future crop protection efforts. Toward this goal, a large-scale metagenomic approach utilizing high throughput sequencing (HTS) was employed to identify novel viruses with the potential to contribute to yield loss of graminaceous species, particularly maize, in North America. RESULTS: Here we present four novel viruses discovered by HTS and individually validated by Sanger sequencing. Three of these viruses are RNA viruses belonging to either the Betaflexiviridae or Tombusviridae families. Additionally, a novel DNA virus belonging to the Geminiviridae family was discovered, the first Mastrevirus identified in North American maize. CONCLUSIONS: Metagenomic studies of crop and crop-related species such as this may be useful for the identification and surveillance of known and novel viral pathogens of crops. Monitoring related species may prove useful in identifying viruses capable of infecting crops due to overlapping insect vectors and viral host-range to protect food security.

Opium Poppy Mosaic Virus Has an Xrn-Resistant, Translated Subgenomic RNA and a BTE 3' CITE.

Opium poppy mosaic virus (OPMV) is a recently discovered umbravirus in the family Tombusviridae OPMV has a plus-sense genomic RNA (gRNA) of 4,241 nucleotides (nt) from which replication protein p35 and p35 extension product p98, the RNA-dependent RNA polymerase (RdRp), are expressed. Movement proteins p27 (long distance) and p28 (cell to cell) are expressed from a 1,440-nt subgenomic RNA (sgRNA2). A highly conserved structure was identified just upstream from the sgRNA2 transcription start site in all umbraviruses, which includes a carmovirus consensus sequence, denoting generation by an RdRp-mediated mechanism. OPMV also has a second sgRNA of 1,554 nt (sgRNA1) that starts just downstream of a canonical exoribonuclease-resistant sequence (xrRNAD). sgRNA1 codes for a 30-kDa protein in vitro that is in frame with p28 and cannot be synthesized in other umbraviruses. Eliminating sgRNA1 or truncating the p30 open reading frame (ORF) without affecting p28 substantially reduced accumulation of OPMV gRNA, suggesting a functional role for the protein. The 652-nt 3' untranslated region of OPMV contains two 3' cap-independent translation enhancers (3' CITEs), a T-shaped structure (TSS) near its 3' end, and a Barley yellow dwarf virus-like translation element (BTE) in the central region. Only the BTE is functional in luciferase reporter constructs containing gRNA or sgRNA2 5' sequences in vivo, which differs from how umbravirus 3' CITEs were used in a previous study. Similarly to most 3' CITEs, the OPMV BTE links to the 5' end via a long-distance RNA-RNA interaction. Analysis of 14 BTEs revealed additional conserved sequences and structural features beyond the previously identified 17-nt conserved sequence.IMPORTANCEOpium poppy mosaic virus (OPMV) is an umbravirus in the family Tombusviridae We determined that OPMV accumulates two similarly sized subgenomic RNAs (sgRNAs), with the smaller known to code for proteins expressed from overlapping open reading frames. The slightly larger sgRNA1 has a 5' end just upstream from a previously predicted xrRNAD site, identifying this sgRNA as an unusually long product produced by exoribonuclease trimming. Although four umbraviruses have similar predicted xrRNAD sites, only sgRNA1 of OPMV can code for a protein that is an extension product of umbravirus ORF4. Inability to generate the sgRNA or translate this protein was associated with reduced gRNA accumulation in vivo We also characterized the OPMV BTE structure, a 3' cap-independent translation enhancer (3' CITE). Comparisons of 13 BTEs with the OPMV BTE revealed additional stretches of sequence similarity beyond the 17-nt signature sequence, as well as conserved structural features not previously recognized in these 3' CITEs.

Carrot mottle virus ORF4 movement protein targets plasmodesmata by interacting with the host cell SUMOylation system.

Plant virus movement proteins (MPs) facilitate virus spread in their plant hosts, and some of them are known to target plasmodesmata (PD). However, how the MPs target PD is still largely unknown. Carrot mottle virus (CMoV) encodes the ORF3 and ORF4 proteins, which are involved in CMoV movement. In this study, we used CMoV as a model to study the PD targeting of a plant virus MP. We showed that the CMoV ORF4 protein, but not the ORF3 protein, modified PD and led to the virus movement. We found that the CMoV ORF4 protein interacts with the host cell small ubiquitin-like modifier (SUMO) 1, 2 and the SUMO-conjugating enzyme SCE1, resulting in the ORF4 protein SUMOylation. Downregulation of mRNAs for NbSCE1 and NbSUMO impaired CMoV infection. The SUMO-interacting motifs (SIMs) LVIVF, VIWV, and a lysine residue at position 78 (K78) are required for the ORF4 protein SUMOylation. The mutation of these motifs prevented the protein to efficiently target PD, and further slowed or completely abolished CMoV systemic movement. Finally, we found that some of these motifs are highly conserved among umbraviruses. Our data suggest that the CMoV ORF4 protein targets PD by interacting with the host cell SUMOylation system.

First report of Ethiopian tobacco bushy top virus and its associated satellite RNA infecting common bean (Phaseolus vulgaris L.) in Zambia.

During surveys for common bean viruses in Central Province of Zambia in April 2018, symptoms of bushy top, deep green curled branches and patchy leaf chlorosis were observed on five plants in a 2-ha farmer's field. Total RNA was isolated from symptomatic leaf samples using the CTAB method (Chang et al. 1993). The RNA from one sample (CP414-1) was used to construct a cDNA library with the Illumina TruSeq RNA Library Prep Kit (Illumina, San Diego, CA), followed by high-throughput sequencing (HTS) on the Illumina MiSeq platform that generated ~3.1M single-end raw reads of ~300 nucleotides (nt) each. A total of 355,885 reads showed hits to Ethiopian tobacco bushy top virus (ETBTV; Umbravirus), ETBTV satellite RNA (satRNA) and peanut mottle virus (PeMoV, Potyvirus) based on BLASTn analysis. The full-length genomes of ETBTV (4239-nt; MT225089), its satRNA (521-nt; MT225092) and PeMoV (9,643-nt) were assembled from the HTS reads using Geneious R11.1.2 (Biomatters, Auckland, New Zealand). The obtained complete genome sequences of ETBTV (MT225089) and ETBTV satRNA (MT225092) shared 88% and 95% nt identities, respectively with the corresponding viral (KJ918748) and satRNA (KJ918747) sequences of isolate 18-2 (Abraham et al. 2014). The near complete PeMoV genome was 89% identical to isolate Liaoning (MH270528). The HTS results were validated by two-step RT-PCR analyses of the five field-collected samples using newly designed primer pairs (data not shown). All five samples gave the expected 988-bp ETBTV-specific and 521-bp satRNA-specific DNA bands while three samples produced the expected 2100-bp PeMoV-specific fragment. The virus specificities of the agent specific PCR fragments were ascertained by Sanger sequencing (ETBTV: MT225090-91; ETBTV satRNA: MT225093-94; PeMoV: MT900843-44) and they shared 98-100% identities with their corresponding HTS-derived sequences. To further probe for the presence of an ETBTV helper virus, the samples were screened by RT-PCR with the degenerate primer pair Lu1-mod-F/C2R3 that was modified from Robertson et al. (1991). The expected 245-bp DNA bands was obtained from all five samples, indicating the presence of a possible luteovirus or polerovirus target in these samples. The BLASTn analyses of the two Sanger sequenced gel-eluted products (MT900845-46) showed that they shared 100% identity with each other and 96% nt identity with cowpea polerovirus 1 (CPPV1, KX599163). Leaf tissue extracts from a common bean plant that was confirmed by RT-PCR to be positive for all four agents were rub-inoculated onto Nicotiana occidentalis and common bean (Sutter Pink) plants (n=5 each) at the three fully expanded leaf stage, with a buffer inoculation as control. Systemic foliar symptoms consisting of leaf deformation, stunting and leaf bushy top were observed on all ten plants, 10 days post-inoculation whereas the control plants remained symptomless. All the test plants were screened by RT-PCR as described above. The results showed that all five N. occidentalis plants were positive for ETBTV+ETBTVsatRNA, the five common bean plants tested positive for ETBTV+satRNA+PeMoV, and all 10 plants of both species were negative for CPPV1. To the best of our knowledge, this is the first report of ETBTV, ETBTV satRNA and CPPV1 infecting common bean in Zambia, and the first molecular based confirmation of PeMoV occurrence in the country. Ongoing studies are focused on determining the extent of the disease spread and assessment of its economic impact.

Expanding the Plant Virome: Umbra-Like Viruses Use Host Proteins for Movement.

Before the very recent discovery of umbra-like viruses (ULVs), the signature defining feature of all plant RNA viruses was the encoding of specialized RNA-binding movement proteins (MPs) for transiting their RNA genomes through gated plasmodesmata to establish systemic infections. The vast majority of ULVs share umbravirus-like RNA-dependent RNA polymerases and 3'-terminal structures, but they differ by not encoding cell-to-cell and long-distance MPs and by not relying on a helper virus for trans-encapsidation and plant-to-plant transmission. The recent finding that two groups of ULVs do not necessarily encode MPs is expanding our understanding of the minimum requirements for modern plant RNA viruses. ULV CY1 from citrus uses host protein PHLOEM PROTEIN 2 (PP2) for systemic movement, and related ULVs encode a capsid protein, thereby providing an explanation for the lack of helper viruses present in many ULV-infected plants. ULVs thus resemble the first viruses that infected plants, which were likely deposited from feeding organisms and would have similarly required the use of host proteins such as PP2 to exit initially infected cells.

Dissecting dynamic plant virus synergism in mixed infections of poleroviruses, umbraviruses, and tombusvirus-like associated RNAs.

Mixed infections of a plant infecting polerovirus, umbravirus, and/or tombusvirus-like associated RNAs (tlaRNAs) produce unique virus disease complexes that exemplify "helper-dependence" interactions, a type of viral synergism that occurs when a "dependent" virus that lacks genes encoding for certain protein products necessary for it to complete its infection cycle can utilize complementary proteins encoded by a co-infecting "helper" virus. While much research has focused on polerovirus-umbravirus or polerovirus-tlaRNA interactions, only recently have umbravirus-tlaRNA interactions begun to be explored. To expand on the limited understanding of umbravirus-tlaRNA interactions in such disease complexes, we established various co-infection pairings of the polerovirus turnip yellows virus (TuYV), the umbravirus carrot mottle virus (CMoV), and three different tlaRNAs-carrot red leaf virus aRNAs (CRLVaRNAs) gamma and sigma, and the TuYVaRNA ST9-in the model plant Nicotiana benthamiana, then investigated the effects of these different co-infections on tlaRNA systemic movement within the host, and on virus accumulation, and aphid and mechanical transmission of each of these viruses. We found that CMoV alone could support systemic movement of each of the tlaRNAs, making this the second report to demonstrate such an interaction between an umbravirus and tlaRNAs. We also report for the first time that CMoV could also impart mechanical transmissibility to the tlaRNAs sigma and ST9, and that co-infections of either of these tlaRNAs with both TuYV and CMoV increased the efficiency with which TuYV could be mechanically co-transmitted with CMoV.

Conserved Structure Associated with Different 3'CITEs Is Important for Translation of Umbraviruses.

The cap-independent translation of plus-strand RNA plant viruses frequently depends on 3' structures to attract translation initiation factors that bind ribosomal subunits or bind directly to ribosomes. Umbraviruses are excellent models for studying 3' cap-independent translation enhancers (3'CITEs), as umbraviruses can have different 3'CITEs in the central region of their lengthy 3'UTRs, and most also have a particular 3'CITE (the T-shaped structure or 3'TSS) near their 3' ends. We discovered a novel hairpin just upstream of the centrally located (known or putative) 3'CITEs in all 14 umbraviruses. These CITE-associated structures (CASs) have conserved sequences in their apical loops and at the stem base and adjacent positions. In 11 umbraviruses, CASs are preceded by two small hairpins joined by a putative kissing loop interaction (KL). Converting the conserved 6-nt apical loop to a GNRA tetraloop in opium poppy mosaic virus (OPMV) and pea enation mosaic virus 2 (PEMV2) enhanced translation of genomic (g)RNA, but not subgenomic (sg)RNA reporter constructs, and significantly repressed virus accumulation in Nicotiana benthamiana. Other alterations throughout OPMV CAS also repressed virus accumulation and only enhanced sgRNA reporter translation, while mutations in the lower stem repressed gRNA reporter translation. Similar mutations in the PEMV2 CAS also repressed accumulation but did not significantly affect gRNA or sgRNA reporter translation, with the exception of deletion of the entire hairpin, which only reduced translation of the gRNA reporter. OPMV CAS mutations had little effect on the downstream BTE 3'CITE or upstream KL element, while PEMV2 CAS mutations significantly altered KL structures. These results introduce an additional element associated with different 3'CITEs that differentially affect the structure and translation of different umbraviruses.

Enhanced Apiaceous Potyvirus Phylogeny, Novel Viruses, and New Country and Host Records from Sequencing Apiaceae Samples.

The family Apiaceae comprises approximately 3700 species of herbaceous plants, including important crops, aromatic herbs and field weeds. Here we report a study of 10 preserved historical or recent virus samples of apiaceous plants collected in the United Kingdom (UK) import interceptions from the Mediterranean region (Egypt, Israel and Cyprus) or during surveys of Australian apiaceous crops. Seven complete new genomic sequences and one partial sequence, of the apiaceous potyviruses apium virus Y (ApVY), carrot thin leaf virus (CaTLV), carrot virus Y (CarVY) and celery mosaic virus (CeMV) were obtained. When these 7 and 16 earlier complete non-recombinant apiaceous potyvirus sequences were subjected to phylogenetic analyses, they split into 2 separate lineages: 1 containing ApVY, CeMV, CarVY and panax virus Y and the other CaTLV, ashitabi mosaic virus and konjac virus Y. Preliminary dating analysis suggested the CarVY population first diverged from CeMV and ApVY in the 17th century and CeMV from ApVY in the 18th century. They also showed the "time to most recent common ancestor" of the sampled populations to be more recent: 1997 CE, 1983 CE and 1958 CE for CarVY, CeMV and ApVY, respectively. In addition, we found a new family record for beet western yellows virus in coriander from Cyprus; a new country record for carrot torradovirus-1 and a tentative novel member of genus Ophiovirus as a co-infection in a carrot sample from Australia; and a novel member of the genus Umbravirus recovered from a sample of herb parsley from Israel.

Discovery and Characterization of a Novel Umbravirus from Paederia scandens Plants Showing Leaf Chlorosis and Yellowing Symptoms.

Umbraviruses are a special class of plant viruses that do not encode any viral structural proteins. Here, a novel umbravirus that has been tentatively named Paederia scandens chlorosis yellow virus (PSCYV) was discovered through RNA-seq in Paederia scandens plants showing leaf chlorosis and yellowing symptoms. The PSCYV genome is a 4301 nt positive-sense, single strand RNA that contains four open reading frames (ORFs), i.e., ORF1-4, that encode P1-P4 proteins, respectively. Together, ORF1 and ORF2 are predicted to encode an additional protein, RdRp, through a -1 frameshift mechanism. The P3 protein encoded by ORF3 was predicted to be the viral long-distance movement protein. P4 was determined to function as the viral cell-to-cell movement protein (MP) and transcriptional gene silencing (TGS) suppressor. Both P1 and RdRp function as weak post-transcriptional gene silencing (PTGS) suppressors of PSCYV. The PVX-expression system indicated that all viral proteins may be symptom determinants of PSCYV. Phylogenetic analysis indicated that PSCYV is evolutionarily related to members of the genus Umbravirus in the family Tombusviridae. Furthermore, a cDNA infectious clone of PSCYV was successfully constructed and used to prove that PSCYV can infect both Paederia scandens and Nicotiana benthamiana plants through mechanical inoculation, causing leaf chlorosis and yellowing symptoms. These findings have broadened our understanding of umbraviruses and their host range.

Novel 3' Proximal Replication Elements in Umbravirus Genomes.

The 3' untranslated regions (UTRs) of positive-strand RNA plant viruses commonly contain elements that promote viral replication and translation. The ~700 nt 3'UTR of umbravirus pea enation mosaic virus 2 (PEMV2) contains three 3' cap-independent translation enhancers (3'CITEs), including one (PTE) found in members of several genera in the family Tombusviridae and another (the 3'TSS) found in numerous umbraviruses and several carmoviruses. In addition, three 3' terminal replication elements are found in nearly every umbravirus and carmovirus. For this report, we have identified a set of three hairpins and a putative pseudoknot, collectively termed "Trio", that are exclusively found in a subset of umbraviruses and are located just upstream of the 3'TSS. Modification of these elements had no impact on viral translation in wheat germ extracts or in translation of luciferase reporter constructs in vivo. In contrast, Trio hairpins were critical for viral RNA accumulation in Arabidopsis thaliana protoplasts and for replication of a non-autonomously replicating replicon using a trans-replication system in Nicotiana benthamiana leaves. Trio and other 3' terminal elements involved in viral replication are highly conserved in umbraviruses possessing different classes of upstream 3'CITEs, suggesting conservation of replication mechanisms among umbraviruses despite variation in mechanisms for translation enhancement.

Structural Analysis and Whole Genome Mapping of a New Type of Plant Virus Subviral RNA: Umbravirus-Like Associated RNAs.

We report the biological and structural characterization of umbravirus-like associated RNAs (ulaRNAs), a new category of coat-protein dependent subviral RNA replicons that infect plants. These RNAs encode an RNA-dependent RNA polymerase (RdRp) following a -1 ribosomal frameshift event, are 2.7-4.6 kb in length, and are related to umbraviruses, unlike similar RNA replicons that are related to tombusviruses. Three classes of ulaRNAs are proposed, with citrus yellow vein associated virus (CYVaV) placed in Class 2. With the exception of CYVaV, Class 2 and Class 3 ulaRNAs encode an additional open reading frame (ORF) with movement protein-like motifs made possible by additional sequences just past the RdRp termination codon. The full-length secondary structure of CYVaV was determined using Selective 2' Hydroxyl Acylation analyzed by Primer Extension (SHAPE) structure probing and phylogenic comparisons, which was used as a template for determining the putative structures of the other Class 2 ulaRNAs, revealing a number of distinctive structural features. The ribosome recoding sites of nearly all ulaRNAs, which differ significantly from those of umbraviruses, may exist in two conformations and are highly efficient. The 3' regions of Class 2 and Class 3 ulaRNAs have structural elements similar to those of nearly all umbraviruses, and all Class 2 ulaRNAs have a unique, conserved 3' cap-independent translation enhancer. CYVaV replicates independently in protoplasts, demonstrating that the reported sequence is full-length. Additionally, CYVaV contains a sequence in its 3' UTR that confers protection to nonsense mediated decay (NMD), thus likely obviating the need for umbravirus ORF3, a known suppressor of NMD. This initial characterization lays down a road map for future investigations into these novel virus-like RNAs.

Biological and Genetic Characterization of Pod Pepper Vein Yellows Virus-Associated RNA From Capsicum frutescens in Wenshan, China.

Tombusvirus-like associated RNAs (tlaRNAs) are positive-sense single-stranded RNAs found in plants co-infected with some viruses of the genus Polerovirus. Pod pepper vein yellows virus (PoPeVYV) was recently reported as a new recombinant polerovirus causing interveinal yellowing, stunting, and leaf rolling in Capsicum frutescens plants at Wenshan city, Yunnan province, China. The complete genome sequence of its associated RNA has now been determined by next-generation sequencing and reverse transcription (RT) polymerase chain reaction (PCR). PoPeVYV-associated RNA (PoPeVYVaRNA) (GenBank Accession No. MW323470) has 2970 nucleotides and is closely related to other group II tlaRNAs, particularly tobacco bushy top disease-associated RNA (TBTDaRNA, GenBank Accession No. EF529625). In infection experiments on Nicotiana benthamiana and C. frutescens plants, synergism between PoPeVYVaRNA and PoPeVYV was demonstrated, leading to severe interveinal yellowing of leaves and stunting of plants. The results provide further information on the genetic and biological properties of the various agents associated with pepper vein yellows disease (PeVYD).

Differential use of 3'CITEs by the subgenomic RNA of Pea enation mosaic virus 2.

The genomic RNA (gRNA) of Pea enation mosaic virus 2 (PEMV2) is the template for p33 and -1 frameshift product p94. The PEMV2 subgenomic RNA (sgRNA) encodes two overlapping ORFs, p26 and p27, which are required for movement and stability of the gRNA. Efficient translation of p33 requires two of three 3' proximal cap-independent translation enhancers (3'CITEs): the kl-TSS, which binds ribosomes and engages in a long-distance interaction with the 5'end; and the adjacent eIF4E-binding PTE. Unlike the gRNA, all three 3'CITEs were required for efficient translation of the sgRNA, which included the ribosome-binding 3'TSS. A hairpin in the 5' proximal coding region of p26/p27 supported translation by the 3'CITEs by engaging in a long-distance RNA:RNA interaction with the kl-TSS. These results strongly suggest that the 5' ends of PEMV2 gRNA and sgRNA connect with the 3'UTR through similar long-distance interactions while having different requirements for 3'CITEs.