Probing of plant transcriptomes reveals the hidden genetic diversity of the family Secoviridae.

Secoviruses are single-stranded RNA viruses that infect plants. In the present study, we identified 61 putative novel secoviral genomes in various plant species by mining publicly available plant transcriptome data. These viral sequences represent the genomes of 13 monopartite and 48 bipartite secovirids. The genome sequences of 52 secovirids were coding-complete, and nine were partial. Except for small open reading frames (ORFs) determined in waikaviral genomes and RNA2 of torradoviruses, all of the recovered genomes/genome segments contained a large ORF encoding a polyprotein. Based on genome organization and phylogeny, all but three of the novel secoviruses were assigned to different genera. The genome organization of two identified waika-like viruses resembled that of the recently identified waika-like virus Triticum aestivum secovirus. Phylogenetic analysis revealed a pattern of host-virus co-evolution in a few waika- and waika-like viruses and increased phylogenetic diversity of nepoviruses. The study provides a basis for further investigation of the biological properties of these novel secoviruses.

Molecular characterisation of a novel sadwavirus infecting cattleya orchids in Australia.

The complete genome sequence of a novel sadwavirus infecting cattleya orchids in South East Queensland is described. Isometric virions of c. 27 nm diameter were observed in sap extracts viewed under a transmission electron microscope, and the genome sequence of this virus was determined by high-throughput sequencing. The viral genome consists of two RNA components, 5,910 and 4,435 nucleotides (nt) in length, each encoding a long polyprotein, with predicted cleavage sites at H/Y, E/G, Q/S, and Q/G for the RNA1 and T/G for the RNA2 translation products, respectively. RNA2 has an additional small ORF of 684 nt near the 3' untranslated region. Phylogenetic analysis based on an amino acid sequence alignment of the Pro-Pol region suggested that this virus is most closely related to pineapple secovirus A, a member of the subgenus Cholivirus, but warrants classification as a member of a new species because it exhibited no more than 64% amino acid identity in pairwise sequence comparisons. Because of the prominent purple ringspots that were observed on the leaves of some of the plants, we propose the name "cattleya purple ringspot virus" for this virus (suggested species name: "Sadwavirus cattleyacola").

Identification of a putative novel cholivirus in the transcriptome of Gymnema sylvestre R. Br.

Gymnema sylvestre is a tropical climber species that is widely used in traditional medicine since ages. In the present study, the transcriptome datasets of G. sylvestre available in public domain were screened for the presence of novel plant viral sequences and a putative novel virus tentatively named as Gymnema sylvestre virus 1 (GysV1) was identified. Coding-complete genome segments of GysV1 that are 6.35 kb (RNA1) and 3.98 kb (RNA2) long possessed a single large open reading frame coding for a polyprotein. BLASTp, sequence identity and phylogenetic analyses revealed the relatedness of GysV1 to the members of the subgenus Cholivirus (genus Sadwavirus; family Secoviridae; order Picornavirales). Based on the species demarcation criteria of the family Secoviridae, GysV1 can be regarded as a new cholivirus member.

Genomic characterization of a novel torradovirus infecting Arctium lappa L. in China.

Burdock (Arctium lappa L.) is not only a popular vegetable crop but also an important medicinal plant. In burdock plants with symptoms of leaf mosaic, a novel torradovirus tentatively named "burdock mosaic virus" (BdMV) was identified by high-throughput sequencing. The complete genomic sequence of BdMV was further determined using RT-PCR and the rapid amplification of cDNA ends (RACE) method. The genome is composed of two positive-sense single-stranded RNAs. RNA1 (6991 nt) encodes a polyprotein of 2186 aa, and RNA2 (4700 nt) encodes a protein of 201 aa and a polyprotein of 1212 aa that is predicted to be processed into one movement protein (MP) and three coat proteins (CPs). The Pro-Pol region of RNA1 and the CP region of RNA2 shared the highest amino acid sequence identity of 74.0% and 70.6%, respectively, with the corresponding sequences of lettuce necrotic leaf curl virus (LNLCV) isolate JG3. Phylogenetic analysis based on the amino acid sequences of the Pro-Pol and CP regions showed that BdMV clustered with other non-tomato-infecting torradoviruses. Taken together, these results suggest that BdMV is a new member of the genus Torradovirus.

Genome sequence of pineapple secovirus B, a second sadwavirus reported infecting Ananas comosus.

The complete genome sequence of pineapple secovirus B (PSV-B), a new virus infecting pineapple (Ananas comosus) on the island of Oahu, Hawaii, was determined by high-throughput sequencing (HTS). The genome comprises two RNAs that are 5,956 and 3,808 nt long, excluding the 3'-end poly-A tails, both coding for a single large polyprotein. The RNA1 polyprotein contains five conserved domains associated with replication, while the RNA2 polyprotein is cleaved into the movement protein and coat protein. PSV-B is representative of a new species in the subgenus Cholivirus (genus Sadwavirus; family Secoviridae), as the level of amino acid sequence identity to recognized members of this subgenus in the Pro-Pol and coat protein regions is below currently valid species demarcation thresholds.

Re-examination of nepovirus polyprotein cleavage sites highlights the diverse specificities and evolutionary relationships of nepovirus 3C-like proteases.

Plant-infecting viruses of the genus Nepovirus (subfamily Comovirinae, family Secoviridae, order Picornavirales) are bipartite positive-strand RNA viruses with each genomic RNA encoding a single large polyprotein. The RNA1-encoded 3C-like protease cleaves the RNA1 polyprotein at five sites and the RNA2 polyprotein at two or three sites, depending on the nepovirus. The specificity of nepovirus 3C-like proteases is notoriously diverse, making the prediction of cleavage sites difficult. In this study, the position of nepovirus cleavage sites was systematically re-evaluated using alignments of the RNA1 and RNA2 polyproteins, phylogenetic relationships of the proteases, and sequence logos to examine specific preferences for the P6 to P1' positions of the cleavage sites. Based on these analyses, the positions of previously elusive cleavage sites, notably the 2a-MP cleavage sites of subgroup B nepoviruses, are now proposed. Distinct nepovirus protease clades were identified, each with different cleavage site specificities, mostly determined by the nature of the amino acid at the P1 and P1' positions of the cleavage sites, as well as the P2 and P4 positions. The results will assist the prediction of cleavage sites for new nepoviruses and help refine the taxonomy of nepoviruses. An improved understanding of the specificity of nepovirus 3C-like proteases can also be used to investigate the cleavage of plant proteins by nepovirus proteases and to understand their adaptation to a broad range of hosts.

Analysis of public domain plant transcriptomes expands the phylogenetic diversity of the family Secoviridae.

Secoviruses are mono-/bipartite plant-infecting, icosahedral RNA viruses that incite economically important diseases in plants. In the present study, nine secoviruses tentatively named as Ananas comosus secovirus (AcSV), Artocarpus altilis secovirus (AaSV), Boehmeria nivea secovirus (BnSV), Gynostemma pentaphyllum secovirus (GpSV), Orobanche cernua secovirus (OcSV), Paris polyphylla secovirus 1 (PpSV1), Paris polyphylla secovirus 2 (PpSV2), Rhododendron delavayi secovirus (RdSV), and Yucca gloriosa secovirus (YgSV) were identified by probing publicly available transcriptomes of eight plant species. Coding-complete genome/genome segments of all the identified viruses encoding a polyprotein were recovered. Two of the nine identified viruses-AcSV and GpSV were discovered in few of the small RNA libraries of respective plant species. Putative cleavage sites were predicted in polyproteins encoded by AcSV, GpSV, PpSV2 and YgSV genome segments. Phylogenetic and sequence identity analyses revealed that AcSV, GpSV and YgSV, PpSV1 and RdSV putatively belong to the genera- Sadwavirus (sub genus: Cholivirus), Fabavirus, Nepovirus and Waikavirus, respectively, while AaSV, BnSV, and PpSV2 may represent a distinct group of viruses within the family Secoviridae as they could not conclusively be assigned to a single genus.

Complete genome sequence of Codonopsis torradovirus A, a novel torradovirus infecting Codonopsis lanceolata in South Korea.

We herein present the complete genome sequence of codonopsis torradovirus A (CoTVA), which was isolated from Codonopsis lanceolata (deodeok) in Gangwon-do, South Korea. The CoTVA genome contains two positive-sense RNA segments, namely RNA1 (6922 nucleotides), which encodes a predicted polyprotein, and RNA2 (4613 nucleotides), which encodes a movement protein and coat proteins (CPs). The proteinase-polymerase (Pro-Pol) and CP amino acid sequences were 75% and 54% identical, respectively, to those of motherwort yellow mosaic virus. Pairwise comparisons of the Pro-Pol and CP sequences revealed that the virus described in this study should be considered a member of a new torradovirus species. Phylogenetic analysis of the Pro-Pol sequence encoded by RNA1 and the CP region encoded by RNA2 indicated that CoTVA is a new member of the genus Torradovirus in the family Secoviridae. CoTVA is the first torradovirus detected in Codonopsis lanceolata.

Development of a New Tomato Torrado Virus-Based Vector Tagged with GFP for Monitoring Virus Movement in Plants.

Green fluorescent protein (GFP)-tagged viruses are basic research tools widely applied in studies concerning molecular determinants of disease during virus infection. Here, we described a new generation of genetically stable infectious clones of tomato torrado virus isolate Kra (ToTVpJL-Kra) that could infect Nicotiana benthamiana and Solanum lycopersicum. Importantly, a modified variant of the viral RNA2-with inserted sGFP (forming, together with virus RNA1, into ToTVpJL-KraGFP)-was engineered as well. RNA2 of ToTVpJL-KraGFP was modified by introducing an additional open reading frame (ORF) of sGFP flanked with an amino acid-coding sequence corresponding to the putative virus protease recognition site. Our further analysis revealed that sGFP-tagged ToTV-Kra was successfully passaged by mechanical inoculation and spread systemically in plants. Therefore, the clone might be applied in studying the in vivo cellular, tissue, and organ-level localization of ToTV during infection. By performing whole-plant imaging, followed by fluorescence and confocal microscopy, the presence of the ToTVpJL-KraGFP-derived fluorescence signal was confirmed in infected plants. All this information was verified by sGFP-specific immunoprecipitation and western blot analysis. The molecular biology of the torradovirus-plant interaction is still poorly characterized; therefore, the results obtained here opened up new possibilities for further research. The application of sGFP-tagged virus infectious clones and their development method can be used for analyzing plant-virus interactions in a wide context of plant pathology.

Apple latent spherical virus (ALSV)-induced gene silencing in a medicinal plant, Lithospermum erythrorhizon.

Lithospermum erythrorhizon is a medicinal plant that produces shikonin, a red lipophilic naphthoquinone derivative that accumulates exclusively in roots. The biosynthetic steps required to complete the naphthalene ring of shikonin and its mechanism of secretion remain unclear. Multiple omics studies identified several candidate genes involved in shikonin production. The functions of these genes can be evaluated using virus-induced gene silencing (VIGS) systems, which have been shown advantageous in introducing iRNA genes into non-model plants. This study describes the development of a VIGS system using an apple latent spherical virus (ALSV) vector and a target gene, phytoene desaturase (LePDS1). Virus particles packaged in Nicotiana benthamiana were inoculated into L. erythrorhizon seedlings, yielding new leaves with albino phenotype but without disease symptoms. The levels of LePDS1 mRNAs were significantly lower in the albino plants than in mock control or escape plants. Virus-derived mRNA was detected in infected plants but not in escape and mock plants. Quantitative PCR and deep sequencing analysis indicated that transcription of another hypothetical PDS gene (LePDS2) also decreased in the defective leaves. Virus infection, however, had no effect on shikonin production. These results suggest that virus-based genetic transformation and the VIGS system silence target genes in soil-grown L. erythrorhizon.

Estimation of the functions of viral RNA silencing suppressors by apple latent spherical virus vector.

Apple latent spherical virus (ALSV) is a latent virus with wide host range of plant species. In the present study, we prepared ALSV vectors expressing RNA silencing suppressors (RSSs) from eight plant viruses: P19 of carnation Italian ring spot virus (tombusvirus), 2b of peanut stunt virus (cucumovirus), NSs of tomato spotted wilt virus (tospovirus), HC-Pro of bean yellow mosaic virus (potyvirus), γb of barley stripe mosaic virus (hordeivirus), P15 of peanut clump virus (pecluvirus), P1 of rice yellow mottle virus (sobemovirus), or P21 of beet yellows virus (closterovirus). These vectors were inoculated to Nicotiana benthamiana to investigate the effects of RSSs on the virulence and accumulation of ALSV. Among the vectors, ALSV expressing NSs (ALSV-NSs) developed severe mosaic symptoms in newly developed leaves followed by plant death. Infection of ALSV-γb induced characteristic concentric ringspot symptoms on leaves, and plants infected with ALSV-HC-Pro showed mosaic and dwarf symptoms. Infection of the other five ALSV vectors did not show symptoms. ELISA and immunoblot assay indicated that virus titer increased in leaves infected with ALSV-NSs, γb, HC-Pro, or P19. RT-qPCR indicated that the amount of ALSV in plants infected with ALSV-NSs was increased by approximately 45 times compared with that of wtALSV without expression of any RSS. When ALSV-P19, NSs, or HC-Pro was inoculated to Cucumis sativus plants, none of these ALSV vectors induced symptoms, but accumulation of ALSV in plants infected with ALSV-NSs was increased, suggesting that functions of RSSs on virulence and accumulation of ALSV depend on host species.

RNA Silencing-Mediated Apple Latent Spherical Virus Vaccine in Plants.

The apple latent spherical virus (ALSV), originally isolated from an apple tree in Japan, is a small spherical virus with a diameter of 25 nm and comprises a bisegmented, single-stranded RNA genome (RNA1 and RNA2) and three different capsid proteins (Vp25, Vp20, and Vp24). The virus can experimentally infect a broad range of plants including, not only model plants (Arabidopsis thaliana and Nicotiana species) but also economically important crops such as cucumber, soybean, tomato, fruit trees, and flowers. ALSV has been used as an effective plant virus vector for virus-induced gene silencing (VIGS) to assess gene functions because the virus infects most of the host plants without showing any symptoms and induces a uniform knockout phenotype in infected plants. Moreover, the VIGS persists throughout plant growth in infected plants. Here, we show that genetically engineered ALSV vectors (ALSV vaccines) containing a partial genome sequence of pathogenic viruses display a high degree of cross-protection against the challenge inoculation of the corresponding pathogenic viruses. Treatment effects can also be expected in virus-infected plants by subsequent inoculation with ALSV vaccine.

Strawberry Mottle Virus (Family Secoviridae, Order Picornavirales) Encodes a Novel Glutamic Protease To Process the RNA2 Polyprotein at Two Cleavage Sites.

Strawberry mottle virus (SMoV) belongs to the family Secoviridae (order Picornavirales) and has a bipartite genome with each RNA encoding one polyprotein. All characterized secovirids encode a single protease related to the picornavirus 3C protease. The SMoV 3C-like protease was previously shown to cut the RNA2 polyprotein (P2) at a single site between the predicted movement protein and coat protein (CP) domains. However, the SMoV P2 polyprotein includes an extended C-terminal region with a coding capacity of up to 70 kDa downstream of the presumed CP domain, an unusual characteristic for this family. In this study, we identified a novel cleavage event at a P↓AFP sequence immediately downstream of the CP domain. Following deletion of the PAFP sequence, the polyprotein was processed at or near a related PKFP sequence 40 kDa further downstream, defining two protein domains in the C-terminal region of the P2 polyprotein. Both processing events were dependent on a novel protease domain located between the two cleavage sites. Mutagenesis of amino acids that are conserved among isolates of SMoV and of the related Black raspberry necrosis virus did not identify essential cysteine, serine, or histidine residues, suggesting that the RNA2-encoded SMoV protease is not related to serine or cysteine proteases of other picorna-like viruses. Rather, two highly conserved glutamic acid residues spaced by 82 residues were found to be strictly required for protease activity. We conclude that the processing of SMoV polyproteins requires two viral proteases, the RNA1-encoded 3C-like protease and a novel glutamic protease encoded by RNA2.IMPORTANCE Many viruses encode proteases to release mature proteins and intermediate polyproteins from viral polyproteins. Polyprotein processing allows regulation of the accumulation and activity of viral proteins. Many viral proteases also cleave host factors to facilitate virus infection. Thus, viral proteases are key virulence factors. To date, viruses with a positive-strand RNA genome are only known to encode cysteine or serine proteases, most of which are related to the cellular papain, trypsin, or chymotrypsin proteases. Here, we characterize the first glutamic protease encoded by a plant virus or by a positive-strand RNA virus. The novel glutamic protease is unique to a few members of the family Secoviridae, suggesting that it is a recent acquisition in the evolution of this family. The protease does not resemble known cellular proteases. Rather, it is predicted to share structural similarities with a family of fungal and bacterial glutamic proteases that adopt a lectin fold.

Gentian (Gentiana triflora) prevents transmission of apple latent spherical virus (ALSV) vector to progeny seeds.

MAIN CONCLUSION: Gentian plants ( Gentiana triflora ) severely restrict apple latent spherical virus (ALSV) invasion to the gametes (pollens and ovules) and block seed transmission to progeny plants. Early flowering of horticultural plants can be induced by infection of ALSV vector expressing Flowering Locus T (FT) gene. In the present study, flowering of gentian plants was induced by infection with an ALSV vector expressing a gentian FT gene and the patterns of seed transmission of ALSV in gentian were compared with those in apple and Nicotiana benthamiana. Infection of gentian progeny plants with ALSV was examined by quantitative reverse transcription-polymerase chain reaction (qRT-PCR), reverse transcription-loop-mediated isothermal amplification (RT-LAMP), and enzyme-linked immunosorbent assay (ELISA). ALSV was not transmitted to the progeny gentian plants, whereas small proportions of apple and N. benthamiana progeny plants are infected with ALSV. The in situ hybridization analyses indicated that ALSVs are not present in gentian pollen and ovules, but detected in most of gametes in apple and N. benthamiana. Collectively, these results suggest that seed transmission of ALSV is blocked in gentian plants through the unknown barriers present in their gametes. On the other hand, apple and N. benthamiana seem to minimize ALSV seed transmission by inhibiting viral propagation in embryos.

Secoviridae: a proposed family of plant viruses within the order Picornavirales that combines the families Sequiviridae and Comoviridae, the unassigned genera Cheravirus and Sadwavirus, and the proposed genus Torradovirus.

The order Picornavirales includes several plant viruses that are currently classified into the families Comoviridae (genera Comovirus, Fabavirus and Nepovirus) and Sequiviridae (genera Sequivirus and Waikavirus) and into the unassigned genera Cheravirus and Sadwavirus. These viruses share properties in common with other picornavirales (particle structure, positive-strand RNA genome with a polyprotein expression strategy, a common replication block including type III helicase, a 3C-like cysteine proteinase and type I RNA-dependent RNA polymerase). However, they also share unique properties that distinguish them from other picornavirales. They infect plants and use specialized proteins or protein domains to move through their host. In phylogenetic analysis based on their replication proteins, these viruses form a separate distinct lineage within the picornavirales branch. To recognize these common properties at the taxonomic level, we propose to create a new family termed "Secoviridae" to include the genera Comovirus, Fabavirus, Nepovirus, Cheravirus, Sadwavirus, Sequivirus and Waikavirus. Two newly discovered plant viruses share common properties with members of the proposed family Secoviridae but have distinct specific genomic organizations. In phylogenetic reconstructions, they form a separate sub-branch within the Secoviridae lineage. We propose to create a new genus termed Torradovirus (type species, Tomato torrado virus) and to assign this genus to the proposed family Secoviridae.

Cheravirus and Sadwavirus: two unassigned genera of plant positive-sense single-stranded RNA viruses formerly considered atypical members of the genus Nepovirus (family Comoviridae).

The genus Nepovirus (family Comoviridae) was known both for a good level of homogeneity and for the presence of atypical members. In particular, the atypical members of the genus differed by the number of capsid protein (CP) subunits. While typical nepoviruses have a single CP subunit with three structural domains, atypical nepoviruses have either three small CP subunits, probably corresponding to the three individual domains, or a large and a small subunit, probably containing two and one structural domains, respectively. These differences are corroborated by hierarchical clustering based on sequences derived from both genomic RNAs. Therefore, these atypical viruses are now classified in two distinct genera, Cheravirus (three CP subunits; type species Cherry rasp leaf virus) and Sadwavirus (two CP subunits; type species Satsuma dwarf virus).

Nucleotide sequence analysis of RNA-2 of a flat apple isolate of Cherry rasp leaf virus with regions showing greater identity to animal picornaviruses than to related plant viruses.

RNA-2 of a flat apple isolate of Cherry rasp leaf virus (CRLV-FA) appears to consist of 3274 nucleotides, excluding a 3' poly (A) tail. The data supports re-classification of CRLV in a new genus in the family Comoviridae. A single open reading frame (ORF) encoding a putative 108 kDa polyprotein was identified. Potential protease cleavage sites were identified which would result in the production of a putative movement protein (41 kDa), and 3 capsid protein subunits (24, 20, and 22 kDa, respectively). A 5'-UTR and 3'-UTR were identified, 248 nt and 146 nt long, respectively. The genome organisation of CRLV-FA RNA-2 is similar to that of Apple latent spherical virus (ALSV) RNA-2, a new member of the family Comoviridae. The Vp25 amino acid sequences were unique to CRLV-FA and ALSV (54% identity), with no relationship identified to any other virus. CRLV-FA Vp20 and Vp24 amino acid sequences were closely related to ALSV (59 and 65%, respectively) but the only other relationships identified were with a range of animal ssRNA positive-strand viruses.

Nucleotide sequence of black currant reversion associated nepovirus RNA1.

The RNA1 of black currant reversion associated nepovirus (BRAV) is 7711 nucleotides (nt) long, excluding the poly-A tail, and contains one long open reading frame (ORF) which is translated into a polyprotein of 2094 amino acids. The 5' NTR of BRAV RNA1 is 66 nt long and 78% identical with RNA2 5' NTR only over the first 57 nucleotides. The 3' non-translated region (3'NTR) is 1360 nucleotides long, and after the first 24 nucleotides 95% identical with the 3'NTR of RNA2. RNA1 3'NTR contains several stretches, 694-24 nucleotides in length, which are 60-80% similar to corresponding areas of the other viruses of the subgroup c of nepoviruses (BLMV, CLRV, PRMV or TomRSV). The 2094 amino acids-long polypeptide encoded by BRAV RNA1 is 33% identical with that of PRMV between amino acids 9 and 2057, and has significant similarity also to those of other nepoviruses and comoviruses. Conserved amino acid motifs, characteristic for the viral protease co-factor, the NTP-binding protein, the cysteine protease and the RdRp core domains, known to occur in the polyproteins of different viruses of the picornavirus-like supergroup, are all detected in the amino acid sequences encoded by BRAV RNA1.