Dicer-Like Protein 4 and RNA-Dependent RNA Polymerase 6 Are Involved in Tomato Torrado Virus Pathogenesis in Nicotiana benthamiana.

Tomato torrado virus (ToTV) is a type member of the Torradovirus genus in the Secoviridae family known to cause severe necrosis in susceptible tomato varieties. ToTV also infects other Solanaceae plants, including Nicotiana benthamiana, where it induces distinctive disease symptoms: plant growth drop with the emergence of spoon-like malformed systemic leaves. Virus-induced post-transcriptional gene silencing (PTGS) is significant among plant defense mechanisms activated upon virus invasion. The PTGS, however, can be counteracted by suppressors of RNA silencing commonly found in viruses, which efficiently disrupt the antiviral defense of their host. Here, we addressed the question of PTGS antiviral activity and its suppression in N. benthamiana during ToTV infection-a phenomenon not described for any representative from the Torradovirus genus so far. First, we showed that neither the Vp26-a necrosis-inducing pathogenicity determinant of ToTV-nor other structural viral proteins limited the locally induced PTGS similar to p19, a well-characterized potent suppressor of RNA silencing of tombusviruses. Moreover, by employing wild-type and transgenic lines of N. benthamiana with suppressed Dicer-like 2 (DCL2), Dicer-like 4 (DCL4), Argonaute 2 and RNA-dependent RNA polymerase 6 (RDR6) proteins, we proved their involvement in anti-ToTV defense. Additionally, we identified DCL4 as the major processor of ToTV-derived siRNA. More importantly, our results indicate the essential role of the Suppressor of Gene Silencing 3 (SGS3)/RDR6 pathway in anti-ToTV defense. Finally, we conclude that ToTV might not require a potent RNA silencing suppressor during infection of the model plant N. benthamiana.

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").

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.

The Resistance of Soybean Variety Heinong 84 to Apple Latent Spherical Virus Is Controlled by Two Genetic Loci.

Apple latent spherical virus (ALSV) is widely used as a virus-induced gene silencing (VIGS) vector for function genome study. However, the application of ALSV to soybeans is limited by the resistance of many varieties. In this study, the genetic locus linked to the resistance of a resistant soybean variety Heinong 84 was mapped by high-throughput sequencing-based bulk segregation analysis (HTS-BSA) using a hybrid population crossed from Heinong 84 and a susceptible variety, Zhonghuang 13. The results showed that the resistance of Heinong 84 to ALSV is controlled by two genetic loci located on chromosomes 2 and 11, respectively. Cleaved amplified polymorphic sequence (CAPS) markers were developed for identification and genotyping. Inheritance and biochemical analyses suggest that the resistance locus on chromosome 2 plays a dominant dose-dependent role, while the other locus contributes a secondary role in resisting ALSV. The resistance locus on chromosome 2 might encode a protein that can directly inhibit viral proliferation, while the secondary resistance locus on chromosome 11 may encode a host factor required for viral proliferation. Together, these data reveal novel insights on the resistance mechanism of Heinong 84 to ALSV, which will benefit the application of ALSV as a VIGS vector.

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.

Identification and complete genome sequence of a novel sadwavirus discovered in chrysanthemum (Chrysanthemum morifolium Ramat.).

The complete genome sequence of a putative novel member of the genus Sadwavirus was determined by high-throughput sequencing of a chrysanthemum from an orchard of the Tongxiang Agricultural Science Institute in Tongxiang, Zhejiang province. The complete genome sequence was confirmed using RT-PCR and the rapid amplification of cDNA ends (RACE) method. The predicted genome of the putative virus is composed of two RNA molecules, 7016 and 6772 nucleotides in length, excluding their poly-A tails. The new virus was tentatively named "chrysanthemum sadwavirus" (ChSV). The Pro-Pol region of RNA1 and the CP region of RNA2 of ChSV shared the highest amino acid sequence identity (53.01% and 36.40%, respectively) with the corresponding sequences of lettuce secovirus 1 (LSV-1). Phylogenetic analysis showed that ChSV clustered with members of the subgenus Stramovirus (genus Sadwavirus). Taken together, these results suggest that ChSV is a new member of the genus Sadwavirus.

Cohombrillo-associated virus: a novel virus infecting Ecballium elaterium plants.

We determined the complete genome sequence of a new virus infecting Ecballium elaterium ('cohombrillo amargo') plants, a weed species common on the borders of cultivated fields in the Mediterranean region. The genome of this virus is composed of two molecules of monocistronic positive-sense RNA, 6,934 and 3,501 nucleotides in length, excluding their poly(A) tails. The highest amino acid sequence similarity (50 % identity) in the Pro-Pol core region encoded by RNA 1 was observed in the corresponding protein of strawberry latent ringspot virus. Based on pairwise comparisons and phylogenetic analysis, this virus, tentatively named "cohombrillo-associated virus" (CoAV), appears to be a member of a new species in the genus Stralarivirus (family Secoviridae), for which the name "Stralarivirus elaterii" is proposed. This new virus has different putative cleavage patterns from members of other species belonging to this genus.

Virome Analysis of Aconitum carmichaelii Reveals Infection by Eleven Viruses, including Two Potentially New Species.

Aconitum carmichaelii is a herbaceous herb indigenous to China that has been cultivated for traditional medicine for centuries. Virus-like symptoms of A. carmichaelii plants were observed on leaves in some A. carmichaelii plantations in Zhanyi and Wuding Counties, Yunnan Province, southwest China. High-throughput sequencing (HTS) was performed on 28 symptomatic plants, and the results revealed infection with 11 viruses, including 2 novel viruses and 9 previously described viruses: Aconitum amalgavirus 1 (AcoAV-1), aconite virus A (AcVA), cucumber mosaic virus (CMV), currant latent virus (CuLV), apple stem grooving virus (ASGV), chilli veinal mottle virus (ChiVMV), tomato spotted wilt orthotospovirus (TSWV), tobacco vein distorting virus (TVDV), and potato leafroll virus (PLRV). Two novel viruses tentatively named Aconitum potyvirus 1 and Aconitum betapartitivirus 1, were supported by sequence and phylogenetic analysis results of their genomes. We proposed the names Potyvirus aconiti and Betapartitivirus aconiti. RT-PCR assays of 142 plants revealed the predominance and widespread distribution of CMV, AcVA, and AcoPV-1 in plantations. The detection of isolates of CuLV, ASGV, ChiVMV, TSWV, TVDV, and PLRV infections for the first time in A. carmichaelii expands their known host ranges.

ICTV Virus Taxonomy Profile: Secoviridae 2022.

Members of the family Secoviridae are non-enveloped plant viruses with mono- or bipartite linear positive-sense ssRNA genomes with a combined genome of 9 to 13.7 kb and icosahedral particles 25-30 nm in diameter. They are related to picornaviruses and are members of the order Picornavirales. Genera in the family are distinguished by the host range, vector, genomic features and phylogeny of the member viruses. Most members infect dicotyledonous plants, and many cause serious disease epidemics. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) report on the family Secoviridae, which is available at ictv.global/report/secoviridae.

Cowpea Mosaic Virus Outperforms Other Members of the Secoviridae as In Situ Vaccine for Cancer Immunotherapy.

In situ vaccination for cancer immunotherapy uses intratumoral administration of small molecules, proteins, nanoparticles, or viruses that activate pathogen recognition receptors (PRRs) to reprogram the tumor microenvironment and prime systemic antitumor immunity. Cowpea mosaic virus (CPMV) is a plant virus that─while noninfectious toward mammals─activates mammalian PRRs. Application of CPMV as in situ vaccine (ISV) results in a potent and durable efficacy in tumor mouse models and canine patients; data indicate that CPMV outperforms small molecule PRR agonists and other nonrelated plant viruses and virus-like particles (VLPs). In this work, we set out to compare the potency of CPMV versus other plant viruses from the Secoviridae. We developed protocols to produce and isolate cowpea severe mosaic virus (CPSMV) and tobacco ring spot virus (TRSV) from plants. CPSMV, like CPMV, is a comovirus with genome and protein homology, while TRSV lacks homology and is from the genus nepovirus. When applied as ISV in a mouse model of dermal melanoma (using B16F10 cells and C57Bl6J mice), CPMV outperformed CPSMV and TRSV─again highlighting the unique potency of CPMV. Mechanistically, the increased potency is related to increased signaling through toll-like receptors (TLRs)─in particular, CPMV signals through TLR2, 4, and 7. Using knockout (KO) mouse models, we demonstrate here that all three plant viruses signal through the adaptor molecule MyD88─with CPSMV and TRSV predominantly activating TLR2 and 4. CPMV induced significantly more interferon ß (IFNß) compared to TRSV and CPSMV; therefore, IFNß released upon signaling through TLR7 may be a differentiator for the observed potency of CPMV-ISV. Additionally, CPMV induced a different temporal pattern of intratumoral cytokine generation characterized by significantly increased inflammatory cytokines 4 days after the second of 2 weekly treatments, as if CPMV induced a "memory response". This higher, longer-lasting induction of cytokines may be another key differentiator that explains the unique potency of CPMV-ISV.

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.

Genomic characterization of a new torradovirus from common fleabane (Erigeron annuus).

A new virus was detected in common fleabane (Erigeron annuus) showing virus-like symptoms including leaf yellowing, mosaic, and mottling. This virus is tentatively named "fleabane yellow mosaic virus" (FbYMV). The complete genome sequence consists of two RNA segments of 7,133 nt (RNA 1) and 4,810 nt (RNA 2), excluding the poly(A) tract. Sequence analysis showed a genome organization comparable to that of members of the genus Torradovirus. The level of sequence identity between FbYMV and known members of the genus Torradovirus was below the cutoff established by the ICTV for species demarcation. Therefore, FbYMV should be classified as a new member of the genus Torradovirus.

Host range and molecular variability of the sadwavirus dioscorea mosaic associated virus.

Dioscorea mosaic associated virus (DMaV) is a member of the genus Sadwavirus, family Secoviridae, that is associated with mosaic symptoms in Dioscorea rotundata in Brazil. The genome of a DMaV isolate detected in D. trifida in Guadeloupe was sequenced by high-throughput sequencing. Using an RT-PCR-based detection assay, we found that DMaV infects D. alata, D. bulbifera, D. cayenensis-rotundata, D. esculenta, and D. trifida accessions conserved in Guadeloupe and Côte d'Ivoire and displays a very high level of molecular diversity in a relatively small region of the genome targeted by the assay. We also provide evidence that DMaV is also present in D. rotundata in Benin and in D. alata in Nigeria.

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.

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.

Proposed revision of the family Secoviridae taxonomy to create three subgenera, "Satsumavirus", "Stramovirus" and "Cholivirus", in the genus Sadwavirus.

We present a taxonomic proposal for revision of the family Secoviridae, a taxon of plant viruses in the order Picornavirales. We propose the reorganization of the genus Sadwavirus to create three new subgenera and to update the classification of five existing species. The proposed subgenera are "Satsumavirus" (one species: Satsuma dwarf virus), "Stramovirus" (two species: Strawberry mottle virus and Black raspberry necrosis virus) and "Cholivirus" (two species: Chocolate lily virus A and Dioscorea mosaic associated virus).

Identification and complete genomic sequence of a novel sadwavirus discovered in pineapple (Ananas comosus).

The complete genomic sequence of a putative novel member of the family Secoviridae was determined by high-throughput sequencing of a pineapple accession obtained from the National Plant Germplasm Repository in Hilo, Hawaii. The predicted genome of the putative virus was composed of two RNA molecules of 6,128 and 4,161 nucleotides in length, excluding the poly-A tails. Each genome segment contained one large open reading frame (ORF) that shares homology and phylogenetic identity with members of the family Secoviridae. The presence of this new virus in pineapple was confirmed using RT-PCR and Sanger sequencing from six samples collected in Oahu, Hawaii. The name "pineapple secovirus A" (PSVA) is proposed for this putative new sadwavirus.