Search

Background: Sugarcane is cultivated globally and affected by more than 125 pathogens, which lead to various plant diseases. In recent years, high-throughput sequencing (HTS)-based genome analyses have been broadly adopted for the discovery of both characterized and un-characterized viruses from plant samples. In this study, the HTS data of sugarcane pooled sample retrieved from sequence read archive (SRA) were de novo re-assembled using CLC Genomic Workbench. Methods and results: The genomic sequence of a novel dsRNA totivirus, 5,384 nucleotides (nt) long, excluding the 5' untranslated region (UTR) and 3' UTR, was discovered and named sugarcane totivirus 1 (STV 1). The genome contains two open reading frames (ORFs): a putative coat protein (CP) encoding 866 amino acids (aa) and RNA-dependent RNA polymerase (RdRp) encoding 824 aa. Phylogenetic studies based on the genomic sequences (nt), and the aa sequences of CP as well as RdRp regions revealed that STV 1 is closely related to other members of the genus Totivirus. Pairwise sequence identity of CP and RdRp aa sequences showed 30.0-51.5% and 26.3–47.2% similarity, respectively with other members of the family Totiviridae. The HTS results were further validated and confirmed through OneStep RT-PCR assay and Sanger sequencing. Conclusion: A novel totivirus (STV 1) in the genus Totivirus, family Totiviridae has been identified. This is the first report of dsRNA totivirus STV 1 associated with sugarcane from India. [ABSTRACT FROM AUTHOR]

Trichomonas vaginalis (Tvag) is a sexually transmitted human pathogen that is commonly infected with strains of one or more of five known species of Trichomonas vaginalis viruses (TVVs), members of genus Trichomonasvirus. TVVs are thought not to have an extracellular phase to their lifecycle and instead to be transmitted vertically from mother to daughter cells. As a result, generation of isogenic virus-positive and virus-negative sets of Tvag clones has been a major barrier to studying interactions between TVVs and their host. Nucleoside analog 2′-C-methylcytidine (2CMC) has been recently reported to clear trichomonads of infections with TVV1, TVV2, and TVV3. We used 2CMC to treat a panel of Tvag isolates that collectively harbor at least one representative strain of each TVV species and thereby provided evidence that infections with TVV4 and TVV5 can also be cleared by 2CMC. Furthermore, our results suggest a newly identified difference in drug susceptibility between TVV species. We took advantage of these susceptibility difference to generate isogenic sets of Tvag clones harboring different combinations of the five TVV species. These results provide both new insight into differences between these species and new avenues for generating tools to study the potential roles of TVVs in Tvag biology. [ABSTRACT FROM AUTHOR]

Double-stranded RNA (dsRNA) molecules are mediators of RNA interference (RNAi) in eukaryotic cells. RNAi is a conserved mechanism of post-transcriptional silencing of genes cognate to the sequences of the applied dsRNA. RNAi-based therapeutics for the treatment of rare hereditary diseases have recently emerged, and the first sprayable dsRNA biopesticide has been proposed for registration. The range of applications of dsRNA molecules will likely expand in the future. Therefore, cost-effective methods for the efficient large-scale production of high-quality dsRNA are in demand. Conventional approaches to dsRNA production rely on the chemical or enzymatic synthesis of single-stranded (ss)RNA molecules with a subsequent hybridization of complementary strands. However, the yield of properly annealed biologically active dsRNA molecules is low. As an alternative approach, we have developed methods based on components derived from bacteriophage phi6, a dsRNA virus encoding RNA-dependent RNA polymerase (RdRp). Phi6 RdRp can be harnessed for the enzymatic production of high-quality dsRNA molecules. The isolated RdRp efficiently synthesizes dsRNA in vitro on a heterologous ssRNA template of any length and sequence. To scale up dsRNA production, we have developed an in vivo system where phi6 polymerase complexes produce target dsRNA molecules inside Pseudomonas cells. [ABSTRACT FROM AUTHOR]

Trichomonas vaginalis (Tvag) is a sexually transmitted human pathogen that is commonly infected with strains of one or more of five known species of Trichomonas vaginalis viruses (TVVs), members of genus Trichomonasvirus. TVVs are thought not to have an extracellular phase to their lifecycle and instead to be transmitted vertically from mother to daughter cells. As a result, generation of isogenic virus-positive and virus-negative sets of Tvag clones has been a major barrier to studying interactions between TVVs and their host. Nucleoside analog 2′-C-methylcytidine (2CMC) has been recently reported to clear trichomonads of infections with TVV1, TVV2, and TVV3. We used 2CMC to treat a panel of Tvag isolates that collectively harbor at least one representative strain of each TVV species and thereby provided evidence that infections with TVV4 and TVV5 can also be cleared by 2CMC. Furthermore, our results suggest a newly identified difference in drug susceptibility between TVV species. We took advantage of these susceptibility difference to generate isogenic sets of Tvag clones harboring different combinations of the five TVV species. These results provide both new insight into differences between these species and new avenues for generating tools to study the potential roles of TVVs in Tvag biology.

Half a century has passed since the discovery of Pseudomonas phage phi6, the first enveloped dsRNA bacteriophage to be isolated. It remained the sole known dsRNA phage for a quarter of a century and the only recognised member of the Cystoviridae family until the year 2018. After the initial discovery of phi6, additional dsRNA phages have been isolated from globally distant locations and identified in metatranscriptomic datasets, suggesting that this virus type is more ubiquitous in nature than previously acknowledged. Most identified dsRNA phages infect Pseudomonas strains and utilise either pilus or lipopolysaccharide components of the host as the primary receptor. In addition to the receptor-mediated strictly lytic lifestyle, an alternative persistent infection strategy has been described for some dsRNA phages. To date, complete genome sequences of fourteen dsRNA phage isolates are available. Despite the high sequence diversity, similar sets of genes can typically be found in the genomes of dsRNA phages, suggesting shared evolutionary trajectories. This review provides a brief overview of the recognised members of the Cystoviridae virus family and related dsRNA phage isolates, outlines the current classification of dsRNA phages, and discusses their relationships with eukaryotic RNA viruses. [ABSTRACT FROM AUTHOR]

Deletion or truncation of transgenes encoded in RNA virus vectors frequently occur. Genetic instability of transgenes is the primary obstacle to establishing RNA virus vectors as tractable gene transduction tools. To improve transgene stability, we designed codon-modified genes that imitated codon usage bias in the genomes of rotavirus (RV) and mammalian orthoreovirus (MRV), both of which have segmented double-stranded (ds) RNA genomes. Synthesized, codon-modified NanoLuc, ZsGreen, AsRed, and Akaluc genes, which had RV genome-like and/or MRV genome-like codon usage biases, were used as transgenes in RV and MRV vectors. Here, we show that the genetic stabilities of these codon-modified genes in RV and MRV vectors were dramatically increased relative to their respective parental genes. Because genetic stabilities were improved independent of transgenes and insertion sites within the viral genome, virus-like codon modification could be essential for constructing dsRNA virus vectors and overcome the primary obstacle of transgene genetic instability. IMPORTANCE The stabilities of transgenes in RNA virus vectors differ between the genes of interest, but the molecular mechanisms determining genetic stability remain unknown. This study demonstrated that the stability of a transgene was affected by the nucleotide composition, and altering the codon usage of transgenes to resemble that of the viral genome significantly increased transgene stability in double-stranded RNA virus vectors. The virus-like codon modification strategy enabled generation of stable rotavirus and mammalian orthoreovirus vectors, which could be developed as machinery for gene delivery to the intestines and/or respiratory organs. This technology has further potential to be expanded to other RNA viruses. [ABSTRACT FROM AUTHOR]

Mycoviruses are widespread and purportedly common throughout the fungal kingdom, although most are known from hosts in the two most recently diverged phyla, Ascomycota and Basidiomycota, together called Dikarya. To augment our knowledge of mycovirus prevalence and diversity in underexplored fungi, we conducted a large-scale survey of fungi in the earlier-diverging lineages, using both culture-based and transcriptome-mining approaches to search for RNA viruses. In total, 21.6% of 333 isolates were positive for RNA mycoviruses. This is a greater proportion than expected based on previous taxonomically broad mycovirus surveys and is suggestive of a strong phylogenetic component to mycoviral infection. Our newly found viral sequences are diverse, composed of double-stranded RNA, positive-sense single-stranded RNA (ssRNA), and negative-sense ssRNA genomes and include novel lineages lacking representation in the public databases. These identified viruses could be classified into 2 orders, 5 families, and 5 genera; however, half of the viruses remain taxonomically unassigned. Further, we identified a lineage of virus-like sequences in the genomes of members of Phycomycetaceae and Mortierellales that appear to be novel genes derived from integration of a viral RNA-dependent RNA polymerase gene. The two screening methods largely agreed in their detection of viruses; thus, we suggest that the culture-based assay is a cost-effective means to quickly assess whether a laboratory culture is virally infected. This study used culture collections and publicly available transcriptomes to demonstrate that mycoviruses are abundant in laboratory cultures of early-diverging fungal lineages. The function and diversity of mycoviruses found here will help guide future studies into mycovirus origins and ecological functions.IMPORTANCE Viruses are key drivers of evolution and ecosystem function and are increasingly recognized as symbionts of fungi. Fungi in early-diverging lineages are widespread, ecologically important, and comprise the majority of the phylogenetic diversity of the kingdom. Viruses infecting early-diverging lineages of fungi have been almost entirely unstudied. In this study, we screened fungi for viruses by two alternative approaches: a classic culture-based method and by transcriptome-mining. The results of our large-scale survey demonstrate that early-diverging lineages have higher infection rates than have been previously reported in other fungal taxa and that laboratory strains worldwide are host to infections, the implications of which are unknown. The function and diversity of mycoviruses found in these basal fungal lineages will help guide future studies into mycovirus origins and their evolutionary ramifications and ecological impacts.

Rotaviruses (RVs) are non-enveloped multilayered dsRNA viruses that are major etiologic agents of diarrheal disease in humans and in the young in a large number of animal species. The viral particle is composed of three different protein layers that enclose the segmented dsRNA genome and the transcriptional complexes. Each layer defines a unique subparticle that is associated with a different phase of the replication cycle. Thus, while single- and double-layered particles are associated with the intracellular processes of selective packaging, genome replication, and transcription, the viral machinery necessary for entry is located in the third layer. This modular nature of its particle allows rotaviruses to control its replication cycle by the disassembly and assembly of its structural proteins. In this review, we examine the significant advances in structural, molecular, and cellular RV biology that have contributed during the last few years to illuminating the intricate details of the RV particle disassembly and assembly processes. [ABSTRACT FROM AUTHOR]

Bluetongue virus (BTV), a major threat to livestock, is a multilayered, nonturreted member of the Reoviridae, a family of segmented dsRNA viruses characterized by endogenous RNA transcription through an RNA-dependent RNA polymerase (RdRp). To date, the structure of BTV RdRp has been unknown, limiting our mechanistic understanding of BTV transcription and hindering rational drug design effort targeting this essential enzyme. Here, we report the in situ structures of BTV RdRp VP1 in both the triple-layered virion and double-layered core, as determined by cryo-electron microscopy (cryoEM) and subparticle reconstruction. BTV RdRp has 2 unique motifs not found in other viral RdRps: a fingernail, attached to the conserved fingers subdomain, and a bundle of 3 helices: 1 from the palm subdomain and 2 from the N-terminal domain. BTV RdRp VP1 is anchored to the inner surface of the capsid shell via 5 asymmetrically arranged N termini of the inner capsid shell protein VP3A around the 5-fold axis. The structural changes of RdRp VP1 and associated capsid shell proteins between BTV virions and cores suggest that the detachment of the outer capsid proteins VP2 and VP5 during viral entry induces both global movements of the inner capsid shell and local conformational changes of the N-terminal latch helix (residues 34 to 51) of 1 inner capsid shell protein VP3A, priming RdRp VP1 within the capsid for transcription. Understanding this mechanism in BTV also provides general insights into RdRp activation and regulation during viral entry of other multilayered, nonturreted dsRNA viruses.

Mitophagy that selectively eliminates damaged mitochondria is an essential mitochondrial quality control mechanism. Recently, mitophagy has been shown to be induced in host cells infected by a few animal viruses. Here, we report that southern rice black-streaked dwarf virus (SRBSDV), a plant nonenveloped double-stranded RNA virus, can also trigger mitophagy in its planthopper vector to prevent mitochondria-dependent apoptosis and promote persistent viral propagation. We find that the fibrillar structures constructed by the nonstructural protein P7-1 of SRBSDV directly target mitochondria via interaction with the mitophagy receptor BNIP3 (BCL2 interacting protein 3), and these mitochondria are then sequestered within autophagosomes to form mitophagosomes. Moreover, SRBSDV infection or P7-1 expression alone can promote BNIP3 dimerization on the mitochondria, and induce autophagy via the P7-1-ATG8 interaction. Furthermore, SRBSDV infection stimulates the phosphorylation of AMP-activated protein kinase (AMPK), resulting in BNIP3 phosphorylation via the AMPKα-BNIP3 interaction. Together, P7-1 induces BNIP3-mediated mitophagy by promoting the formation of phosphorylated BNIP3 dimers on the mitochondria. Silencing of ATG8, BNIP3, or AMPKα significantly reduces virus-induced mitophagy and viral propagation in insect vectors. These data suggest that in planthopper, SRBSDV-induced mitophagosomes are modified to accommodate virions and facilitate persistent viral propagation. In summary, our results demonstrate a previously unappreciated role of a viral protein in the induction of BNIP3-mediated mitophagy by bridging autophagosomes and mitochondria and reveal the functional importance of virus-induced mitophagy in maintaining persistent viral infection in insect vectors.Abbreviations: AMPK: AMP-activated protein kinase; ATG: autophagy related; BNIP3: BCL2 interacting protein 3; CASP3: caspase 3; dsRNA: double strand RNA; ER: endoplasmic reticulum; FITC: fluorescein isothiocyanate; FKBP8: FKBP prolyl isomerase 8; FUNDC1: FUN14 domain containing 1; GFP: green fluorescent protein; GST: glutathione S-transferase; padp: post-first access to diseased plants; Phos-tag: Phosphate-binding tag; PINK1: PTEN induced kinase 1; Sf9: Spodoptera frugiperda; SQSTM1: sequestosome 1; SRBSDV: southern rice black-streaked dwarf virus; STK11/LKB1: serine/threonine kinase 11; TOMM20: translocase of outer mitochondrial membrane 20; RBSDV: rice black-streaked dwarf virus; TUNEL: terminal deoxynucleotidyl dUTP nick end labeling; ULK1: unc-51 like autophagy activating kinase 1; VDAC1: voltage dependent anion channel 1. [ABSTRACT FROM AUTHOR]

AQUAREOVIRUS: a genus of within the family Spinareoviridae, order Reovirales, infects aquatic animals. Their genomes comprise 11 segments of double-stranded RNA, which function directly as mRNAs upon release into the cytoplasm of infected cells. Here, liquid chromatography-tandem mass spectrometry was employed to annotate small coding ORFs in the Aquareovirus-C genome. Its plus-strand RNA of segment 8 (S8) contains a novel protein-coding frame (NS15), and S5 seems to has an additional reading frame (NS18) with a putative non-AUG initiation codon. Among them, NS15 polypeptide has been proved by immunoblotting assay. Remarkably, the S4 and S11 minus-strand mRNAs may encode polypeptides, suggesting ambisense polarity of the two segmented RNAs. And the newly discovered NS12 ORF in 2019, from viral tricistronic S7 mRNA, was also confirmed by this mass-spectrometry data. Taken together, these identified new ORFs reveal the genome-coding complexity of Aquareovirus-C., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Polymycoviridae is a relatively new viral family that was established nearly 5 years ago, but their viral morphologies (naked or encapsidated) remain controversial since only one member namely, Colletotrichum camelliae filamentous virus 1 (CcFV1), was identified as being encapsidated in filamentous virions. Here, three novel double-stranded RNA (dsRNA) viruses belonging to the family Polymycoviridae were identified in three phytopathogenic fungal strains and tentatively named Pseudopestalotiopsis camelliae -sinensis polymycovirus 1 (PcsPmV1), and Phyllosticta capitalensis polymycovirus 1 and 2 (PhcPmV1 and 2), respectively. PcsPmV1 and PhcPmVs have five or six genomic dsRNAs, ranging from 1,055 to 2,405 bp, encoding five or seven putative open reading frames (ORFs), of which ORF1 encodes an RNA-dependent RNA polymerase, ORF5 encodes a prolein-alanine-serine-rich (P-A-S-rich) protein behaving as coat protein (CP); and dsRNAs 4 and 6 encode putative proteins with unknown functions and share no detectable identities with known viral sequences. Upon examination under transmission electron microscopy after purification from fungal mycelia, PcsPmV1 and PhcPmVs were found to be encapsidated in filamentous particles, as was a known polymycovirus, Botryosphaeria dothidea RNA virus 1 (BdRV1), which was previously assumed to likely have no conventional virions. The morphology of PcsPmV1 was further supported by the observation that its particles could be decorated by polyclonal antibodies against its CP and bound by immuno-gold particles conjugated to the specific CP antibody. Together with CcFV1, BdRV1, PcsPmV1, and PhcPmVs, these provide strong evidence to support the notion that polymycoviruses are encapsidated in filamentous virions constituted by P-A-S-rich CPs. Moreover, their biological effects on their fungal hosts were assessed, suggesting that PcsPmV1 infection could enhance growth and virulence.IMPORTANCE Polymycoviridae , a recently established viral family, has raised questions about encapsidation. Here, we identify and characterize three novel polymycoviral double-stranded RNA (dsRNA) viruses in phytopathogenic fungal strains, tentatively named Pseudopestalotiopsis camelliae -sinensis polymycovirus 1, and Phyllosticta capitalensis polymycovirus 1 and 2, respectively. These polymycoviruses possess five or six genomic dsRNAs, ranging from 1,055 to 2,405 bp, with two encoding putative proteins of unknown functions and sharing no detectable identities with known viral sequences. Their morphologies indicate filamentous virions constituted by proline-alanine-serine-rich coat proteins, observed using immunosorbent electron microscopy combined with immune-gold labeling techniques. Additionally, Botryosphaeria dothidea RNA virus 1, previously assumed to lack conventional virions, is also shown to be encapsidated in filamentous particles. This study provides new evidence supporting the encapsidation of polymycoviruses into elongated and flexuous virions, significantly contributing to our understanding of the evolutionary particle architecture within the virosphere and expanding our knowledge of viral diversity and evolution. Moreover, this is the first report of a polymycovirus enhancing the virulence and growth of a phytopathogenic fungus.

Totivirus-like viruses are a group of non-segmented double-stranded (ds)RNA viruses with two open reading frames, which were recently discovered and provisionally assigned to the Totiviridae family. Unlike yeast and protozoan Totiviridae viruses, these totivirus-like viruses infect a diverse spectrum of metazoan hosts and currently have enormous impacts on fisheries and agriculture. We developed the first infectious full-length cDNA clone of a totivirus-like virus, the Omono River virus (OmRV), and produced infectious particles using an RNA-transcript-based method. Compared with the parent wild-type particles from nature, the infectious-cloning OmRV particles have presented strong cytopathic effects, infectivity and similar morphology. Thus far, the established system is one of the few reported systems for generating a non-segmented dsRNA virus cDNA clone. • The first synthetic transcript of a metazoan dsRNA totivirus-like virus. • The synthetic particles were intensively assessed on their morphology and infectivity. • One function-deficient mutant T365A reveals the importance of the protrusion protein. [ABSTRACT FROM AUTHOR]

Talaromyces amestolkiae is an important fungal species owing to its ubiquity in soils, plants, air, and food. In this study, we identified a novel six-segmented polymycovirus, Talaromyces amestolkiae polymycovirus 1 (TaPmV-1). Each of the double-stranded (ds) RNA segments of TaPmV-1 contained a single open reading frame, and the proteins encoded by dsRNA1, dsRNA2, dsRNA3, and dsRNA 5 shared significant amino acid identities of 56, 40, 47, and 43%, respectively, with the corresponding proteins of Aspergillus fumigatus polymycovirus-1(AfuPmV-1). DsRNA1, dsRNA3, and dsRNA5 of TaPmV-1 encoded an RNA-dependent RNA polymerase (RdRp), a viral methyltransferase, and a PAS-rich protein, respectively. The functions of the proteins encoded by dsRNA2, dsRNA4, and dsRNA6 have not been elucidated. Comparison of the virus-infected strain LSH3 with virus-cured strain LSHVF revealed that infection with TaPmV-l may reduce the production of red pigments and induce the clustering of fungal sclerotia. Furthermore, transcriptomic analyses demonstrated that infection with TaPmV-l downregulated the expression of transcripts related to metabolism, and may correlate with the reduced production of red pigments and clustering of sclerotia in T. amestolkiae. These results of this study provide novel insights into the mechanism of fungal gene regulation by polymycovirus infections at the transcriptome level, and this study is the first to report a novel polymycovirus of T. amestolkiae. [ABSTRACT FROM AUTHOR]

Abstract Vector‐borne parasites often manipulate hosts to attract uninfected vectors. For example, parasites causing malaria alter host odor to attract mosquitoes. Here, we discuss the ecology and evolution of fruit‐colonizing yeast in a tripartite symbiosis—the so‐called “killer yeast” system. “Killer yeast” consists of Saccharomyces cerevisiae yeast hosting two double‐stranded RNA viruses (M satellite dsRNAs, L‐A dsRNA helper virus). When both dsRNA viruses occur in a yeast cell, the yeast converts to lethal toxin‑producing “killer yeast” phenotype that kills uninfected yeasts. Yeasts on ephemeral fruits attract insect vectors to colonize new habitats. As the viruses have no extracellular stage, they depend on the same insect vectors as yeast for their dispersal. Viruses also benefit from yeast dispersal as this promotes yeast to reproduce sexually, which is how viruses can transmit to uninfected yeast strains. We tested whether insect vectors are more attracted to killer yeasts than to non‑killer yeasts. In our field experiment, we found that killer yeasts were more attractive to Drosophila than non‐killer yeasts. This suggests that vectors foraging on yeast are more likely to transmit yeast with a killer phenotype, allowing the viruses to colonize those uninfected yeast strains that engage in sexual reproduction with the killer yeast. Beyond insights into the basic ecology of the killer yeast system, our results suggest that viruses could increase transmission success by manipulating the insect vectors of their host.

Diplodia seriata in the family Botryosphaeriaceae is a cosmopolitan phytopathogenic fungus and is responsible for causing cankers, fruit rot and leaf spots on economically important plants. In this study, we characterized the virome of a single Pakistani strain (L3) of D. seriata. Several viral-like contig sequences were obtained via a previously conducted next-generation sequencing analysis. Multiple infection of the L3 strain by eight RNA mycoviruses was confirmed through RT-PCR using total RNA samples extracted from this strain; the entire genomes were determined via Sanger sequencing of RT-PCR and RACE clones. A BLAST search and phylogenetic analyses indicated that these eight mycoviruses belong to seven different viral families. Four identified mycoviruses belong to double-stranded RNA viral families, including Polymycoviridae, Chrysoviridae, Totiviridae and Partitiviridae, and the remaining four identified mycoviruses belong to single-stranded RNA viral families, i.e., Botourmiaviridae, and two previously proposed families "Ambiguiviridae" and "Splipalmiviridae". Of the eight, five mycoviruses appear to represent new virus species. A morphological comparison of L3 and partially cured strain L3ht1 suggested that one or more of the three viruses belonging to Polymycoviridae, "Splipalmiviridae" and "Ambiguiviridae" are involved in the irregular colony phenotype of L3. To our knowledge, this is the first report of diverse virome characterization from D. seriata. [ABSTRACT FROM AUTHOR]

In this study, a novel positive-sense single-stranded RNA (1ssRNA) mycovirus, tentatively named Colletotrichum fructicola RNA virus 1 (CfRV1), was identified in the phytopathogenic fungus Colletotrichum fructicola. CfRV1 has seven genomic components, encoding seven proteins from open reading frames (ORFs) flanked by highly conserved untranslated regions (UTRs). Proteins encoded by ORFs 1, 2, 3, 5, and 6 are more similar to the putative RNA-dependent RNA polymerase (RdRp), hypothetical protein (P2), methyltransferase, and two hypothetical proteins of Hadaka virus 1 (HadV1), a capsidless 10- or 11-segmented 1ssRNA virus, while proteins encoded by ORFs 4 and 7 showed no detectable similarity to any known proteins. Notably, proteins encoded by ORFs 1 to 3 also share considerably high similarity with the corresponding proteins of polymycoviruses. Phylogenetic analysis conducted based on the amino acid sequence of CfRV1 RdRp and related viruses placed CfRV1 and HadV1 together in the same clade, close to polymycoviruses and astroviruses. CfRV1-infected C. fructicola strains demonstrate a moderately attenuated growth rate and virulence compared to uninfected isolates. CfRV1 is capsidless and potentially encapsulated in vesicles inside fungal cells, as revealed by transmission electron microscopy. CfRV1 and HadV1 are 1ssRNA mycoviruses closely related to polymycoviruses and astroviruses, represent a new linkage between 1ssRNA viruses and the intermediate double-stranded RNA (dsRNA) polymycoviruses, and expand our understanding of virus diversity, taxonomy, evolution, and biological traits. [ABSTRACT FROM AUTHOR]

The advancement of high throughput sequencing has greatly facilitated the exploration of viruses that infect marine hosts. For example, a number of putative virus genomes belonging to the Totiviridae family have been described in crustacean hosts. However, there has been no characterization of the most newly discovered putative viruses beyond description of their genomes. In this study, two novel double-stranded RNA (dsRNA) virus genomes were discovered in the Atlantic blue crab (Callinectes sapidus) and further investigated. Sequencing of both virus genomes revealed that they each encode RNA dependent RNA polymerase proteins (RdRps) with similarities to toti-like viruses. The viruses were tentatively named Callinectes sapidus toti-like virus 1 (CsTLV1) and Callinectes sapidus toti-like virus 2 (CsTLV2). Both genomes have typical elements required for −1 ribosomal frameshifting, which may induce the expression of an encoded ORF1–ORF2 (gag-pol) fusion protein. Phylogenetic analyses of CsTLV1 and CsTLV2 RdRp amino acid sequences suggested that they are members of two new genera in the family Totiviridae. The CsTLV1 and CsTLV2 genomes were detected in muscle, gill, and hepatopancreas of blue crabs by real-time reverse transcription quantitative PCR (RT-qPCR). The presence of ~40 nm totivirus-like viral particles in all three tissues was verified by transmission electron microscopy, and pathology associated with CsTLV1 and CsTLV2 infections were observed by histology. PCR assays showed the prevalence and geographic range of these viruses, to be restricted to the northeast United States sites sampled. The two virus genomes co-occurred in almost all cases, with the CsTLV2 genome being found on its own in 8.5% cases, and the CsTLV1 genome not yet found on its own. To our knowledge, this is the first report of toti-like viruses in C. sapidus. The information reported here provides the knowledge and tools to investigate transmission and potential pathogenicity of these viruses. [ABSTRACT FROM AUTHOR]

Trichomonas vaginalis is the most common non-viral cause of sexually transmitted infections globally. Infection by this protozoan parasite results in the clinical syndrome trichomoniasis, which manifests as an inflammatory disease with acute and chronic consequences. Half or more isolates of this parasite are themselves infected with one or more dsRNA viruses that can exacerbate the inflammatory syndrome. At least four distinct viruses have been identified in T. vaginalis to date, constituting species Trichomonas vaginalis virus 1 through Trichomonas vaginalis virus 4 in genus Trichomonasvirus. Despite the global prevalence of these viruses, few complete coding sequences have been reported. We conducted viral sequence mining in publicly available transcriptomes across 60 RNA-Seq accessions representing at least 13 distinct T. vaginalis isolates. The results led to sequence assemblies for 27 novel trichomonasvirus strains across all four recognized species. Using a strategy of de novo sequence assembly followed by taxonomic classification, we additionally discovered six strains of a newly identified fifth species, for which we propose the name Trichomonas vaginalis virus 5, also in genus Trichomonasvirus. These additional strains exhibit high sequence identity to each other, but low sequence identity to strains of the other four species. Phylogenetic analyses corroborate the species-level designations. These results substantially increase the number of trichomonasvirus genome sequences and demonstrate the utility of mining publicly available transcriptomes for virus discovery in a critical human pathogen. [ABSTRACT FROM AUTHOR]

The advancement of high throughput sequencing has greatly facilitated the exploration of viruses that infect marine hosts. For example, a number of putative virus genomes belonging to the Totiviridae family have been described in crustacean hosts. However, there has been no characterization of the most newly discovered putative viruses beyond description of their genomes. In this study, two novel double-stranded RNA (dsRNA) virus genomes were discovered in the Atlantic blue crab (Callinectes sapidus) and further investigated. Sequencing of both virus genomes revealed that they each encode RNA dependent RNA polymerase proteins (RdRps) with similarities to toti-like viruses. The viruses were tentatively named Callinectes sapidus toti-like virus 1 (CsTLV1) and Callinectes sapidus toti-like virus 2 (CsTLV2). Both genomes have typical elements required for −1 ribosomal frameshifting, which may induce the expression of an encoded ORF1–ORF2 (gag-pol) fusion protein. Phylogenetic analyses of CsTLV1 and CsTLV2 RdRp amino acid sequences suggested that they are members of two new genera in the family Totiviridae. The CsTLV1 and CsTLV2 genomes were detected in muscle, gill, and hepatopancreas of blue crabs by real-time reverse transcription quantitative PCR (RT-qPCR). The presence of ~40 nm totivirus-like viral particles in all three tissues was verified by transmission electron microscopy, and pathology associated with CsTLV1 and CsTLV2 infections were observed by histology. PCR assays showed the prevalence and geographic range of these viruses, to be restricted to the northeast United States sites sampled. The two virus genomes co-occurred in almost all cases, with the CsTLV2 genome being found on its own in 8.5% cases, and the CsTLV1 genome not yet found on its own. To our knowledge, this is the first report of toti-like viruses in C. sapidus. The information reported here provides the knowledge and tools to investigate transmission and potential pathogenicity of these viruses.

We report here the identification of a dsRNA virus, obtained from Fusarium solani strain Newher-7, tentatively named F. solani partitivirus 3 (FsPV3). It consists of four dsRNA segments (dsRNA1-4) with lengths of 1961, 1900, 1830 and 1830 bp, respectively. Sequence analysis showed that dsRNA1 encodes an RNA-dependent RNA polymerase (RdRp), dsRNA2 encodes a capsid protein (CP), dsRNA3 encodes a hypothetical protein of unknown function and dsRNA4 encodes two hypothetical proteins of unknown function. Amino acid sequence comparisons showed that the RdRp of FsPV3 is most similar to that of Hulunbuir Parti tick virus 1. In contrast, the CP of FsPV3, as well as the hypothetical protein encoded by ORF3 of dsRNA3, was most similar to cognate proteins encoded by Colletotrichum -associated partitivirus 2. However, the two hypothetical proteins encoded by dsRNA4 showed no significant similarity to the available sequences in the National Center for Biotechnology Information database and encoded no apparent conserved domains. Phylogenetic analysis of the RdRp and CP showed that FsPV3 clustered together with other species in the genus Alphapartitivirus . Given that proteins encoded by FsPV3 are not sufficiently highly homologous to a single known virus and that it encodes two novel proteins, we suggest that FsPV3 should be regarded as a new member of the genus Alphapartitivirus in the family Partitiviridae . This is the first report of FsPV3 infecting F. solani .

While RNAi is often heralded as a promising new strategy for insect pest control, a major obstacle that still remains is the efficient delivery of dsRNA molecules within the cells of the targeted insects. However, it seems overlooked that dsRNA viruses already have developed efficient strategies for transport of dsRNA molecules across tissue barriers and cellular membranes. Besides protecting their dsRNA genomes in a protective shell, dsRNA viruses also display outer capsid layers that incorporate sophisticated mechanisms to disrupt the plasma membrane layer and to translocate core particles (with linear dsRNA genome fragments) within the cytoplasm. Because of the perceived efficiency of the translocation mechanism, it is well worth analyzing in detail the molecular processes that are used to achieve this feat. In this review, the mechanism of cell entry by dsRNA viruses belonging to the Reoviridae family is discussed in detail. Because of the large amount of progress in mammalian versus insect models, the mechanism of infections of reoviruses in mammals (orthoreoviruses, rotaviruses, orbiviruses) will be treated as a point of reference against which infections of reoviruses in insects (orbiviruses in midges, plant viruses in hemipterans, insect-specific cypoviruses in lepidopterans) will be compared. The goal of this discussion is to uncover the basic principles by which dsRNA viruses cross tissue barriers and translocate their cargo to the cellular cytoplasm; such knowledge subsequently can be incorporated into the design of dsRNA virus-based viral-like particles for optimal delivery of RNAi triggers in targeted insect pests.

Alternaria alternata virus 1 (AaV1) has been identified in the saprophytic fungus Alternaria alternata strain EGS 35–193. AaV1 has four genomic double-stranded (ds)RNA segments (dsRNA1–4) packaged in isometric particles. The 3' end of each coding strand is polyadenylated (36–50nt), but the presence of a cap structure at each 5' end has not previously been investigated. Here, we have characterized the AaV1 genome and found that it has unique features among the mycoviruses. We confirmed the existence of cap structures on the 5' ends of the AaV1 genomic dsRNAs using RNA dot blots with anti-cap antibodies and the oligo-capping method. Polyclonal antibodies against purified AaV1 particles specifically bound to an 82kDa protein, suggesting that this protein is the major capsid component. Subsequent Edman degradation indicated that the AaV1 dsRNA3 segment encodes the major coat protein. Two kinds of defective AaV1 dsRNA2, which is 2,794bp (844 aa) in length when intact, appeared in EGS 35–193 during subculturing, as confirmed by RT-PCR and northern hybridization. Sequence analysis revealed that one of the two defective dsRNA2s contained a 231bp deletion, while the other carried both the 231bp deletion and an additional 465bp deletion in the open reading frame. Both deletions occurred in-frame, resulting in predicted proteins of 767 aa and 612 aa. The fungal isolates carrying virions with the defective dsRNA2s showed impaired growth and abnormal pigmentation. To our best knowledge, AaV1 is the first dsRNA virus to be identified with both 5' cap and 3'poly(A) structures on its genomic segments, as well as the specific deletions of dsRNA2.

While RNAi is often heralded as a promising new strategy for insect pest control, a major obstacle that still remains is the efficient delivery of dsRNA molecules within the cells of the targeted insects. However, it seems overlooked that dsRNA viruses already have developed efficient strategies for transport of dsRNA molecules across tissue barriers and cellular membranes. Besides protecting their dsRNA genomes in a protective shell, dsRNA viruses also display outer capsid layers that incorporate sophisticated mechanisms to disrupt the plasma membrane layer and to translocate core particles (with linear dsRNA genome fragments) within the cytoplasm. Because of the perceived efficiency of the translocation mechanism, it is well worth analyzing in detail the molecular processes that are used to achieve this feat. In this review, the mechanism of cell entry by dsRNA viruses belonging to the Reoviridae family is discussed in detail. Because of the large amount of progress in mammalian versus insect models, the mechanism of infections of reoviruses in mammals (orthoreoviruses, rotaviruses, orbiviruses) will be treated as a point of reference against which infections of reoviruses in insects (orbiviruses in midges, plant viruses in hemipterans, insect-specific cypoviruses in lepidopterans) will be compared. The goal of this discussion is to uncover the basic principles by which dsRNA viruses cross tissue barriers and translocate their cargo to the cellular cytoplasm; such knowledge subsequently can be incorporated into the design of dsRNA virus-based viral-like particles for optimal delivery of RNAi triggers in targeted insect pests. [ABSTRACT FROM AUTHOR]

Alternaria alternata virus 1 (AaV1) has been identified in the saprophytic fungus Alternaria alternata strain EGS 35–193. AaV1 has four genomic double-stranded (ds)RNA segments (dsRNA1–4) packaged in isometric particles. The 3' end of each coding strand is polyadenylated (36–50nt), but the presence of a cap structure at each 5' end has not previously been investigated. Here, we have characterized the AaV1 genome and found that it has unique features among the mycoviruses. We confirmed the existence of cap structures on the 5' ends of the AaV1 genomic dsRNAs using RNA dot blots with anti-cap antibodies and the oligo-capping method. Polyclonal antibodies against purified AaV1 particles specifically bound to an 82kDa protein, suggesting that this protein is the major capsid component. Subsequent Edman degradation indicated that the AaV1 dsRNA3 segment encodes the major coat protein. Two kinds of defective AaV1 dsRNA2, which is 2,794bp (844 aa) in length when intact, appeared in EGS 35–193 during subculturing, as confirmed by RT-PCR and northern hybridization. Sequence analysis revealed that one of the two defective dsRNA2s contained a 231bp deletion, while the other carried both the 231bp deletion and an additional 465bp deletion in the open reading frame. Both deletions occurred in-frame, resulting in predicted proteins of 767 aa and 612 aa. The fungal isolates carrying virions with the defective dsRNA2s showed impaired growth and abnormal pigmentation. To our best knowledge, AaV1 is the first dsRNA virus to be identified with both 5' cap and 3'poly(A) structures on its genomic segments, as well as the specific deletions of dsRNA2. [ABSTRACT FROM AUTHOR]

Vector‐borne parasites often manipulate hosts to attract uninfected vectors. For example, parasites causing malaria alter host odor to attract mosquitoes. Here, we discuss the ecology and evolution of fruit‐colonizing yeast in a tripartite symbiosis—the so‐called "killer yeast" system. "Killer yeast" consists of Saccharomyces cerevisiae yeast hosting two double‐stranded RNA viruses (M satellite dsRNAs, L‐A dsRNA helper virus). When both dsRNA viruses occur in a yeast cell, the yeast converts to lethal toxin‑producing "killer yeast" phenotype that kills uninfected yeasts. Yeasts on ephemeral fruits attract insect vectors to colonize new habitats. As the viruses have no extracellular stage, they depend on the same insect vectors as yeast for their dispersal. Viruses also benefit from yeast dispersal as this promotes yeast to reproduce sexually, which is how viruses can transmit to uninfected yeast strains. We tested whether insect vectors are more attracted to killer yeasts than to non‑killer yeasts. In our field experiment, we found that killer yeasts were more attractive to Drosophila than non‐killer yeasts. This suggests that vectors foraging on yeast are more likely to transmit yeast with a killer phenotype, allowing the viruses to colonize those uninfected yeast strains that engage in sexual reproduction with the killer yeast. Beyond insights into the basic ecology of the killer yeast system, our results suggest that viruses could increase transmission success by manipulating the insect vectors of their host. [ABSTRACT FROM AUTHOR]

ABSTRACT Mycoviruses are widespread and purportedly common throughout the fungal kingdom, although most are known from hosts in the two most recently diverged phyla, Ascomycota and Basidiomycota, together called Dikarya. To augment our knowledge of mycovirus prevalence and diversity in underexplored fungi, we conducted a large-scale survey of fungi in the earlier-diverging lineages, using both culture-based and transcriptome-mining approaches to search for RNA viruses. In total, 21.6% of 333 isolates were positive for RNA mycoviruses. This is a greater proportion than expected based on previous taxonomically broad mycovirus surveys and is suggestive of a strong phylogenetic component to mycoviral infection. Our newly found viral sequences are diverse, composed of double-stranded RNA, positive-sense single-stranded RNA (ssRNA), and negative-sense ssRNA genomes and include novel lineages lacking representation in the public databases. These identified viruses could be classified into 2 orders, 5 families, and 5 genera; however, half of the viruses remain taxonomically unassigned. Further, we identified a lineage of virus-like sequences in the genomes of members of Phycomycetaceae and Mortierellales that appear to be novel genes derived from integration of a viral RNA-dependent RNA polymerase gene. The two screening methods largely agreed in their detection of viruses; thus, we suggest that the culture-based assay is a cost-effective means to quickly assess whether a laboratory culture is virally infected. This study used culture collections and publicly available transcriptomes to demonstrate that mycoviruses are abundant in laboratory cultures of early-diverging fungal lineages. The function and diversity of mycoviruses found here will help guide future studies into mycovirus origins and ecological functions. IMPORTANCE Viruses are key drivers of evolution and ecosystem function and are increasingly recognized as symbionts of fungi. Fungi in early-diverging lineages are widespread, ecologically important, and comprise the majority of the phylogenetic diversity of the kingdom. Viruses infecting early-diverging lineages of fungi have been almost entirely unstudied. In this study, we screened fungi for viruses by two alternative approaches: a classic culture-based method and by transcriptome-mining. The results of our large-scale survey demonstrate that early-diverging lineages have higher infection rates than have been previously reported in other fungal taxa and that laboratory strains worldwide are host to infections, the implications of which are unknown. The function and diversity of mycoviruses found in these basal fungal lineages will help guide future studies into mycovirus origins and their evolutionary ramifications and ecological impacts.

Partitiviruses (dsRNA viruses, family Partitiviridae) are ubiquitously detected in plants and fungi. Although previous surveys suggested their omnipresence in the white root rot fungus, Rosellinia necatrix, only a few of them have been molecularly and biologically characterized thus far. We report the characterization of a total of 20 partitiviruses from 16 R. necatrix strains belonging to 15 new species, for which “Rosellinia necatrix partitivirus 11–Rosellinia necatrix partitivirus 25” were proposed, and 5 previously reported species. The newly identified partitiviruses have been taxonomically placed in two genera, Alphapartitivirus, and Betapartitivirus. Some partitiviruses were transfected into reference strains of the natural host, R. necatrix, and an experimental host, Cryphonectria parasitica, using purified virions. A comparative analysis of resultant transfectants revealed interesting differences and similarities between the RNA accumulation and symptom induction patterns of R. necatrix and C. parasitica. Other interesting findings include the identification of a probable reassortment event and a quintuple partitivirus infection of a single fungal strain. These combined results provide a foundation for further studies aimed at elucidating mechanisms that underly the differences observed.

Despite their diversity, most double-stranded-RNA (dsRNA) viruses share a specialized T 1 capsid built from dimers of a single protein that provides a platform for genome transcription and replication. This ubiquitous capsid remains structurally undisturbed throughout the viral cycle, isolating the genome to avoid triggering host defense mechanisms. Human picobirnavirus (hPBV) is a dsRNA virus frequently associated with gastroenteritis, although its pathogenicity is yet undefined. Here, we report the cryoelectron microscopy (cryo-EM) structure of hPBV at 2.6-Å resolution. The capsid protein (CP) is arranged in a single-shelled, ~380-Å-diameter T 1 capsid with a rough outer surface similar to that of dsRNA mycoviruses. The hPBV capsid is built of 60 quasisymmetric CP dimers (A and B) stabilized by domain swapping, and only the CP-A N-terminal basic region interacts with the packaged nucleic acids. hPBV CP has an helical domain with a fold similar to that of fungal partitivirus CP, with many domain insertions in its C-terminal half. In contrast to dsRNA mycoviruses, hPBV has an extracellular life cycle phase like complex reoviruses, which indicates that its own CP probably participates in cell entry. Using an in vitro reversible assembly/disassembly system of hPBV, we isolated tetramers as possible assembly intermediates. We used atomic force microscopy to characterize the biophysical properties of hPBV capsids with different cargos (host nucleic acids or proteins) and found that the CP N-terminal segment not only is involved in nucleic acid interaction/packaging but also modulates the mechanical behavior of the capsid in conjunction with the cargo. [ABSTRACT FROM AUTHOR]

Freshwater planarians, flatworms from order Tricladida, are experimental models of stem cell biology and tissue regeneration. An aspect of their biology that remains less well studied is their relationship with viruses that may infect them. In this study, we identified a taxon of monosegmented double-stranded RNA (dsRNA) viruses in five planarian species, including the well-characterized model Schmidtea mediterranea. Sequences for the S. mediterranea virus (abbreviated SmedTV for S. mediterranea tricladivirus) were found in public transcriptome data from multiple institutions, indicating that SmedTV is prevalent in S. mediterranea lab colonies, though without causing evident disease. The presence of SmedTV in discrete cells was shown through in situ hybridization methods for detecting the viral RNA. SmedTV-staining cells were found to be concentrated in neural structures (eyes and brain) but were also scattered in other worm tissues as well. In contrast, few SmedTV-staining cells were seen in stem cell compartments (also consistent with RNA sequencing data) or early blastema tissue. RNA interference (RNAi) targeted to the SmedTV sequence led to apparent cure of infection, though effects on worm health or behavior were not observed. Efforts to transmit SmedTV horizontally through microinjection were unsuccessful. Based on these findings, we conclude that SmedTV infects S. mediterranea in a persistent manner and undergoes vertical transmission to progeny worms during serial passage in lab colonies. The utility of S. mediterranea as a regeneration model, coupled with the apparent capacity of SmedTV to evade normal host immune/RNAi defenses under standard conditions, argues that further studies are warranted to explore this newly recognized virus-host system. [ABSTRACT FROM AUTHOR]

Background: The Totiviridae family includes a number of double-stranded RNA viruses that can infect Trichomonas vaginalis. Some T. vaginalis isolates are infected with one or more double-stranded RNA (dsRNA) viruses. In this study, different strains of double-stranded RNA virus in Iranian isolates of T. vaginalis were evaluated for the first time in Iran. Methods: Vaginal swabs were collected from 1550 participants who were referred to hospitals associated with Iran University of Medical Sciences, Tehran,Iran from June to November 2018. T. vaginalis isolates were cultured in Diamond's modified medium. After the extraction of nucleic acids using a DNA/RNA extraction kit, RT-PCR was performed and PCR products were purified and sequenced. Results: In general 9 (0.6%) isolates were confirmed as T. vaginalis among 1550 collected vaginal samples. Among 9 isolates of T. vaginalis, three of them were infected with TVV1. One isolate has multiple infections with T. vaginalis virus (TVV1, TVV2 and TVV3) as coinfection. The nucleotide BLAST indicated that the T. vaginalis virus 1(TVV1) isolates were most closely related to TVV1-OC5, TVV1-UR1-1.The T. vaginalis virus 2 (TVV2) sequence had also a similarity with TVV2-UR1-1, TVV2-UR1 and TVV2-OC3. The sequence of T. vaginalis virus 3(TVV3) had similarity with TVV3-OC5, TVV3-UR1-1 and TVV3-UR1.Conclusion: Three ds RNA viruses T. vaginalis virus (TVV1, TVV2 and TVV3) were detected using RT-PCR in T. vaginalis Iranian isolates. The coinfection of TVV1, TVV2 and TVV3 in one isolate of T.vaginalis was observed for the first time in Iran. [ABSTRACT FROM AUTHOR]

The Birnaviridae family, responsible for major economic losses to poultry and aquaculture, are non-enveloped viruses with a segmented double-stranded (ds)RNA genome that replicate in discrete cytoplasmic virus factories (VFs). Reassortment is common, however, the underlying mechanism remains unknown given that VFs may act as a barrier to genome mixing. In order to provide new information on VF trafficking during dsRNA virus co-infection, we rescued two recombinant infectious bursal disease viruses (IBDVs) of strain PBG98 containing either a split GFP11- or Tetracysteine (TC)- tag fused to the VP1 polymerase (PBG98-VP1-GFP11 and PBG98- VP1-TC). DF-1 cells transfected with GFP1-10 prior to PBG98-VP1-GFP11 infection, or stained with ReAsH following PBG98-VP1-TC infection, had green or red foci in the cytoplasm respectively that co-localised with VP3 and dsRNA, consistent with VFs. The average number of VFs decreased from a mean of 60 to 5 per cell between 10 and 24 hours post infection (hpi) (p<0.0001), while the average area increased from 1.24 µm² to 45.01µm² (p<0.0001), and live cell imaging revealed that the VFs were highly dynamic structures that coalesced in the cytoplasm. Small VFs moved faster than large (average 0.57μm/s at 16 hpi compared to 0.22 μm/s at 22 hpi), and VF coalescence was dependent on an intact microtubule network and actin cytoskeleton. During co-infection with PBG98-VP1-GFP11 and PBG98-VP1-TC viruses, discrete VFs initially formed from each input virus that subsequently coalesced 10-16 hpi, and we speculate that Birnaviridae reassortment requires VF coalescence. Importance Reassortment is common in viruses with segmented double stranded (ds)RNA genomes. However, these viruses typically replicate within discrete cytoplasmic virus factories (VFs) that may represent a barrier to genome mixing. We generated the first replication competent tagged reporter birnaviruses, infectious bursal disease viruses (IBDVs) containing a split GFP11 or tetracysteine (TC) tag and used the viruses to track the location and movement of IBDV VFs, in order to better understand the intracellular dynamics of VFs during a co-infection. Discrete VFs initially formed from each virus that subsequently coalesced from 10 hours pi. We hypothesise that VF coalescence is required for the reassortment of the Birnaviridae. This study provides new information that adds to our understanding of dsRNA virus VF trafficking. [ABSTRACT FROM AUTHOR]

Partitiviruses (dsRNA viruses, family Partitiviridae) are ubiquitously detected in plants and fungi. Although previous surveys suggested their omnipresence in the white root rot fungus, Rosellinia necatrix , only a few of them have been molecularly and biologically characterized thus far. We report the characterization of a total of 20 partitiviruses from 16 R. necatrix strains belonging to 15 new species, for which " Rosellinia necatrix partitivirus 11 – Rosellinia necatrix partitivirus 25 " were proposed, and 5 previously reported species. The newly identified partitiviruses have been taxonomically placed in two genera, Alphapartitivirus , and Betapartitivirus. Some partitiviruses were transfected into reference strains of the natural host, R. necatrix , and an experimental host, Cryphonectria parasitica , using purified virions. A comparative analysis of resultant transfectants revealed interesting differences and similarities between the RNA accumulation and symptom induction patterns of R. necatrix and C. parasitica. Other interesting findings include the identification of a probable reassortment event and a quintuple partitivirus infection of a single fungal strain. These combined results provide a foundation for further studies aimed at elucidating mechanisms that underly the differences observed. [ABSTRACT FROM AUTHOR]

Totiviridae, a viral family of double-stranded RNA (dsRNA) viruses, contain a single dsRNA genome 4.6–7.0 kb in length. Totiviridae were initially only known to infect fungi and other eukaryotes as well as plants, but an increase in totiviruses has been detected in insects, mosquitoes, and bats. Here, we describe the isolation and characterization of a strain belonging to the family Totiviridae isolated from Culex tritaeniorhynchus in Kenli, China, in 2016. We isolated a totivirus from field-collected mosquitoes in China by cell culture in Aedes albopictus C6/36 cells, identified the virus by morphological observation and complete genome sequencing, and characterized it by phylogenetic analysis. Transmission electron microscopy identified icosahedral, non-enveloped virus particles with a mean diameter of 35–40 nm. The genome was 7612 bp in length, including two open reading frames (ORFs). ORF1 (5058 nt) encodes the capsid protein, while ORF2 (2216 nt) encodes the viral RNA-dependent RNA polymerase (RdRp). Nucleotide and amino acid homology analysis of isolate showed higher levels of sequence identity with isolate CTV_NJ2 (China, 2010) with 94.87% nucleic acid identity and 97.32% amino acid identity. The isolate was designated C. tritaeniorhynchus totivirus KL (CTV-KL). This is the first identification of a totivirus in a C. tritaeniorhynchus in northern China. Analysis of the virus's morphology, characteristic and genome organization will further enrich our understanding of the molecular and biological characteristics of dsRNA Totiviridae viruses. [ABSTRACT FROM AUTHOR]

Unlike those of double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), and ssRNA viruses, the mechanism of genome packaging of dsRNA viruses is poorly understood. Here, we combined the techniques of high-resolution cryoelectron microscopy (cryo-EM), cellular cryoelectron tomography (cryo-ET), and structure-guided mutagenesis to investigate genome packaging and capsid assembly of bluetongue virus (BTV), a member of the Reoviridae family of dsRNA viruses. A total of eleven assembly states of BTV capsid were captured, with resolutions up to 2.8 Å, with most visualized in the host cytoplasm. ATPase VP6 was found underneath the vertices of capsid shell protein VP3 as an RNA-harboring pentamer, facilitating RNA packaging. RNA packaging expands the VP3 shell, which then engages middle- and outer-layer proteins to generate infectious virions. These revealed "duality" characteristics of the BTV assembly mechanism reconcile previous contradictory co-assembly and core-filling models and provide insights into the mysterious RNA packaging and capsid assembly of Reoviridae members and beyond. [Display omitted] • Capsid assembly of BTV in host cells visualized by cryo-FIB-based cellular cryo-ET • High-resolution structures of BTV assembly intermediates by single-particle cryo-EM • ATPase VP6 pentamer bound underneath the VP3 shell of the pre-subcore for RNA threading • Duality model for BTV RNA packaging with features of both dsDNA and ssRNA viruses Integrated structural analyses captured a total of eleven assembly states of bluetongue virus (BTV), revealing insights into the mysterious RNA packaging and capsid assembly of dsRNA viruses. [ABSTRACT FROM AUTHOR]

The Fusarium graminearum virus China 9 (FgV-ch9) is a member of the genus Betachrysovirus in the Chrysoviridae family and causes hypovirulence in its host, Fusarium graminearum , the causal agent of Fusarium head blight. Although insights into viral biology of FgV-ch9 have expanded in recent years, questions regarding the function of virus-encoded proteins, cis-acting elements, and virus transmission are yet to be answered. Therefore, we developed a tool for the establishment of an artificial 6th segment of FgV-ch9, which encodes a GFP gene flanked by the non-translated regions of FgV-ch9 segment 1. Subsequently, we have proved successful encapsidation of this artificial segment into virus particles as well as its horizontal transmission. Expression of GFP was further verified via immunoassay and life cell imaging. Thus far, we were able to establish for the first time a mini-replicon system for segmented dsRNA viruses replicating in fungi. • An artificial virus transcript is converted to dsRNA by the chrysovirus FgV-ch9. • The artificial dsRNA segment is encapsidated into virus particles. • The artificial dsRNA is horizontally transmitted. • The artificial dsRNA is suitable for subcellular localization studies. • The artificial dsRNA is suitable for the investigation of cis -acting elements. [ABSTRACT FROM AUTHOR]

Heterobasidion RNA virus 6 (HetRV6) is a double-stranded (ds)RNA mycovirus and a member of the recently established genus Orthocurvulavirus within the family Orthocurvulaviridae. The purpose of the study was to determine the biochemical requirements for RNA synthesis catalyzed by HetRV6 RNA-dependent RNA polymerase (RdRp). HetRV6 RdRp was expressed in Escherichia coli and isolated to near homogeneity using liquid chromatography. The enzyme activities were studied in vitro using radiolabeled UTP. The HetRV6 RdRp was able to initiate RNA synthesis in a primer-independent manner using both virus-related and heterologous single-stranded (ss)RNA templates, with a polymerization rate of about 46 nt/min under optimal NTP concentration and temperature. NTPs with 2′-fluoro modifications were also accepted as substrates in the HetRV6 RdRp-catalyzed RNA polymerization reaction. HetRV6 RdRp transcribed viral RNA genome via semi-conservative mechanism. Furthermore, the enzyme demonstrated terminal nucleotidyl transferase (TNTase) activity. Presence of Mn2+ was required for the HetRV6 RdRp catalyzed enzymatic activities. In summary, our study shows that HetRV6 RdRp is an active replicase in vitro that can be potentially used in biotechnological applications, molecular biology, and biomedicine.

A Japanese isolate of Magnaporthe oryzae is infected by Magnaporthe oryzae chrysovirus 1-D (MoCV1-D), which is classified in cluster II of the family Chrysoviridae. The genome of MoCV1-D consists of five dsRNAs. dsRNAs 1–4 show high identity with those of related MoCV1 viruses, whereas dsRNA5 shows relatively low identity and is sometimes deleted during virus propagation. MoCV1-D causes growth inhibition of its host fungus, and the protein encoded by its dsRNA4 impairs cell growth when expressed in yeast cells. It also causes abnormal pigmentation and colony albinization, and we showed that these phenotypes are associated with reduced accumulation of the melanin biosynthesis intermediate scylatone. MoCV1-D exhibits multiform viral structural proteins during prolonged culture. The original host isolate is co-infected with MoCV1-D, a victorivirus, and a partitivirus, and these mycoviruses are detected in cell-free supernatant fractions after prolonged liquid culturing. Hyphal fusion experiments demonstrated that MoCV1-D is transmissible via anastomosis. • Rice blast in Japan was co-infected by chrysovirus, totivirus and partitiviruses. • Rice blast chrysovirus was transmissible to other individuals via anastomosis. • Nutrient starvation induced regular degradation of structural viral proteins. • Expression of mycoviral protein in S. cerevisiae induces cytological damage. • Viral genomic dsRNAs were released into extra-cellular phase after long-term culture. [ABSTRACT FROM AUTHOR]

Bluetongue virus (BTV), a major threat to livestock, is a multilayered, nonturreted member of the Reoviridae, a family of segmented dsRNA viruses characterized by endogenous RNA transcription through an RNA-dependent RNA polymerase (RdRp). To date, the structure of BTV RdRp has been unknown, limiting our mechanistic understanding of BTV transcription and hindering rational drug design effort targeting this essential enzyme. Here, we report the in situ structures of BTV RdRp VP1 in both the triple-layered virion and double-layered core, as determined by cryo-electron microscopy (cryoEM) and subparticle reconstruction. BTV RdRp has 2 unique motifs not found in other viral RdRps: a fingernail, attached to the conserved fingers subdomain, and a bundle of 3 helices: 1 from the palm subdomain and 2 from the N-terminal domain. BTV RdRp VP1 is anchored to the inner surface of the capsid shell via 5 asymmetrically arranged N termini of the inner capsid shell protein VP3A around the 5-fold axis. The structural changes of RdRp VP1 and associated capsid shell proteins between BTV virions and cores suggest that the detachment of the outer capsid proteins VP2 and VP5 during viral entry induces both global movements of the inner capsid shell and local conformational changes of the N-terminal latch helix (residues 34 to 51) of 1 inner capsid shell protein VP3A, priming RdRp VP1 within the capsid for transcription. Understanding this mechanism in BTV also provides general insights into RdRp activation and regulation during viral entry of other multilayered, nonturreted dsRNA viruses. [ABSTRACT FROM AUTHOR]

Endornaviruses are unique, persistent, double-stranded RNA (dsRNA) viruses with symbiotic properties that infect diverse eukaryotes, such as plants, fungi, and oomycetes. Endornaviruses contain a linear dsRNA genome of approximately 10 to 17 kbp in length and are classified in the family Endornaviridae, which consists of two genera, Alphaendornavirus and Betaendornavirus. The endornaviruses encode a single long open reading frame encoding approximately 3200 to 5800 amino acid residues of conserved viral RNA helicase and RNA-dependent RNA polymerase domains, and some endornaviruses contain a site-specific nick in the coding strand of their dsRNA genome. Acute plant viruses propagate rapidly and systemically, eventually killing the host plant, and are then transmitted horizontally. In contrast, plant endornaviruses have several common persistent (symbiotic) properties: a stable low copy number in the host plant, no obvious effect on the host plant, and efficient vertical transmission via gametes. Plant endornaviruses are likely maintained within host plants for hundreds of generations, so the host must stringently regulate their propagation. Whereas RNA silencing functions as a defense system against acute viruses in plants, it may be necessary for the persistent infection (symbiotic life cycle) of endornaviruses. This process includes the stringent regulation of low virus copy number (steady replication before every host cell division) and efficient vertical transmission of the virus to the next generation. [ABSTRACT FROM AUTHOR]

ABSTRACT In contrast to well-established internal ribosomal entry site (IRES)-mediated translational initiation in animals and plants, no IRESs were established in fungal viral or cellular RNAs. To identify IRES elements in mycoviruses, we developed a luciferase-based dual-reporter detection system in Cryphonectria parasitica, a model filamentous fungus for virus-host interactions. A bicistronic construct entails a codon-optimized Renilla and firefly luciferase (ORluc and OFluc, respectively) gene, between which potential IRES sequences can be inserted. In this system, ORluc serves as an internal control, while OFluc represents IRES activity. Virus sequences in the 5′ untranslated regions (UTRs) of the genomes of diverse positive-sense single-stranded RNA and double-stranded RNA (dsRNA) viruses were analyzed. The results show relatively high IRES activities for Cryphonectria hypovirus 1 (CHV1) and CHV2 and faint but measurable activity for CHV3. The weak IRES signal of CHV3 may be explained by its monocistronic nature, differing from the bicistronic nature of CHV1 and CHV2. This would allow these three hypoviruses to have similar rates of translation of replication-associated protein per viral mRNA molecule. The importance of 24 5′-proximal codons of CHV1 as well as the 5′ UTR for IRES function was confirmed. Furthermore, victoriviruses and chrysoviruses tested IRES positive, whereas mycoreoviruses, partitiviruses, and quadriviruses showed similar Fluc activities as the negative controls. Overall, this study represents the first development of an IRES identification system in filamentous fungi based on the codon-optimized dual-luciferase assay and provides evidence for IRESs in filamentous fungi. IMPORTANCE Cap-independent, internal ribosomal entry site (IRES)-mediated translational initiation is often used by virus mRNAs and infrequently by cellular mRNAs in animals and plants. However, no IRESs have been established in fungal virus RNAs or cellular RNAs in filamentous fungi. Here, we report the development of a dual-luciferase assay system and measurement of the IRES activities of fungal RNA viruses in a model filamentous fungal host, Cryphonectria parasitica. Viruses identified as IRES positive include hypoviruses (positive-sense RNA viruses, members of the expanded Picornavirus supergroup), totiviruses (nonsegmented dsRNA viruses), and chrysoviruses (tetrasegmented dsRNA viruses). No IRES activities were observed in the 5′ untranslated regions of mycoreoviruses (11-segmented dsRNA viruses), quadriviruses (tetrasegmented dsRNA viruses), or partitiviruses (bisegmented dsRNA viruses). This study provides the first evidence for IRES activities in diverse RNA viruses in filamentous fungi and is a first step toward identifying trans-acting host factors and cis-regulatory viral RNA elements.

The International Committee for Taxonomy of Viruses (ICTV) recognizes four species of tick-borne orbiviruses (TBOs): Chenuda virus, Chobar Gorge virus, Wad Medani virus and Great Island virus (genus Orbivirus, family Reoviridae). Nucleotide (nt) and amino acid (aa) sequence comparisons provide a basis for orbivirus detection and classification, however full genome sequence data were only available for the Great Island virus species. We report representative genome-sequences for the three other TBO species (virus isolates: Chenuda virus (CNUV); Chobar Gorge virus (CGV) and Wad Medani virus (WMV)). Phylogenetic comparisons show that TBOs cluster separately from insect-borne orbiviruses (IBOs). CNUV, CGV, WMV and GIV share low level aa/nt identities with other orbiviruses, in ‘conserved’ Pol, T2 and T13 proteins/genes, identifying them as four distinct virus-species. The TBO genome segment encoding cell attachment, outer capsid protein 1 (OC1), is approximately half the size of the equivalent segment from insect-borne orbiviruses, helping to explain why tick-borne orbiviruses have a ~1 kb smaller genome.

Horizontal transmission of virulence attenuating hypoviruses of Cryphonectria parasitica is restricted by an allorecognition system termed vegetative incompatibility (vic). A super donor formulation of two engineered C. parasitica strains (SD328/SD82) with gene disruptions at four of six vic loci transmitted hypovirus to strains in the laboratory independent of vic genotype. We now report the transmission of hypovirus by the SD328/82 formulation to a diverse, natural C. parasitica population infecting American chestnut in a forest setting. Hypovirulent (HV) isolates were recovered from 94% of cankers treated with the hypovirus-infected SD328/82 formulation compared to 51% of cankers treated with a hypovirus-infected EU5/6 formulation (strains having the same vic genotypes as SD strains but lacking vic gene disruptions). Overall, the SD328/82 formulation transmitted hypovirus into more divergent vic genotypes compared to the EU5/6 formulation. These results demonstrate the SD328/82 formulation can serve as an enhanced hypovirus vector for highly divergent C. parasitica populations. • First release of C. parasitica engineered to bypass hypovirus transmission barriers. • Enhanced hypovirus transmission by SD328/82 formulation in a forest setting. • SD328/82 formulation can serve as an important tool for restoring American chestnut. [ABSTRACT FROM AUTHOR]