Identification and molecular characterization of a novel totivirus from Mangifera indica.

In this study, we determined the complete genome sequence of a novel totivirus, tentatively named "Mangifera indica totivirus 1" (MiTV1), identified in 'Apple' mango in China. The double-stranded RNA genome of MiTV1 is 4800 base pairs (bp) in length and contains two open reading frames (ORFs) encoding a putative coat protein (CP) and an RNA-dependent RNA polymerase (RdRp). Phylogenetic analysis based on RdRp and CP amino acid sequences showed that MiTV1 is closely related to members of the genus Totivirus in the family Totiviridae. To our knowledge, this is the first report of a totivirus found in Mangifera indica.

Viromes of Tabanids from Russia.

Advances in sequencing technologies and bioinformatics have greatly enhanced our knowledge of virus biodiversity. Currently, the viromes of hematophagous invertebrates, such as mosquitoes and ixodid ticks, are being actively studied. Tabanidae (Diptera) are a widespread family, with members mostly known for their persistent hematophagous behavior. They transmit viral, bacterial, and other pathogens, both biologically and mechanically. However, tabanid viromes remain severely understudied. In this study, we used high-throughput sequencing to describe the viromes of several species in the Hybomitra, Tabanus, Chrysops, and Haematopota genera, which were collected in two distant parts of Russia: the Primorye Territory and Ryazan Region. We assembled fourteen full coding genomes of novel viruses, four partial coding genomes, as well as several fragmented viral sequences, which presumably belong to another twelve new viruses. All the discovered viruses were tested for their ability to replicate in mammalian porcine embryo kidney (PEK), tick HAE/CTVM8, and mosquito C6/36 cell lines. In total, 16 viruses were detected in at least one cell culture after three passages (for PEK and C6/36) or 3 weeks of persistence in HAE/CTVM8. However, in the majority of cases, qPCR showed a decline in virus load over time.

Complete genome sequence of a novel double-stranded RNA virus infecting the phytopathogenic fungus Rhizopus stolonifer.

In this study, we report the complete genome sequence of a novel toti-like virus, tentatively named "Rhizopus stolonifer double-stranded RNA virus 1" (RsDSV1), identified from a phytopathogenic fungal agent of apple fruit rot disease, Rhizopus stolonifer strain A2-1. RsDSV1 has a double-stranded RNA genome. The complete RsDSV1 genome is 5178 nucleotides (nt) in length and contains two open reading frames (ORFs) encoding a putative coat protein (CP) and an RNA-dependent RNA polymerase (RdRp). Phylogenetic analysis based on RdRp and CP amino acid sequences revealed that RsDSV1 is closely related to unclassified members of the family Totiviridae. In stress-inducing Vogel's minimal and sodium dodecyl sulfate-containing media, hyphal growth of A2-1 was suppressed, but the accumulation of RsDSV1 RNA increased, indicating that stresses promote RsDSV1 replication. To our knowledge, this is the first report of a mycovirus found in R. stolonifer.

A Capsid Protein Fragment of a Fusagra-like Virus Found in Carica papaya Latex Interacts with the 50S Ribosomal Protein L17.

Papaya sticky disease is caused by the association of a fusagra-like and an umbra-like virus, named papaya meleira virus (PMeV) and papaya meleira virus 2 (PMeV2), respectively. Both viral genomes are encapsidated in particles formed by the PMeV ORF1 product, which has the potential to encode a protein with 1563 amino acids (aa). However, the structural components of the viral capsid are unknown. To characterize the structural proteins of PMeV and PMeV2, virions were purified from Carica papaya latex. SDS-PAGE analysis of purified virus revealed two major proteins of ~40 kDa and ~55 kDa. Amino-terminal sequencing of the ~55 kDa protein and LC-MS/MS of purified virions indicated that this protein starts at aa 263 of the deduced ORF1 product as a result of either degradation or proteolytic processing. A yeast two-hybrid assay was used to identify Arabidopsis proteins interacting with two PMeV ORF1 product fragments (aa 321-670 and 961-1200). The 50S ribosomal protein L17 (AtRPL17) was identified as potentially associated with modulated translation-related proteins. In plant cells, AtRPL17 co-localized and interacted with the PMeV ORF1 fragments. These findings support the hypothesis that the interaction between PMeV/PMeV2 structural proteins and RPL17 is important for virus-host interactions.

Aedes aegypti Totivirus identified in mosquitoes in the Brazilian Amazon region.

The totiviridae family contains viruses with double-stranded RNA genomes of 4.6-7.0 kpb, which encode a capsid protein (CP) and RNA-dependent RNA polymerase (RdRp), and they are approximately 40 nm in diameter with icosahedral symmetry. Totiviruses were first isolated from mosquitoes collected in Shaanxi Province (China). Here, we report a new Aedes aegypti Totivirus (AaTV) identified in mosquitoes from the Amazon rainforest. Mosquitoes (Diptera: Culicidae) were collected from a forest reserve belonging to the Amazon forest in the city of Macapá, Amapá state, Northern Brazil. A viral sequence with a 5748 nucleotide length that was nearly identical to Aedes aegypti Totivirus (AaTV), here named Aedes aegypti Totivirus BR59AP, was detected. A detailed molecular analysis was performed and shows that AaTV-BR59AP is highly related to the AaTV strain from the Caribbean region. We emphasize the importance of the characterization of new viruses in mosquitoes to deepen our understanding of viral diversity in insects and their potential role in disease.

First report of putative Leishmania RNA virus 2 (LRV2) in Leishmania infantum strains from canine and human visceral leishmaniasis cases in the southeast of Brazil

BACKGROUND Leishmania RNA virus 1 (LRV1) is commonly found in South American Leishmania parasites belonging to the subgenus Viannia, whereas Leishmania RNA virus 2 (LRV2) was previously thought to be restricted to the Old-World pathogens of the subgenus Leishmania. OBJECTIVES In this study, we investigated the presence of LRV2 in strains of Leishmania (L.) infantum, the causative agent of visceral leishmaniasis (VL), originating from different hosts, clinical forms, and geographical regions. METHODS A total of seventy-one isolates were screened for LRV2 using semi-nested reverse transcription-polymerase chain reaction (RT-PCR) targeting the RNA-dependent RNA polymerase (RdRp) gene. FINDINGS We detected LRV2 in two L. infantum isolates (CUR268 and HP-EMO) from canine and human cases, respectively. MAIN CONCLUSIONS To the best of our knowledge, this is the first detection of LRV2 in the New World.

Molecular Characterization of Novel Mycoviruses in Seven Umbelopsis Strains.

The presence of viruses is less explored in Mucoromycota as compared to other fungal groups such as Ascomycota and Basidiomycota. Recently, more and more mycoviruses are identified from the early-diverging lineages of fungi. We have determined the genome of 11 novel dsRNA viruses in seven different Umbelopsis strains with next-generation sequencing (NGS). The identified viruses were named Umbelopsis ramanniana virus 5 (UrV5), 6a (UrV6a); 6b (UrV6b); 7 (UrV7); 8a (UrV8a); 8b (UrV8b); Umbelopsis gibberispora virus 1 (UgV1); 2 (UgV2) and Umbelopsis dimorpha virus 1a (UdV1a), 1b (UdV1b) and 2 (UdV2). All the newly identified viruses contain two open reading frames (ORFs), which putatively encode the coat protein (CP) and the RNA-dependent RNA polymerase (RdRp), respectively. Based on the phylogeny inferred from the RdRp sequences, eight viruses (UrV7, UrV8a, UrV8b, UgV1, UgV2, UdV1a, UdV1b and UdV2) belong to the genus Totivirus, while UrV5, UrV6a and UrV6b are placed into a yet unclassified but well-defined Totiviridae-related group. In UrV5, UgV1, UgV2, UrV8b, UdV1a, UdV2 and UdV1b, ORF2 is predicted to be translated as a fusion protein via a rare +1 (or -2) ribosomal frameshift, which is not characteristic to most members of the Totivirus genus. Virus particles 31 to 32 nm in diameter could be detected in the examined fungal strains by transmission electron microscopy. Through the identification and characterization of new viruses of Mucoromycota fungi, we can gain insight into the diversity of mycoviruses, as well as into their phylogeny and genome organization.

Association of ScV-LA Virus with Host Protein Metabolism Determined by Proteomics Analysis and Cognate RNA Sequencing.

Saccharomyces yeasts are highly dispersed in the environment and microbiota of higher organisms. The yeast killing phenotype, encoded by the viral system, was discovered to be a significant property for host survival. Minor alterations in transcription patterns underpin the reciprocal relationship between LA and M viruses and their hosts, suggesting the fine-tuning of the transcriptional landscape. To uncover the principal targets of both viruses, we performed proteomics analysis of virus-enriched subsets of host proteins in virus type-specific manner. The essential pathways of protein metabolism-from biosynthesis and folding to degradation-were found substantially enriched in virus-linked subsets. The fractionation of viruses allowed separation of virus-linked host RNAs, investigated by high-content RNA sequencing. Ribosomal RNA was found to be inherently associated with LA-lus virus, along with other RNAs essential for ribosome biogenesis. This study provides a unique portrayal of yeast virions through the characterization of the associated proteome and cognate RNAs, and offers a background for understanding ScV-LA viral infection persistency.

Virion structure of Leishmania RNA virus 1.

The presence of Leishmania RNA virus 1 (LRV1) enables Leishmania protozoan parasites to cause more severe disease than the virus-free strains. The structure of LRV1 virus-like particles has been determined previously, however, the structure of the LRV1 virion has not been characterized. Here we used cryo-electron microscopy and single-particle reconstruction to determine the structures of the LRV1 virion and empty particle isolated from Leishmania guyanensis to resolutions of 4.0 Å and 3.6 Å, respectively. The capsid of LRV1 is built from sixty dimers of capsid proteins organized with icosahedral symmetry. RNA genomes of totiviruses are replicated inside the virions by RNA polymerases expressed as C-terminal extensions of a sub-population of capsid proteins. Most of the virions probably contain one or two copies of the RNA polymerase, however, the location of the polymerase domains in LRV1 capsid could not be identified, indicating that it varies among particles. Importance. Every year over 200 000 people contract leishmaniasis and more than five hundred people die of the disease. The mucocutaneous form of leishmaniasis produces lesions that can destroy the mucous membranes of the nose, mouth, and throat. Leishmania parasites carrying Leishmania RNA virus 1 (LRV1) are predisposed to cause aggravated symptoms in the mucocutaneous form of leishmaniasis. Here, we present the structure of the LRV1 virion determined using cryo-electron microscopy.

Potential origins of fish toti-like viruses in invertebrates.

The virus family Totiviridae had originally been considered to include only viruses which infected fungal and protist hosts, but since 2006 a growing number of viruses found in invertebrates and fish have been shown to cluster phylogenetically within this family. These Totiviridae-like, or toti-like, viruses do not appear to belong within any existing genera of Totiviridae, and whilst a number of new genus names have been suggested, none has yet been universally accepted. Within this growing number of toti-like viruses from animal hosts, there exists emerging viral threats particularly to aquaculture, namely Infectious myonecrosis virus in whiteleg shrimp and Piscine myocarditis virus (PMCV) in Atlantic salmon (Salmo salar). PMCV in particular continues to be an issue in salmon aquaculture as a number of questions remain unanswered about how the virus is transmitted and the route of entry into host fish. Using a phylogenetic approach, this study shows how PMCV and the other fish toti-like viruses probably have deeper origins in an arthropod host. Based on this, it is hypothesized that sea lice could be acting as a vector for PMCV, as seen with other RNA viruses in Atlantic salmon aquaculture and in the toti-like Cucurbit yellows-associated virus which is spread by the greenhouse whitefly Trialeurodes vaporariorum.

Detection and Characterization of RNA Viruses in Red Macroalgae (Bangiaceae) and Their Food Product (Nori Sheets).

Persistent RNA viruses, which have been suggested to form symbiotic relationships with their hosts, have been reported to occur in eukaryotes, such as plants, fungi, and algae. Based on empirical findings, these viruses may also be present in commercially cultivated macroalgae. Accordingly, the present study aimed to screen red macroalgae (family Bangiaceae conchocelis and Neopyropia yezoensis thallus) and processed nori sheets (N. yezoensis) for persistent RNA viruses using fragmented and primer-ligated dsRNA sequencing (FLDS) and targeted reverse transcription PCR (RT-PCR). A Totiviridae-related virus was detected in the conchocelis of Neoporphyra haitanensis, which is widely cultivated in China, while two Mitoviridae-related viruses were found in several conchocelis samples and all N. yezoensis-derived samples (thallus and nori sheets). Mitoviridae-related viruses in N. yezoensis are widespread among cultivated species and not expected to inhibit host growth. Mitoviridae-related viruses were also detected in several phylogenetically distant species in the family Bangiaceae, which suggests that these viruses persisted and coexist in the family Bangiaceae over a long period of time. The present study is the first to report persistent RNA viruses in nori sheets and their raw materials.

First Evidence of Past and Present Interactions between Viruses and the Black Soldier Fly, Hermetia illucens.

Black soldier flies (BSFs, Hermetia illucens) are becoming a prominent research model encouraged by the insect as food and feed and waste bioconversion industries. Insect mass-rearing facilities are at risk from the spread of viruses, but so far, none have been described in BSFs. To fill this knowledge gap, a bioinformatic approach was undertaken to discover viruses specifically associated with BSFs. First, BSF genomes were screened for the presence of endogenous viral elements (EVEs). This led to the discovery and mapping of seven orthologous EVEs integrated into three BSF genomes originating from five viral families. Secondly, a virus discovery pipeline was used to screen BSF transcriptomes. This led to detecting a new exogenous totivirus that we named hermetia illucens totivirus 1 (HiTV1). Phylogenetic analyses showed this virus belongs to a clade of insect-specific totiviruses and is closely related to the largest EVE located on chromosome 1 of the BSF genome. Lastly, this EVE was found to express a small transcript in some BSFs infected by HiTV1. Altogether, this data mining study showed that far from being unscathed from viruses, BSFs bear traces of past interactions with several viral families and of present interactions with the exogenous HiTV1.

Adaptive Response of Saccharomyces Hosts to Totiviridae L-A dsRNA Viruses Is Achieved through Intrinsically Balanced Action of Targeted Transcription Factors.

Totiviridae L-A virus is a widespread yeast dsRNA virus. The persistence of the L-A virus alone appears to be symptomless, but the concomitant presence of a satellite M virus provides a killer trait for the host cell. The presence of L-A dsRNA is common in laboratory, industrial, and wild yeasts, but little is known about the impact of the L-A virus on the host's gene expression. In this work, based on high-throughput RNA sequencing data analysis, the impact of the L-A virus on whole-genome expression in three different Saccharomyces paradoxus and S. cerevisiae host strains was analyzed. In the presence of the L-A virus, moderate alterations in gene expression were detected, with the least impact on respiration-deficient cells. Remarkably, the transcriptional adaptation of essential genes was limited to genes involved in ribosome biogenesis. Transcriptional responses to L-A maintenance were, nevertheless, similar to those induced upon stress or nutrient availability. Based on these data, we further dissected yeast transcriptional regulators that, in turn, modulate the cellular L-A dsRNA levels. Our findings point to totivirus-driven fine-tuning of the transcriptional landscape in yeasts and uncover signaling pathways employed by dsRNA viruses to establish the stable, yet allegedly profitless, viral infection of fungi.

Discovery of a Novel Species of Trichomonasvirus in the Human Parasite Trichomonas vaginalis Using Transcriptome Mining.

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.

Molecular characterization of three novel mycoviruses in the plant pathogenic fungus Exobasidium.

The plant pathogen Exobasidium gracile, which belongs to the basidiomycetous genus Exobasidium, can lead to swollen and thicker leaves of C. oleifera. To our knowledge, there have been no reports of mycoviruses infecting Exobasidium gracile. This study characterized three mycoviruses coinfecting the plant pathogen Exobasidium gracile strain Z-1. Based on phylogenetic and genomic analyses, E. gracile strain Z-1 was infected two putative Totiviruses designated Exobasidium gracile Totivirus 1 (EgTV1) and Exobasidium gracile Totivirus 2 (EgTV2) and a putative Zybavirus of the family Amalgaviridae defined Exobasidium gracile Zybavirus 1 (EgZV1). Similar to the genomic organization of other Totiviruses, the EgTV1 and EgTV2 genomes are composed of one dsRNA segment that exhibits two large ORFs encoding a CP (capsid protein) and an RdRp (RNA-dependent RNA polymerase), respectively. Moreover, EgTV1 and EgTV2 genomes with a candidate -1 slippery heptamer sequence were discovered between CP and RdRp, respectively. Similar to other Zybaviruses of the family Amalgaviridae, the EgZV1 genome is composed of one dsRNA segment that contains two large ORFs encoding an unknown protein and an RdRp. In addition, the EgZV1 genome has a candidate +1 slippery heptamer sequence between an unknown protein and RdRp.

Metagenomic Identification of Viral Sequences in Laboratory Reagents.

Metagenomic next-generation sequencing has transformed the discovery and diagnosis of infectious disease, with the power to characterise the complete 'infectome' (bacteria, viruses, fungi, parasites) of an individual host organism. However, the identification of novel pathogens has been complicated by widespread microbial contamination in commonly used laboratory reagents. Using total RNA sequencing ("metatranscriptomics") we documented the presence of contaminant viral sequences in multiple 'blank' negative control sequencing libraries that comprise a sterile water and reagent mix. Accordingly, we identified 14 viral sequences in 7 negative control sequencing libraries. As in previous studies, several circular replication-associated protein encoding (CRESS) DNA virus-like sequences were recovered in the blank control libraries, as well as contaminating sequences from the Totiviridae, Tombusviridae and Lentiviridae families of RNA virus. These data suggest that viral contamination of common laboratory reagents is likely commonplace and can comprise a wide variety of viruses.

2A and 2A-like Sequences: Distribution in Different Virus Species and Applications in Biotechnology.

2A is an oligopeptide sequence that mediates a ribosome "skipping" effect and can mediate a co-translation cleavage of polyproteins. These sequences are widely distributed from insect to mammalian viruses and could act by accelerating adaptive capacity. These sequences have been used in many heterologous co-expression systems because they are versatile tools for cleaving proteins of biotechnological interest. In this work, we review and update the occurrence of 2A/2A-like sequences in different groups of viruses by screening the sequences available in the National Center for Biotechnology Information database. Interestingly, we reported the occurrence of 2A-like for the first time in 69 sequences. Among these, 62 corresponded to positive single-stranded RNA species, six to double stranded RNA viruses, and one to a negative-sense single-stranded RNA virus. The importance of these sequences for viral evolution and their potential in biotechnological applications are also discussed.

Comparative Molecular Characterization of Novel and Known Piscine Toti-Like Viruses.

Totiviridae is a virus family well known to infect uni-cellular organisms like fungi and protozoa. In more recent years, viruses characterized as toti-like viruses, have been found in primarily arthropods, but also a couple in planarians and piscine species. These toti-like viruses share phylogenetic similarities to totiviruses; however, their genomes also includes additional coding sequences in either 5' or 3' ends expected to relate to more advanced infection mechanisms in more advanced hosts. Here, we applied next generation sequencing (NGS) technologies and discovered three new toti-like viruses, one in wild common carp and one in bluegill from the USA and one in farmed lumpsucker from Norway. These are named common carp toti-like virus 1 (CCTLV-1), bluegill toti-like virus 1 (BGTLV-1), and Cyclopterus lumpus toti-like virus (CLuTLV), respectively. The genomes of these viruses have been characterized and compared to the three previously known piscine toti-like viruses, piscine myocarditis virus (PMCV) found in Atlantic salmon and the two from golden shiner, now named golden shiner toti-like virus 1 and 2 (GSTLV-1 and -2), and also to totiviruses and other toti-like viruses. We found that four piscine toti-like viruses had additional gene(s) in the 3' end of the genome, and also clustered phylogenetically based on both capsid and RdRp-genes. This cluster constituted a distant branch in the Totiviridae, and we suggest this should be defined as a separate genus named Pistolvirus, to reflect this major cluster of piscine toti-like viruses. The remaining two piscine toti-like viruses differentiated from these by lacking any additional 3' end genes and also by phylogenetical relation, but were both clustering with arthropod viruses in two different clusters.

Multiple Viral Infections Detected in Phytophthora condilina by Total and Small RNA Sequencing.

Marine oomycetes have recently been shown to be concurrently infected by (-)ssRNA viruses of the order Bunyavirales. In this work, even higher virus variability was found in a single isolate of Phytophthora condilina, a recently described member of Phytophthora phylogenetic Clade 6a, which was isolated from brackish estuarine waters in southern Portugal. Using total and small RNA-seq the full RdRp of 13 different potential novel bunya-like viruses and two complete toti-like viruses were detected. All these viruses were successfully confirmed by reverse transcription polymerase chain reaction (RT-PCR) using total RNA as template, but complementarily one of the toti-like and five of the bunya-like viruses were confirmed when dsRNA was purified for RT-PCR. In our study, total RNA-seq was by far more efficient for de novo assembling of the virus sequencing but small RNA-seq showed higher read numbers for most viruses. Two main populations of small RNAs (21 nts and 25 nts-long) were identified, which were in accordance with other Phytophthora species. To the best of our knowledge, this is the first study using small RNA sequencing to identify viruses in Phytophthora spp.

Atomic Structure of the Trichomonas vaginalis Double-Stranded RNA Virus 2.

Trichomonas vaginalis, the causative pathogen for the most common nonviral sexually transmitted infection worldwide, is itself frequently infected with one or more of the four types of small double-stranded RNA (dsRNA) Trichomonas vaginalis viruses (TVV1 to 4, genus Trichomonasvirus, family Totiviridae). Each TVV encloses a nonsegmented genome within a single-layered capsid and replicates entirely intracellularly, like many dsRNA viruses, and unlike those in the Reoviridae family. Here, we have determined the structure of TVV2 by cryo-electron microscopy (cryoEM) at 3.6 Å resolution and derived an atomic model of its capsid. TVV2 has an icosahedral, T = 2*, capsid comprised of 60 copies of the icosahedral asymmetric unit (a dimer of the two capsid shell protein [CSP] conformers, CSP-A and CSP-B), typical of icosahedral dsRNA virus capsids. However, unlike the robust CSP-interlocking interactions such as the use of auxiliary "clamping" proteins among Reoviridae, only lateral CSP interactions are observed in TVV2, consistent with an assembly strategy optimized for TVVs' intracellular-only replication cycles within their protozoan host. The atomic model reveals both a mostly negatively charged capsid interior, which is conducive to movement of the loosely packed genome, and channels at the 5-fold vertices, which we suggest as routes of mRNA release during transcription. Structural comparison of TVV2 to the Saccharomyces cerevisiae L-A virus reveals a conserved helix-rich fold within the CSP and putative guanylyltransferase domain along the capsid exterior, suggesting conserved mRNA maintenance strategies among Totiviridae This first atomic structure of a TVV provides a framework to guide future biochemical investigations into the interplay between Trichomonas vaginalis and its viruses.IMPORTANCETrichomonas vaginalis viruses (TVVs) are double-stranded RNA (dsRNA) viruses that cohabitate in Trichomonas vaginalis, the causative pathogen of trichomoniasis, the most common nonviral sexually transmitted disease worldwide. Featuring an unsegmented dsRNA genome encoding a single capsid shell protein (CSP), TVVs contrast with multisegmented dsRNA viruses, such as the diarrhea-causing rotavirus, whose larger genome is split into 10 dsRNA segments encoding 5 unique capsid proteins. To determine how TVVs incorporate the requisite functionalities for viral replication into their limited proteome, we derived the atomic model of TVV2, a first for TVVs. Our results reveal the intersubunit interactions driving CSP association for capsid assembly and the properties that govern organization and maintenance of the viral genome. Structural comparison between TVV2 capsids and those of distantly related dsRNA viruses indicates conserved strategies of nascent RNA release and a putative viral guanylyltransferase domain implicated in the cytoplasmic maintenance of viral messenger and genomic RNA.