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Extended abstract of a paper presented at Microscopy and Microanalysis 2013 in Indianapolis, Indiana, USA, August 4 – August 8, 2013.

The flagellated protozoan Trichomonas vaginalis is an obligate human genitourinary parasite and the most frequent cause of sexually transmitted disease worldwide. Most clinical isolates of T. vaginalis are persistently infected with one or more double-stranded RNA (dsRNA) viruses from the genus Trichomonasvirus, family Totiviridae, which appear to influence not only protozoan biology but also human disease. Here we describe the three-dimensional structure of Trichomonas vaginalis virus 1 (TVV1) virions, as determined by electron cryomicroscopy and icosahedral image reconstruction. The structure reveals a T=1 capsid comprising 120 subunits, 60 in each of two nonequivalent positions, designated A and B, as previously observed for fungal Totiviridae family members. The putative protomer is identified as an asymmetric AB dimer consistent with either decamer or tetramer assembly intermediates. The capsid surface is notable for raised plateaus around the icosahedral 5-fold axes, with canyons connecting the 2- and 3-fold axes. Capsid-spanning channels at the 5-fold axes are unusually wide and may facilitate release of the viral genome, promoting dsRNA-dependent immunoinflammatory responses, as recently shown upon the exposure of human cervicovaginal epithelial cells to either TVV-infected T. vaginalis or purified TVV1 virions. Despite extensive sequence divergence, conservative features of the capsid reveal a helix-rich fold probably derived from an ancestor shared with fungal Totiviridae family members. Also notable are mass spectrometry results assessing the virion proteins as a complement to structure determination, which suggest that translation of the TVV1 RNA-dependent RNA polymerase in fusion with its capsid protein involves-2, and not+1, ribosomal frameshifting, an uncommonly found mechanism to date. © 2013 Parent et al.

Three structural forms of type 1 Lang reovirus (virions, intermediate subviral particles [ISVPs], and cores) have been examined by cryoelectron microscopy (cryoEM) and image reconstruction at 27 to 32-A resolution. Analysis of the three-dimensional maps and known biochemical composition allows determination of capsid protein location, globular shape, stoichiometry, quaternary organization, and interactions with adjacent capsid proteins. Comparisons of the virion, ISVP and core structures and examination of difference maps reveal dramatic changes in supra-molecular structure and protein conformation that are related to the early steps of reovirus infection. The intact virion (approximately 850-A diam) is designed for environmental stability in which the dsRNA genome is protected not only by tight sigma 3-mu 1, lambda 2-sigma 3, and lambda 2-mu 1 interactions in the outer capsid but also by a densely packed core shell formed primarily by lambda 1 and sigma 2. The segmented genome appears to be packed in a liquid crystalline fashion at radii < 240 A. Depending on viral growth conditions, virions undergo cleavage by enteric or endosomal/lysosomal proteases, to generate the activated ISVP (approximately 800-A diam). This transition involves the release of an outer capsid layer spanning radii from 360 to 427 A that is formed by 60 tetrameric and 60 hexameric clusters of ellipsoidal subunits of sigma 3. The vertex-associated cell attachment protein, sigma 1, also undergoes a striking change from a poorly visualized, more compact form, to an extended, flexible fiber. This conformational change may maximize interactions of sigma 1 with cell surface receptors. Transcription of viral mRNAs is mediated by the core particle (approximately 600-A diam), generated from the ISVP after penetration and uncoating. The transition from ISVP to core involves release of the 12 sigma 1 fibers and the remaining outer capsid layer formed by 200 trimers of rod-shaped mu 1 subunits that span radii from 306 to 395 A. In the virion and ISVP, flower-shaped pentamers of the lambda 2 protein are centered at the vertices. In the ISVP-to-core transition, domains of the lambda 2 subunits rotate and swing upward and outward to form a turret-like structure extending from radii 305 to 400 A, with a diameter of 184 A, and a central channel 84 A wide. This novel conformational change allows the potential diffusion of substrates for transcription and exit of newly synthesized mRNA segments.(ABSTRACT TRUNCATED AT 400 WORDS)

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.

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.

This article reports the changes to virus taxonomy approved and ratified by the International Committee on Taxonomy of Viruses (ICTV) in March 2021. The entire ICTV was invited to vote on 290 taxonomic proposals approved by the ICTV Executive Committee at its meeting in October 2020, as well as on the proposed revision of the International Code of Virus Classification and Nomenclature (ICVCN). All proposals and the revision were ratified by an absolute majority of the ICTV members. Of note, ICTV mandated a uniform rule for virus species naming, which will follow the binomial 'genus-species' format with or without Latinized species epithets. The Study Groups are requested to convert all previously established species names to the new format. ICTV has also abolished the notion of a type species, i.e., a species chosen to serve as a name-bearing type of a virus genus. The remit of ICTV has been clarified through an official definition of 'virus' and several other types of mobile genetic elements. The ICVCN and ICTV Statutes have been amended to reflect these changes., (© 2021. This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply.)

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. IMPORTANCE Planarians are freshwater flatworms, related more distantly to tapeworms and flukes, and have been developed as models to study the molecular mechanisms of stem cell biology and tissue regeneration. These worms live in aquatic environments, where they are likely to encounter a variety of viruses, bacteria, and eukaryotic organisms with pathogenic potential. How the planarian immune system has evolved to cope with these potential pathogens is not well understood, and only two types of planarian viruses have been described to date. Here, we report discovery and inaugural studies of a novel taxon of dsRNA viruses in five different planarian species. The virus in the best-characterized model species, Schmidtea mediterranea , appears to persist long term in that host while avoiding endogenous antiviral or RNAi mechanisms. The S. mediterranea virus-host system thus seems to offer opportunity for gaining new insights into host defenses and their evolution in an important lab model., (© Crown copyright 2020.)

The article Binomial nomenclature for virus species: a consultation, written by Stuart G. Siddell, Peter J. Walker, Elliot J. Lefkowitz, Arcady R. Mushegian, Bas E. Dutilh.

The Executive Committee of the International Committee on Taxonomy of Viruses (ICTV) recognizes the need for a standardized nomenclature for virus species. This article sets out the case for establishing a binomial nomenclature and presents the advantages and disadvantages of different naming formats. The Executive Committee understands that adopting a binomial system would have major practical consequences, and invites comments from the virology community before making any decisions to change the existing nomenclature. The Executive Committee will take account of these comments in deciding whether to approve a standardized binomial system at its next meeting in October 2020. Note that this system would relate only to the formal names of virus species and not to the names of viruses.

Recent results indicate that mitoviruses, which replicate persistently in host mitochondria, are not restricted to fungi, but instead are found also in plants. Beta vulgaris mitovirus 1 (BevuMV1) is an example first discovered in sugar beet cultivars. For the current study, complete coding sequences of 42 BevuMV1 strains were newly determined, derived from not only sugar beet but also fodder beet, table beet, and Swiss chard cultivars of Beta vulgaris, as well as wild sea beet. BevuMV1 is thus a common phytobiome component of this valuable crop species. Most of the new BevuMV1 sequences originated from RNA extracted from B. vulgaris seed clusters, consistent with vertical transmission of this virus. Results suggest that BevuMV1 entered the B. vulgaris lineage prior to human cultivation and also provides a marker for tracing the maternal ancestry of B. vulgaris cultivars. Especially notable is the monophyletic relationship and limited sequence divergence among BevuMV1 strains from cultivars that are thought or shown to share the "Owen" trait for cytoplasmic male sterility, which is transmitted by maternal mitochondria and has been broadly established in commercial breeding lines of B. vulgaris since the mid-20 th century., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Fungi constituting the Entomophthora muscae species complex (members of subphylum Entomophthoromycotina , phylum Zoopagamycota ) commonly kill their insect hosts and manipulate host behaviors in the process. In this study, we made use of public transcriptome data to identify and characterize eight new species of mitoviruses associated with several different E. muscae isolates. Mitoviruses are simple RNA viruses that replicate in host mitochondria and are frequently found in more phylogenetically apical fungi (members of subphylum Glomeromyoctina , phylum Mucoromycota , phylum Basidiomycota and phylum Ascomycota ) as well as in plants. E. muscae is the first fungus from phylum Zoopagomycota , and thereby the most phylogenetically basal fungus, found to harbor mitoviruses to date. Multiple UGA (Trp) codons are found not only in each of the new mitovirus sequences from E. muscae but also in mitochondrial core-gene coding sequences newly assembled from E. muscae transcriptome data, suggesting that UGA (Trp) is not a rarely used codon in the mitochondria of this fungus. The presence of mitoviruses in these basal fungi has possible implications for the evolution of these viruses.

This article lists the changes to virus taxonomy approved and ratified by the International Committee on Taxonomy of Viruses in February 2018. A total of 451 species, 69 genera, 11 subfamilies, 9 families and one new order were added to the taxonomy. The current totals at each taxonomic level now stand at 9 orders, 131 families, 46 subfamilies, 803 genera and 4853 species. A change was made to the International Code of Virus Classification and Nomenclature to allow the use of the names of people in taxon names under appropriate circumstances. An updated Master Species List incorporating the approved changes was released in March 2018 ( https://talk.ictvonline.org/taxonomy/ ).

Because so few viruses in the family Barnaviridae have been reported, we searched for more of them in public sequence databases. Here, we report the complete coding sequence of Colobanthus quitensis associated barnavirus 1, mined from a transcriptome of the Antarctic pearlwort Colobanthus quitensis. The 4.2-kb plus-strand sequence of this virus encompasses four main open reading frames (ORFs), as expected for barnaviruses, including ORFs for a protease-containing polyprotein, an RNA-dependent RNA polymerase whose translation appears to rely on - 1 ribosomal frameshifting, and a capsid protein that is likely to be translated from a subgenomic RNA. The possible derivation of this virus from a fungus associated with C. quitensis is discussed.

Mammalian reovirus viral factories (VFs) form filamentous or globular structures depending on the viral strain. In this study, we attempt to characterize the dynamics of both filamentous and globular VFs. Here, we present evidence demonstrating that globular VFs are dynamic entities coalescing between them, thereby gaining in size and concomitantly decreasing in numbers during the course of the infection. Additionally, both kinds of VFs condense into a perinuclear position. Our results show that globular VFs rely on an intact MT-network for dynamic motion, structural assembly, and maintenance and for perinuclear condensation. Interestingly, dynein localizes in both kinds of VFs, having a role at least in large globular VFs formation. To study filamentous VF dynamics, we used different transfection ratios of µNS with filamentous µ2. We found a MT-network dependency for VF-like structures perinuclear condensation. Also, µNS promotes VFLSs perinuclear positioning as well as an increase in acetylated tubulin levels., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Mitoviruses have small RNA(+) genomes, replicate in mitochondria, and have been shown to infect only fungi to date. For this report, sequences that appear to represent nearly complete plant mitovirus genomes were recovered from publicly available transcriptome data. Twenty of the refined sequences, 2684-2898 nt long and derived from 10 different species of land plants, appear to encompass the complete coding regions of contemporary plant mitoviruses, which furthermore constitute a monophyletic cluster within genus Mitovirus. Complete coding sequences of several of these viruses were recovered from multiple transcriptome (but not genome) studies of the same plant species and also from multiple plant tissues. Crop plants among implicated hosts include beet and hemp. Other new results suggest that such genuine plant mitoviruses were immediate ancestors to endogenized mitovirus elements now widespread in land plant genomes. Whether these mitoviruses are wholly cryptic with regard to plant health remains to be investigated., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Mammalian orthoreovirus protein μ2 is a component of the viral core particle. Its activities include RNA binding and hydrolysis of the γ-phosphate from NTPs and RNA 5´-termini, suggesting roles as a cofactor for the viral RNA-dependent RNA polymerase, λ3, first enzyme in 5´-capping of viral plus-strand RNAs, and/or prohibitory of RNA-5´-triphosphate-activated antiviral signaling. Within infected cells, μ2 also contributes to viral factories, cytoplasmic structures in which genome replication and particle assembly occur. By associating with both microtubules (MTs) and viral factory matrix protein μNS, μ2 can anchor the factories to MTs, the full effects of which remain unknown. In this study, a protease-hypersensitive region allowed μ2 to be dissected into two large fragments corresponding to residues 1-282 and 283-736. Fusions with enhanced green fluorescent protein revealed that these amino- and carboxyl-terminal regions of μ2 associate in cells with either MTs or μNS, respectively. More exhaustive deletion analysis defined μ2 residues 1-325 as the minimal contiguous region that associates with MTs in the absence of the self-associating tag. A region involved in μ2 self-association was mapped to residues 283-325, and self-association involving this region was essential for MT-association as well. Likewise, we mapped that μNS-binding site in μ2 relates to residues 290-453 which is independent of μ2 self-association. These findings suggest that μ2 monomers or oligomers can bind to MTs and μNS, but that self-association involving μ2 residues 283-325 is specifically relevant for MT-association during viral factories formation.

Bisegmented dsRNA viruses that infect most or all isolates of apicomplexan parasite Cryptosporidium parvum are currently assigned to a single species, Cryptosporidium parvum virus 1, in genus Cryspovirus, family Partitiviridae. An analysis of existing sequence data suggested that the complete sequences of both cryspovirus genome segments, dsRNA1 and dsRNA2, had yet to be determined. We therefore set out to accomplish this for the virus strain that infects C. parvum isolate Iowa. The results suggest that several previous cryspovirus sequences are indeed truncated at one or both segment termini and also identify sequences at or near the termini that are conserved in both segments. Complete sequences of other cryspovirus strains, including ones from other Cryptosporidium species, are needed for refining their classification into one or more virus species.

Mitoviruses replicate in mitochondria of their host fungi. They have small RNA genomes that encompass a single ORF encoding the viral RdRp. Since UGA codons encode Trp in fungal mitochondria, the RdRp ORF of a typical mitovirus includes multiple UGA codons. In some mitoviruses, however, the ORF has no such codons, suggesting that these particular viruses may be under selective pressure to exclude them. In this report, new evidence is presented that host fungi whose mitoviruses have no or few UGA codons are distinctive in also having no or few UGA codons in their core mitochondrial genes. Thus, the relative exclusion of such codons in a subset of mitoviruses appears to reflect most fundamentally that UGA(Trp) is a rare mitochondrial codon in their particular hosts. The fact that UGA(Trp) is a rare mitochondrial codon in many fungi appears not to have been widely discussed to date., (Copyright © 2017 Elsevier Inc. All rights reserved.)

We mark the 50th anniversary of the International Committee on Taxonomy of Viruses (ICTV) by presenting a brief history of the organization since its foundation, showing how it has adapted to advancements in our knowledge of virus diversity and the methods used to characterize it. We also outline recent developments, supported by a grant from the Wellcome Trust (UK), that are facilitating substantial changes in the operations of the ICTV and promoting dialogue with the virology community. These developments will generate improved online resources, including a freely available and regularly updated ICTV Virus Taxonomy Report. They also include a series of meetings between the ICTV and the broader community focused on some of the major challenges facing virus taxonomy, with the outcomes helping to inform the future policy and practice of the ICTV.

A previously reported Expressed Sequence Tag (EST) library from spores of microsporidian Antonospora locustae includes a number of clones with sequence similarities to plant amalgaviruses. Reexamining the sequence accessions from that library, we found additional such clones, contributing to a 3247-nt contig that approximates the length of an amalga-like virus genome. Using A. locustae spores stored from that previous study, and new ones obtained from the same source, we newly visualized the putative dsRNA genome of this virus and obtained amplicons yielding a 3387-nt complete genome sequence. Phylogenetic analyses suggested it as prototype strain of a new genus in family Amalgaviridae. The genome contains two partially overlapping long ORFs, with downstream ORF2 in the +1 frame relative to ORF1 and a proposed motif for +1 ribosomal frameshifting in the region of overlap. Subsequent database searches using the predicted fusion protein sequence of this new amalga-like virus identified related sequences in the transcriptome of a basal hexapod, the springtail species Tetrodontophora bielanensis. We speculate that this second new amalga-like virus (contig length, 3475 nt) likely also derived from a microsporidian, or related organism, which was associated with the springtail specimens at the time of sampling for transcriptome analysis. Other findings of interest include evidence that the ORF1 translation products of these two new amalga-like viruses contain a central region of predicted α-helical coiled coil, as recently reported for plant amalgaviruses, and transcriptome-based evidence for another new amalga-like virus in the transcriptome of another basal hexapod, the two-pronged bristletail species Campodea augens., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The number and diversity of viral sequences that are identified in metagenomic data far exceeds that of experimentally characterized virus isolates. In a recent workshop, a panel of experts discussed the proposal that, with appropriate quality control, viruses that are known only from metagenomic data can, and should be, incorporated into the official classification scheme of the International Committee on Taxonomy of Viruses (ICTV). Although a taxonomy that is based on metagenomic sequence data alone represents a substantial departure from the traditional reliance on phenotypic properties, the development of a robust framework for sequence-based virus taxonomy is indispensable for the comprehensive characterization of the global virome. In this Consensus Statement article, we consider the rationale for why metagenomic sequence data should, and how it can, be incorporated into the ICTV taxonomy, and present proposals that have been endorsed by the Executive Committee of the ICTV.

Sequence accessions attributable to novel plant amalgaviruses have been found in the Transcriptome Shotgun Assembly database. Sixteen accessions, derived from 12 different plant species, appear to encompass the complete protein-coding regions of the proposed amalgaviruses, which would substantially expand the size of genus Amalgavirus from 4 current species. Other findings include evidence for UUU&#95;CGN as a +1 ribosomal frameshifting motif prevalent among plant amalgaviruses; for a variant version of this motif found thus far in only two amalgaviruses from solanaceous plants; for a region of α-helical coiled coil propensity conserved in a central region of the ORF1 translation product of plant amalgaviruses; and for conserved sequences in a C-terminal region of the ORF2 translation product (RNA-dependent RNA polymerase) of plant amalgaviruses, seemingly beyond the region of conserved polymerase motifs. These results additionally illustrate the value of mining the TSA database and others for novel viral sequences for comparative analyses., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Zygosaccharomyces bailii virus Z (ZbV-Z) is a monosegmented dsRNA virus that infects the yeast Zygosaccharomyces bailii and remains unclassified to date despite its discovery >20years ago. The previously reported nucleotide sequence of ZbV-Z (GenBank AF224490) encompasses two nonoverlapping long ORFs: upstream ORF1 encoding the putative coat protein and downstream ORF2 encoding the RNA-dependent RNA polymerase (RdRp). The lack of overlap between these ORFs raises the question of how the downstream ORF is translated. After examining the previous sequence of ZbV-Z, we predicted that it contains at least one sequencing error to explain the nonoverlapping ORFs, and hence we redetermined the nucleotide sequence of ZbV-Z, derived from the same isolate of Z. bailii as previously studied, to address this prediction. The key finding from our new sequence, which includes several insertions, deletions, and substitutions relative to the previous one, is that ORF2 in fact overlaps ORF1 in the +1 frame. Moreover, a proposed sequence motif for +1 programmed ribosomal frameshifting, previously noted in influenza A viruses, plant amalgaviruses, and others, is also present in the newly identified ORF1-ORF2 overlap region of ZbV-Z. Phylogenetic analyses provided evidence that ZbV-Z represents a distinct taxon most closely related to plant amalgaviruses (genus Amalgavirus, family Amalgaviridae). We conclude that ZbV-Z is the prototype of a new species, which we propose to assign as type species of a new genus of monosegmented dsRNA mycoviruses in family Amalgaviridae. Comparisons involving other unclassified mycoviruses with RdRps apparently related to those of plant amalgaviruses, and having either mono- or bisegmented dsRNA genomes, are also discussed., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Recent advances in biosensing technologies present great potential for medical diagnostics, thus improving clinical decisions. However, creating a label-free general sensing platform capable of detecting multiple biotargets in various clinical specimens over a wide dynamic range, without lengthy sample-processing steps, remains a considerable challenge. In practice, these barriers prevent broad applications in clinics and at patients' homes. Here, we demonstrate the nanoplasmonic electrical field-enhanced resonating device (NE(2)RD), which addresses all these impediments on a single platform. The NE(2)RD employs an immunodetection assay to capture biotargets, and precisely measures spectral color changes by their wavelength and extinction intensity shifts in nanoparticles without prior sample labeling or preprocessing. We present through multiple examples, a label-free, quantitative, portable, multitarget platform by rapidly detecting various protein biomarkers, drugs, protein allergens, bacteria, eukaryotic cells, and distinct viruses. The linear dynamic range of NE(2)RD is five orders of magnitude broader than ELISA, with a sensitivity down to 400 fg/mL This range and sensitivity are achieved by self-assembling gold nanoparticles to generate hot spots on a 3D-oriented substrate for ultrasensitive measurements. We demonstrate that this precise platform handles multiple clinical samples such as whole blood, serum, and saliva without sample preprocessing under diverse conditions of temperature, pH, and ionic strength. The NE(2)RD's broad dynamic range, detection limit, and portability integrated with a disposable fluidic chip have broad applications, potentially enabling the transition toward precision medicine at the point-of-care or primary care settings and at patients' homes.

New sequencing studies of the nonsegmented dsRNA genome of penaeid shrimp infectious myonecrosis virus (IMNV), a tentatively assigned member of the family Totiviridae, identified previously unread sequences at both genome termini in three previously analyzed IMNV strains, one from Brazil (the prototype strain of IMNV) and two from Indonesia. The new sequence determinations add >600 nt to the 5' end of the genomic plus strand of each strain, increasing the length of the 5' nontranslated region to at least 469-472 nt and the length of the upstream open reading frame (ORF1) translation product by at least 48 aa. These new findings are similar to recent ones for two other IMNV strains (GenBank KF836757.1 and KJ556923.1) and thereby corroborate important amendments to the full-length IMNV genome sequence.

Mycoviruses are widespread in all major taxa of fungi. They are transmitted intracellularly during cell division, sporogenesis, and/or cell-to-cell fusion (hyphal anastomosis), and thus their life cycles generally lack an extracellular phase. Their natural host ranges are limited to individuals within the same or closely related vegetative compatibility groups, although recent advances have established expanded experimental host ranges for some mycoviruses. Most known mycoviruses have dsRNA genomes packaged in isometric particles, but an increasing number of positive- or negative-strand ssRNA and ssDNA viruses have been isolated and characterized. Although many mycoviruses do not have marked effects on their hosts, those that reduce the virulence of their phytopathogenic fungal hosts are of considerable interest for development of novel biocontrol strategies. Mycoviruses that infect endophytic fungi and those that encode killer toxins are also of special interest. Structural analyses of mycoviruses have promoted better understanding of virus assembly, function, and evolution., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Unlabelled: Giardia lamblia virus (GLV) is a small, nonenveloped, nonsegmented double-stranded RNA (dsRNA) virus infecting Giardia lamblia, the most common protozoan pathogen of the human intestine and a major agent of waterborne diarrheal disease worldwide. GLV (genus Giardiavirus) is a member of family Totiviridae, along with several other groups of protozoal or fungal viruses, including Leishmania RNA viruses and Trichomonas vaginalis viruses. Interestingly, GLV is more closely related than other Totiviridae members to a group of recently discovered metazoan viruses that includes penaeid shrimp infectious myonecrosis virus (IMNV). Moreover, GLV is the only known protozoal dsRNA virus that can transmit efficiently by extracellular means, also like IMNV. In this study, we used transmission electron cryomicroscopy and icosahedral image reconstruction to examine the GLV virion at an estimated resolution of 6.0 Å. Its outermost diameter is 485 Å, making it the largest totivirus capsid analyzed to date. Structural comparisons of GLV and other totiviruses highlighted a related "T=2" capsid organization and a conserved helix-rich fold in the capsid subunits. In agreement with its unique capacity as a protozoal dsRNA virus to survive and transmit through extracellular environments, GLV was found to be more thermoresistant than Trichomonas vaginalis virus 1, but no specific protein machinery to mediate cell entry, such as the fiber complexes in IMNV, could be localized. These and other structural and biochemical findings provide a basis for future work to dissect the cell entry mechanism of GLV into a "primitive" (early-branching) eukaryotic host and an important enteric pathogen of humans., Importance: Numerous pathogenic bacteria, including Corynebacterium diphtheriae, Salmonella enterica, and Vibrio cholerae, are infected with lysogenic bacteriophages that contribute significantly to bacterial virulence. In line with this phenomenon, several pathogenic protozoa, including Giardia lamblia, Leishmania species, and Trichomonas vaginalis are persistently infected with dsRNA viruses, and growing evidence indicates that at least some of these protozoal viruses can likewise enhance the pathogenicity of their hosts. Understanding of these protozoal viruses, however, lags far behind that of many bacteriophages. Here, we investigated the dsRNA virus that infects the widespread enteric parasite Giardia lamblia. Using electron cryomicroscopy and icosahedral image reconstruction, we determined the virion structure of Giardia lamblia virus, obtaining new information relating to its assembly, stability, functions in cell entry and transcription, and similarities and differences with other dsRNA viruses. The results of our study set the stage for further mechanistic work on the roles of these viruses in protozoal virulence., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Prototype victorivirus HvV190S employs stop/restart translation to express its RdRp from the downstream ORF in its bicistronic mRNA. The signals for this activity appear to include a predicted RNA pseudoknot directly upstream of the CP stop and RdRp start codons, which overlap in the motif AUGA. Here we used a dual-fluorescence system to further define which HvV190S sequences are important for stop/restart translation and found that the AUGA motif plus 38 nt directly upstream are both necessary and sufficient for this activity. This RNA cassette encompasses the predicted pseudoknot, and indeed substitutions that disrupted the pseudoknot disrupted the activity whereas complementary substitutions that restored the pseudoknot restored the activity. Replacement of this RNA cassette with those from other victoriviruses with a predicted pseudoknot in comparable position also supported stop/restart translation. To our knowledge, this is the first example of stop/restart translation regulated by an RNA pseudoknot., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Infectious myonecrosis virus (IMNV) is a pathogen of penaeid shrimp, most notably the whiteleg shrimp Litopenaeus vannamei. First discovered in L. vannamei from Brazilian aquaculture farms in 2003, IMNV was additionally confirmed in L. vannamei from Indonesian farms in 2006 and has since been found in numerous provinces there. Only two complete sequences of IMNV strains have been reported to date, one strain from the Brazilian state of Piauí collected in 2003 and another from the Indonesian province of East Java collected in 2006. In this study, we determined the complete sequences of two additional Indonesian strains, one from Lampung province collected in 2011 and another from East Java province collected in 2012. We also determined partial sequences for six other strains to enhance phylogenetic comparisons, which have heretofore been limited by the small number of reported sequences, including only one for an Indonesian strain. The new results demonstrate clear genetic diversification of IMNV between Indonesia and Brazil, as well as within Indonesia. Analyses of conserved sequence motifs suggest a revised RNA pseudoknot prediction for ribosomal frameshifting., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Phylogenetic analyses have prompted a taxonomic reorganization of family Partitiviridae (encapsidated, bisegmented dsRNA viruses that infect plants, fungi, or protozoa), the focus of this review. After a brief introduction to partitiviruses, the taxonomic changes are discussed, including replacement of former genera Partitivirus, Alphacryptovirus, and Betacryptovirus, with new genera Alphapartitivirus, Betapartitivirus, Gammapartitivirus, and Deltapartitivirus, as well as redistribution of species among these new genera. To round out the review, other recent progress of note in partitivirus research is summarized, including discoveries of novel partitivirus sequences by metagenomic approaches and mining of sequence databases, determinations of fungal partitivirus particle structures, demonstrations of fungal partitivirus transmission to new fungal host species, evidence for other aspects of partitivirus-host interactions and host effects, and identification of other fungal or plant viruses with some similarities to partitiviruses. Some outstanding questions are also discussed., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Objectives: Complex interactions of vaginal microorganisms with the genital tract epithelium shape mucosal innate immunity, which holds the key to sexual and reproductive health. Bacterial vaginosis (BV), a microbiome-disturbance syndrome prevalent in reproductive-age women, occurs commonly in concert with trichomoniasis, and both are associated with increased risk of adverse reproductive outcomes and viral infections, largely attributable to inflammation. To investigate the causative relationships among inflammation, BV and trichomoniasis, we established a model of human cervicovaginal epithelial cells colonised by vaginal Lactobacillus isolates, dominant in healthy women, and common BV species (Atopobium vaginae, Gardnerella vaginalis and Prevotella bivia)., Methods: Colonised epithelia were infected with Trichomonas vaginalis (TV) or exposed to purified TV virulence factors (membrane lipophosphoglycan (LPG), its ceramide-phosphoinositol-glycan core (CPI-GC) or the endosymbiont Trichomonas vaginalis virus (TVV)), followed by assessment of bacterial colony-forming units, the mucosal anti-inflammatory microbicide secretory leucocyte protease inhibitor (SLPI), and chemokines that drive pro-inflammatory, antigen-presenting and T cells., Results: TV reduced colonisation by Lactobacillus but not by BV species, which were found inside epithelial cells. TV increased interleukin (IL)-8 and suppressed SLPI, likely via LPG/CPI-GC, and upregulated IL-8 and RANTES, likely via TVV as suggested by use of purified pathogenic determinants. BV species A vaginae and G vaginalis induced IL-8 and RANTES, and also amplified the pro-inflammatory responses to both LPG/CPI-GC and TVV, whereas P bivia suppressed the TV/TVV-induced chemokines., Conclusions: These molecular host-parasite-endosymbiont-bacteria interactions explain epidemiological associations and suggest a revised paradigm for restoring vaginal immunity and preventing BV/TV-attributable inflammatory sequelae in women.

Family Reoviridae, subfamily Spinareovirinae, includes nine current genera. Two of these genera, Aquareovirus and Orthoreovirus, comprise members that are closely related and consistently share nine homologous proteins. Orthoreoviruses have 10 dsRNA genome segments and infect reptiles, birds, and mammals, whereas aquareoviruses have 11 dsRNA genome segments and infect fish. Recently, the first 10-segmented fish reovirus, piscine reovirus (PRV), has been identified and shown to be phylogenetically divergent from the 11-segmented viruses constituting genus Aquareovirus. We have recently extended results for PRV by showing that it does not encode a fusion-associated small transmembrane (FAST) protein, but does encode an outer-fiber protein containing a long N-terminal region of predicted α-helical coiled coil. Three recently characterized 11-segmented fish reoviruses, obtained from grass carp in China and sequenced in full, are also divergent from the viruses now constituting genus Aquareovirus, though not to the same extent as PRV. In the current study, we reexamined the sequences of these three recent isolates of grass carp reovirus (GCRV)-HZ08, GD108, and 104-for further clues to their evolution relative to other aqua- and orthoreoviruses. Structure-based fiber motifs in their encoded outer-fiber proteins were characterized, and other bioinformatics analyses provided evidence against the presence of a FAST protein among their encoded nonstructural proteins. Phylogenetic comparisons showed the combination of more distally branching, approved Aquareovirus and Orthoreovirus members, plus more basally branching isolates GCRV104, GCRV-HZ08/GD108, and PRV, constituting a larger, monophyletic taxon not suitably recognized by the current taxonomic hierarchy. Phylogenetics also suggested that the last common ancestor of all these viruses was a fiber-encoding, nonfusogenic virus and that the FAST protein family arose from at least two separate gain-of-function events. In addition, an apparent evolutionary correlation was found between the gain or loss of NS-FAST and outer-fiber proteins among more distally branching members of this taxon.

We tested a strategy for engineering recombinant mammalian reoviruses (rMRVs) to express exogenous polypeptides. One important feature is that these rMRVs are designed to propagate autonomously and can therefore be tested in animals as potential vaccine vectors. The strategy has been applied so far to three of the 10 MRV genome segments: S3, M1, and L1. To engineer the modified segments, a 5' or 3' region of the essential, long ORF in each was duplicated, and then exogenous sequences were inserted between the repeats. The inner repeat and exogenous insert were positioned in frame with the native protein-encoding sequences but were separated from them by an in-frame "2A-like" sequence element that specifies a cotranslational "stop/continue" event releasing the exogenous polypeptide from the essential MRV protein. This design preserves a terminal region of the MRV genome segment with essential activities in RNA packaging, assortment, replication, transcription, and/or translation and alters the encoded MRV protein to a limited degree. Recovery of rMRVs with longer inserts was made more efficient by wobble-mutagenizing both the inner repeat and the exogenous insert, which possibly helped via respective reductions in homologous recombination and RNA structure. Immunogenicity of a 300-aa portion of the simian immunodeficiency virus Gag protein expressed in mice by an L1-modified rMRV was confirmed by detection of Gag-specific T-cell responses. The engineering strategy was further used for mapping the minimal 5'-terminal region essential to MRV genome segment S3.

Piscine reovirus (PRV) is a tentative new member of the family Reoviridae and has been linked to heart and skeletal muscle inflammation in farmed Atlantic salmon (Salmo salar L.). Recent sequence-based evidence suggests that PRV is about equally related to members of the genera Orthoreovirus and Aquareovirus. Sequence similarities have also suggested that PRV might encode a fusion-associated small transmembrane (FAST) protein, which in turn suggests that PRV might be the prototype of a new genus with syncytium-inducing potential. In previous support of this designation has been the absence of identifiable PRV-encoded homologues of either the virion outer-clamp protein of ortho- and aquareoviruses or the virion outer-fibre protein of most orthoreoviruses. In the current report, we have provided experimental evidence that the putative p13 FAST protein of PRV lacks the defining feature of the FAST protein family - the ability to induce syncytium formation. Instead, p13 is the first example of a cytosolic, integral membrane protein encoded by ortho- or aquareoviruses, and induces cytotoxicity in the absence of cell-cell fusion. Sequence analysis also identified signature motifs of the outer-clamp and outer-fibre proteins of other reoviruses in two of the predicted PRV gene products. Based on these findings, we conclude that PRV does not encode a FAST protein and is therefore unlikely to be a new fusogenic reovirus. The presence of a novel integral membrane protein and two previously unrecognized, essential outer-capsid proteins has important implications for the biology, evolution and taxonomic classification of this virus.

Polarized epithelial cells that line the digestive, respiratory, and genitourinary tracts form a barrier that many viruses must breach to infect their hosts. Current understanding of cell entry by mammalian reovirus (MRV) virions and infectious subvirion particles (ISVPs), generated from MRV virions by extracellular proteolysis in the digestive tract, are mostly derived from in vitro studies with nonpolarized cells. Recent live-cell imaging advances allow us for the first time to visualize events at the apical surface of polarized cells. In this study, we used spinning-disk confocal fluorescence microscopy with high temporal and spatial resolution to follow the uptake and trafficking dynamics of single MRV virions and ISVPs at the apical surface of live polarized Madin-Darby canine kidney cells. Both types of particles were internalized by clathrin-mediated endocytosis, but virions and ISVPs exhibited strikingly different trafficking after uptake. While virions reached early and late endosomes, ISVPs did not and instead escaped the endocytic pathway from an earlier location. This study highlights the broad advantages of using live-cell imaging combined with single-particle tracking for identifying key steps in cell entry by viruses.

Double-stranded (ds)RNA fungal viruses are currently assigned to six different families. Those from the family Totiviridae are characterized by nonsegmented genomes and single-layer capsids, 300-450 Å in diameter. Helminthosporium victoriae virus 190S (HvV190S), prototype of recently recognized genus Victorivirus, infects the filamentous fungus Helminthosporium victoriae (telomorph: Cochliobolus victoriae), which is the causal agent of Victoria blight of oats. The HvV190S genome is 5179 bp long and encompasses two large, slightly overlapping open reading frames that encode the coat protein (CP, 772 aa) and the RNA-dependent RNA polymerase (RdRp, 835 aa). To our present knowledge, victoriviruses uniquely express their RdRps via a coupled termination-reinitiation mechanism that differs from the well-characterized Saccharomyces cerevisiae virus L-A (ScV-L-A, prototype of genus Totivirus), in which the RdRp is expressed as a CP/RdRp fusion protein due to ribosomal frameshifting. Here, we used transmission electron cryomicroscopy and three-dimensional image reconstruction to determine the structures of HvV190S virions and two types of virus-like particles (capsids lacking dsRNA and capsids lacking both dsRNA and RdRp) at estimated resolutions of 7.1, 7.5, and 7.6 Å, respectively. The HvV190S capsid is thin and smooth, and contains 120 copies of CP arranged in a "T = 2" icosahedral lattice characteristic of ScV-L-A and other dsRNA viruses. For aid in our interpretations, we developed and used an iterative segmentation procedure to define the boundaries of the two, chemically identical CP subunits in each asymmetric unit. Both subunits have a similar fold, but one that differs from ScV-L-A in many details except for a core α-helical region that is further predicted to be conserved among many other totiviruses. In particular, we predict the structures of other victoriviruses to be highly similar to HvV190S and the structures of most if not all totiviruses including, Leishmania RNA virus 1, to be similar as well.

Encapsidated dsRNA viruses, most of which are nonenveloped, infect a wide variety of hosts, from bacteria to vertebrates, and are currently grouped into 9 families comprising 33 genera. Given this range, it is not surprising that substantial diversity is seen in their transmission strategies and cell-entry machineries. One interesting set of recent findings is that several of these viruses, otherwise closely related, exhibit major differences in their entry machineries without comparably major differences in their capsid organizations. Examples are presence or absence of receptor-binding fibers among orthoreoviruses and aquareoviruses and presence or absence of both binding and membrane-penetration modules among totiviruses and between picobirnaviruses and partitiviruses. Evolved differences in cell-entry components among these viruses are therefore not only common but also seemingly straightforward from a structural standpoint., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Partitiviruses constitute one of the nine currently recognized families of viruses with encapsidated, double-stranded (ds)RNA genomes. The partitivirus genome is bisegmented, and each genome segment is packaged inside a separate viral capsid. Different partitiviruses infect plants, fungi, or protozoa. Recent studies have shed light on the three-dimensional structures of the virions of three representative fungal partitiviruses. These structures include a number of distinctive features, allowing informative comparisons with the structures of dsRNA viruses from other families. The results and comparisons suggest several new conclusions about the functions, assembly, and evolution of these viruses., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Wide-spread protozoan parasites carry endosymbiotic dsRNA viruses with uncharted implications to the human host. Among them, Trichomonas vaginalis, a parasite adapted to the human genitourinary tract, infects globally ∼250 million each year rendering them more susceptible to devastating pregnancy complications (especially preterm birth), HIV infection and HPV-related cancer. While first-line antibiotic treatment (metronidazole) commonly kills the protozoan pathogen, it fails to improve reproductive outcome. We show that endosymbiotic Trichomonasvirus, highly prevalent in T. vaginalis clinical isolates, is sensed by the human epithelial cells via Toll-like receptor 3, triggering Interferon Regulating Factor -3, interferon type I and proinflammatory cascades previously implicated in preterm birth and HIV-1 susceptibility. Metronidazole treatment amplified these proinflammatory responses. Thus, a new paradigm targeting the protozoan viruses along with the protozoan host may prevent trichomoniasis-attributable inflammatory sequelae.

The viral factories of mammalian reovirus (MRV) are cytoplasmic structures that serve as sites of viral genome replication and particle assembly. A 721-aa MRV non-structural protein, µNS, forms the factory matrix and recruits other viral proteins to these structures. In this report, we show that µNS contains a conserved C-proximal sequence (711-LIDFS-715) that is similar to known clathrin-box motifs and is required for recruitment of clathrin to viral factories. Clathrin recruitment by µNS occurs independently of infecting MRV particles or other MRV proteins. Ala substitution for a single Leu residue (mutation L711A) within the putative clathrin-binding motif of µNS inhibits clathrin recruitment, but does not prevent formation or expansion of viral factories. Notably, clathrin-dependent cellular functions, including both endocytosis and secretion, are disrupted in cells infected with MRV expressing wild-type, but not L711A, µNS. These results identify µNS as a novel adaptor-like protein that recruits cellular clathrin to viral factories, disrupting normal functions of clathrin in cellular membrane trafficking. To our knowledge, this is the only viral or bacterial protein yet shown to interfere with clathrin functions in this manner. The results additionally establish a new approach for studies of clathrin functions, based on µNS-mediated sequestration., (© 2011 John Wiley & Sons A/S.)

The dsRNA genome of mammalian reovirus (MRV), like the dsDNA genomes of herpesviruses and many bacteriophages, is packed inside its icosahedral capsid in liquid-crystalline form, with concentrations near or more than 400 mg/ml. Viscosity in such environments must be high, but the relevance of viscosity for the macromolecular processes occurring there remains poorly characterized. Here, we describe the use of simple viscogens, glycerol and sucrose, to examine their effects on RNA transcription inside MRV core particles. Transcription inside MRV cores was strongly inhibited by these agents and to a greater extent than either predicted by theory or exhibited by a nonencapsidated transcriptase, suggesting that RNA transcription inside MRV cores is unusually sensitive to viscogen effects. The elongation phase of transcription was found to be a primary target of this inhibition. Similar results were obtained with particles of a second dsRNA virus, rhesus rotavirus, from a divergent taxonomic subfamily. Polymeric viscogens such as polyethylene glycol also inhibited RNA transcription inside MRV cores, but in a size-limited manner, suggesting that diffusion through channels in the MRV core is required for their activity. Modeling of the data suggested that the inherent intracapsid viscosity of both reo- and rotavirus is indeed high, two to three times the viscosity of water. The capacity for quantitative comparisons of intracapsid viscosity and effects of viscogens on macromolecular processes in confined spaces should be similarly informative in other systems.

Baboon reovirus (BRV) is a member of the fusogenic subgroup of orthoreoviruses. Unlike most other members of its genus, BRV lacks S-segment coding sequences for the outer fiber protein that binds to cell surface receptors. It shares this lack with aquareoviruses, which constitute a related genus and are also fusogenic. We used electron cryomicroscopy and three-dimensional image reconstruction to determine the BRV virion structure at 9.0-Å resolution. The results show that BRV lacks a protruding fiber at its icosahedral 5-fold axes or elsewhere. The results also show that BRV is like nonfusogenic mammalian and fusogenic avian orthoreoviruses in having 150 copies of the core clamp protein, not 120 as in aquareoviruses. On the other hand, there are no hub-and-spoke complexes attributable to the outer shell protein in the P2 and P3 solvent channels of BRV, which makes BRV like fusogenic avian orthoreoviruses and aquareoviruses but unlike nonfusogenic mammalian orthoreoviruses. The outermost "flap" domains of the BRV core turret protein appear capable of conformational variability within the virion, a trait previously unseen among other ortho- and aquareoviruses. New cDNA sequence determinations for the BRV L1 and M2 genome segments, encoding the core turret and outer shell proteins, were helpful for interpreting the structural features of those proteins. Based on these findings, we conclude that the evolution of ortho- and aquareoviruses has included a series of discrete gains or losses of particular components, several of which cross taxonomic boundaries. Gain or loss of adhesion fibers is one of several common themes in double-stranded RNA virus evolution.

The genome-length, dicistronic mRNA of the double-stranded RNA fungal virus Helminthosporium victoriae virus 190S (genus Victorivirus, family Totiviridae) contains two long open reading frames (ORFs) that overlap in the tetranucleotide AUGA. Translation of the downstream ORF, which encodes the RNA-dependent RNA polymerase (RdRp), has been proposed to depend on ribosomal reinitiation following termination of the upstream ORF, which encodes the capsid protein. In the current study, we examined the RNA sequence determinants for RdRp translation in this virus and demonstrated that a coupled termination-reinitiation (stop-restart) strategy is indeed used. Signals for termination-reinitiation are found within a 32-nucleotide stretch of RNA immediately upstream of the AUGA motif, including a predicted pseudoknot structure. The close proximity in which this predicted structure is followed by the upstream ORF's stop codon appears to be especially important for promoting translation of the downstream ORF. The normal strong preferences for an AUG start codon and the canonical sequence context to favor translation initiation appear somewhat relaxed for the downstream ORF. Similar sequence motifs and predicted RNA structures in other victoriviruses suggest that they all share a related stop-restart strategy for RdRp translation. Members of the genus Victorivirus thus provide new and unique opportunities for exploring the molecular mechanisms of translational coupling, which remain only partly understood in this and other systems.

Trichomonas vaginalis, which causes the most common nonviral sexually transmitted disease worldwide, is itself commonly infected by nonsegmented double-stranded RNA (dsRNA) viruses from the genus Trichomonasvirus, family Totiviridae. To date, cDNA sequences of one or more strains of each of three trichomonasvirus species have been reported, and gel electrophoresis showing several different dsRNA molecules obtained from a few T. vaginalis isolates has suggested that more than one virus strain might concurrently infect the same parasite cell. Here, we report the complete cDNA sequences of 3 trichomonasvirus strains, one from each of the 3 known species, infecting a single, agar-cloned clinical isolate of T. vaginalis, confirming the natural capacity for concurrent (in this case, triple) infections in this system. We furthermore report the complete cDNA sequences of 11 additional trichomonasvirus strains, from 4 other clinical isolates of T. vaginalis. These additional strains represent the three known trichomonasvirus species, as well as a newly identified fourth species. Moreover, 2 of these other T. vaginalis isolates are concurrently infected by strains of all 4 trichomonasvirus species (i.e., quadruple infections). In sum, the full-length cDNA sequences of these 14 new trichomonasviruses greatly expand the existing data set for members of this genus and substantiate our understanding of their genome organizations, protein-coding and replication signals, diversity, and phylogenetics. The complexity of this virus-host system is greater than has been previously well recognized and suggests a number of important questions relating to the pathogenesis and disease outcomes of T. vaginalis infections of the human genital mucosa.

Filamentous fungus Fusarium poae is a worldwide cause of the economically important disease Fusarium head blight of cereal grains. The fungus is itself commonly infected with a bisegmented dsRNA virus from the family Partitiviridae. For this study, we determined the structure of partitivirus Fusarium poae virus 1 (FpV1) to a resolution of 5.6Å or better by electron cryomicroscopy and three-dimensional image reconstruction. The main structural features of FpV1 are consistent with those of two other fungal partitiviruses for which high-resolution structures have been recently reported. These shared features include a 120-subunit T=1 capsid comprising 60 quasisymmetrical capsid protein dimers with both shell and protruding domains. Distinguishing features are evident throughout the FpV1 capsid, however, consistent with its more massive subunits and its greater phylogenetic divergence relative to the other two structurally characterized partitiviruses. These results broaden our understanding of conserved and variable elements of fungal partitivirus structure, as well as that of vertebrate picobirnavirus, and support the suggestion that a phylogenetic subcluster of partitiviruses closely related to FpV1 should constitute a separate taxonomic genus., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Most dsRNA viruses have a genome-enclosing capsid that comprises 120 copies of a single coat protein (CP). These 120 CP subunits are arranged as asymmetrical dimers that surround the icosahedral fivefold axes, forming pentamers of dimers that are thought to be assembly intermediates. This scheme is violated, however, in recent structures of two dsRNA viruses, a fungal virus from family Partitiviridae and a rabbit virus from family Picobirnaviridae, both of which have 120 CP subunits organized as dimers of quasisymmetrical dimers. In this study, we report the CP backbone trace of a second fungal partitivirus, determined in this case by electron cryomicroscopy and homology modeling. This virus also exhibits quasisymmetrical CP dimers that are connected by prominent surface arches and stabilized by domain swapping between the two CP subunits. The CP fold is dominated by alpha-helices, although beta-strands mediate several important contacts. A dimer-of-dimers assembly intermediate is again implicated. The disordered N-terminal tail of each CP subunit protrudes into the particle interior and likely interacts with the genome during packaging and/or transcription. These results broaden our understanding of conserved and variable aspects of partitivirus structure and reflect the growing use of electron cryomicroscopy for atomic modeling of protein folds., (Copyright (c) 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Peroxisomes have long been established to play a central role in regulating various metabolic activities in mammalian cells. These organelles act in concert with mitochondria to control the metabolism of lipids and reactive oxygen species. However, while mitochondria have emerged as an important site of antiviral signal transduction, a role for peroxisomes in immune defense is unknown. Here, we report that the RIG-I-like receptor (RLR) adaptor protein MAVS is located on peroxisomes and mitochondria. We find that peroxisomal and mitochondrial MAVS act sequentially to create an antiviral cellular state. Upon viral infection, peroxisomal MAVS induces the rapid interferon-independent expression of defense factors that provide short-term protection, whereas mitochondrial MAVS activates an interferon-dependent signaling pathway with delayed kinetics, which amplifies and stabilizes the antiviral response. The interferon regulatory factor IRF1 plays a crucial role in regulating MAVS-dependent signaling from peroxisomes. These results establish that peroxisomes are an important site of antiviral signal transduction., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)