Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

The filamentous fungus Neurospora crassa is used as a model organism for genetics, developmental biology and molecular biology. Remarkably, it is not known to host or to be susceptible to infection with any viruses. Here, we identify diverse RNA viruses in N. crassa and other Neurospora species, and show that N. crassa supports the replication of these viruses as well as some viruses from other fungi. Several encapsidated double-stranded RNA viruses and capsid-less positive-sense single-stranded RNA viruses can be experimentally introduced into N. crassa protoplasts or spheroplasts. This allowed us to examine viral replication and RNAi-mediated antiviral responses in this organism. We show that viral infection upregulates the transcription of RNAi components, and that Dicer proteins (DCL-1, DCL-2) and an Argonaute (QDE-2) participate in suppression of viral replication. Our study thus establishes N. crassa as a model system for the study of host-virus interactions.

DOI： 10.1038/s41467-020-19355-y

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

The search for viruses infecting fungi, or mycoviruses, has extended our knowledge about the diversity of RNA viruses, as exemplified by the discovery of polymycoviruses, a phylogenetic group of multisegmented RNA viruses with unusual forms. The genomic RNAs of known polymycoviruses, which show a phylogenetic affinity for animal positive-sense single-stranded RNA [(+)RNA] viruses such as caliciviruses, are comprised of four conserved segments with an additional zero to four segments. The double-stranded form of polymycovirus genomic RNA is assumed to be associated with a virally encoded protein (proline-alanine-serine-rich protein [PASrp]) in either of two manners: a capsidless colloidal form or a filamentous encapsidated form. Detailed molecular characterizations of polymycoviruses, however, have been conducted for only a few strains. Here, a novel polymyco-related virus named Hadaka virus 1 (HadV1), from the phytopathogenic fungus Fusarium oxysporum, was characterized. The genomic RNA of HadV1 consisted of an 11-segmented positive-sense RNA with highly conserved terminal nucleotide sequences. HadV1 shared the three conserved segments with known polymycoviruses but lacked the PASrp-encoding segment. Unlike the known polymycoviruses and encapsidated viruses, HadV1 was not pelleted by conventional ultracentrifugation, possibly due to the lack of PASrp. This result implied that HadV1 exists only as a soluble form with naked RNA. Nevertheless, the 11 genomic segments of HadV1 have been stably maintained through host subculturing and conidiation. Taken together, the results of this study revealed a virus with a potential novel virus lifestyle, carrying many genomic segments without typical capsids or PASrp-associated forms. IMPORTANCE Fungi collectively host various RNA viruses. Examples include encapsidated double-stranded RNA (dsRNA) viruses with diverse numbers of genomic segments (from 1 to 12) and capsidless viruses with nonsegmented (+)RNA genomes. Recently, viruses with unusual intermediate features of an infectious entity between encapsidated dsRNA viruses and capsidless (+)RNA viruses were found. They are called polymycoviruses, which typically have four to eight dsRNA genomic segments associated with one of the virus-encoded proteins and are phylogenetically distantly related to animal (+)RNA caliciviruses. Here, we identified a novel virus phylogenetically related to polymycoviruses, from the phytopathogenic fungus Fusarium oxysporum. The virus, termed Hadaka virus 1 (HadV1), has 11 (+)RNA genomic segments, the largest number in known (+)RNA viruses. Nevertheless, HadV1 lacked a typical structural protein of polymycoviruses and was not pelleted by standard ultracentrifugation, implying an unusual capsidless nature of HadV1. This study reveals a potential novel lifestyle of multisegmented RNA viruses.

DOI： 10.1128/mBio.00450-20

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DOI： 10.1073/pnas.1812407116

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATL ACAD SCIENCES  

Pathogen recognition and transcriptional activation of defense-related genes are crucial steps in cellular defense responses. RNA silencing (RNAi) functions as an antiviral defense in eukaryotic organisms. Several RNAi-related genes are known to be transcriptionally up-regulated upon virus infection in some host organisms, but little is known about their induction mechanism. A phytopathogenic ascomycete, Cryphonectria parasitica (chestnut blight fungus), provides a particularly advantageous system to study RNAi activation, because its infection by certain RNA viruses induces the transcription of dicer-like 2 (dcl2) and argonaute-like 2 (agl2), two major RNAi players. To identify cellular factors governing activation of antiviral RNAi in C. parasitica, we developed a screening protocol entailing multiple transformations of the fungus with cDNA of a hypovirus mutant lacking the RNAi suppressor (CHV1-Delta p69), a reporter construct with a GFP gene driven by the dcl2 promoter, and a random mutagenic construct. Screening for GFP-negative colonies allowed the identification of sgf73, a component of the SAGA (Spt-Ada-Gcn5 acetyltransferase) complex, a well-known transcriptional coactivator. Knockout of other SAGA components showed that the histone acetyltransferase module regulates transcriptional induction of dcl2 and agl2, whereas histone deubiquitinase mediates regulation of agl2 but not dcl2. Interestingly, full-scale induction of agl2 and dcl2 by CHV1 Delta p69 required both DCL2 and AGL2, whereas that by another RNA virus, mycoreovirus 1, required only DCL2, uncovering additional roles for DCL2 and AGL2 in viral recognition and/or RNAi activation. Overall, these results provide insight into the mechanism of RNAi activation.

DOI： 10.1073/pnas.1701196114

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATL ACAD SCIENCES  

As sessile organisms, plants have to accommodate to rapid changes in their surrounding environment. Reactive oxygen species (ROS) act as signaling molecules to transduce biotic and abiotic stimuli into plant stress adaptations. It is established that a respiratory burst oxidase homolog B of Nicotiana benthamiana (NbRBOHB) produces ROS in response to microbe-associated molecular patterns to inhibit pathogen infection. Plant viruses are also known as causative agents of ROS induction in infected plants; however, the function of ROS in plant-virus interactions remains obscure. Here, we show that the replication of red clover necrotic mosaic virus (RCNMV), a plant positive-strand RNA [(+) RNA] virus, requires NbRBOHB-mediated ROS production. The RCNMV replication protein p27 plays a pivotal role in this process, redirecting the subcellular localization of NbRBOHB and a subgroup II calcium-dependent protein kinase of N. benthamiana (NbCDPKiso2) from the plasma membrane to the p27-containing intracellular aggregate structures. p27 also induces an intracellular ROS burst in an RBOH-dependent manner. NbCDPKiso2 was shown to be an activator of the p27-triggered ROS accumulations and to be required for RCNMV replication. Importantly, this RBOH-derived ROS is essential for robust viral RNA replication. The need for RBOHderived ROS was demonstrated for the replication of another (+) RNA virus, brome mosaic virus, suggesting that this characteristic is true for plant (+) RNA viruses. Collectively, our findings revealed a hitherto unknown viral strategy whereby the host ROS-generating machinery is diverted for robust viral RNA replication.

DOI： 10.1073/pnas.1610212114

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Nature Publishing Group  

Viruses typically encode the capsid that encases their genome, while satellite viruses do not encode a replicase and depend on a helper virus for their replication 1. Here, we report interplay between two RNA viruses, yado-nushi virus 1 (YnV1) and yado-kari virus 1 (YkV1), in a phytopathogenic fungus, Rosellinia necatrix2. YkV1 has a close phylogenetic affinity to positive-sense, single-stranded (+)ssRNA viruses such as animal caliciviruses3, while YnV1 has an undivided double-stranded (ds) RNA genome with a resemblance to fungal totiviruses4. Virion transfection and infectious full-length cDNA transformation has shown that YkV1 depends on YnV1 for viability, although it probably encodes functional RNA-dependent RNA polymerase (RdRp). Immunological and molecular analyses have revealed trans-encapsidation of not only YkV1 RNA but also RdRp by the capsid protein of the other virus (YnV1), and enhancement of YnV1 accumulation by YkV1. This study demonstrates interplay in which the capsidless (+)ssRNA virus (YkV1), hijacks the capsid protein of the dsRNA virus (YnV1), and replicates as if it were a dsRNA virus.

DOI： 10.1038/nmicrobiol.2015.1

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Viruses often coinfect single host organisms in nature. Depending on the combination of viruses in such coinfections, the interplay between them may be synergistic, apparently neutral with no effect on each other, or antagonistic. RNA silencing is responsible for many cases of interference or cross-protection between viruses, but such antagonistic interactions are usually restricted to closely related strains of the same viral species. In this study, we present an unprecedented example of RNA silencing-mediated one-way interference between unrelated viruses in a filamentous model fungus, Cryphonectria parasitica. The replication of Rosellinia necatrix victorivirus 1 (RnVV1; Totiviridae) was strongly impaired by coinfection with the prototypic member of the genus Mycoreovirus (MyRV1) or a mutant of the prototype hypovirus (Cryphonectria hypovirus 1, CHV1) lacking the RNA silencing suppressor (CHV1-Delta p69). This interference was associated with marked transcriptional induction of key genes in antiviral RNA silencing, dicer-like 2 (dcl2) and argonaute-like 2 (agl2), following MyRV1 or CHV1-Delta p69 infection. Interestingly, the inhibition of RnVV1 replication was reproduced when the levels of dcl2 and agl2 transcripts were elevated by transgenic expression of a hairpin construct of an endogenous C. parasitica gene. Disruption of dcl2 completely abolished the interference, whereas that of agl2 did not always lead to its abolishment, suggesting more crucial roles of dcl2 in antiviral defense. Taken altogether, these results demonstrated the susceptible nature of RnVV1 to the antiviral silencing in C. parasitica activated by distinct viruses or transgene-derived double-stranded RNAs and provide insight into the potential for broad-spectrum virus control mediated by RNA silencing.

DOI： 10.1073/pnas.1509151112

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

Mycoreovirus 1 (MyRV1) has 11 double-stranded RNA genome segments (S1 to S11) and confers hypovirulence to the chestnut blight fungus, Cryphonectria parasitica. MyRV1 genome rearrangements are frequently generated by a multifunctional protein, p29, encoded by a positive-strand RNA virus, Cryphonectria hypovirus 1. One of its functional roles is RNA silencing suppression. Here, we explored a possible link between MyRV1 genome rearrangements and the host RNA silencing pathway using wild-type (WT) and mutant strains of both MyRV1 and the host fungus. Host strains included deletion mutants of RNA silencing components such as dicer-like (dcl) and argonaute-like (agl) genes, while virus strains included an S4 internal deletion mutant MyRV1/S4ss. Consequently, intragenic rearrangements with nearly complete duplication of the three largest segments, i.e. S1, S2 and S3, were observed even more frequently in the RNA silencing-deficient strains Delta dcl2 and Delta agl2 infected with MyRV1/S4ss, but not with any other viral/host strain combinations. An interesting difference was noted between genome rearrangement events in the two host strains, i.e. generation of the rearrangement required prolonged culture for Delta agl2 in comparison with Delta dcl2. These results suggest a role for RNA silencing that suppresses genome rearrangements of a dsRNA virus.

DOI： 10.1093/nar/gkv239

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Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

Abstract

This article reports changes to virus taxonomy and taxon nomenclature that were approved and ratified by the International Committee on Taxonomy of Viruses (ICTV) in April 2024. The entire ICTV membership was invited to vote on 203 taxonomic proposals that had been approved by the ICTV Executive Committee (EC) in July 2023 at the 55th EC meeting in Jena, Germany, or in the second EC vote in November 2023. All proposals were ratified by online vote. Taxonomic additions include one new phylum (Ambiviricota), one new class, nine new orders, three new suborders, 51 new families, 18 new subfamilies, 820 new genera, and 3547 new species (excluding taxa that have been abolished). Proposals to complete the process of species name replacement to the binomial (genus + species epithet) format were ratified. Currently, a total of 14,690 virus species have been established.

DOI： 10.1007/s00705-024-06143-y

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Other Link： https://link.springer.com/article/10.1007/s00705-024-06143-y/fulltext.html

Authorship：Last author   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.virusres.2024.199493

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.tim.2024.09.010

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Language：English   Publishing type：Research paper (scientific journal)  

Rhizopus microsporus is a species in the order Mucorales that is known to cause mucormycosis, but it is poorly understood as a host of viruses. Here, we examined 25 clinical strains of R. microsporus for viral infection with a conventional double-stranded RNA (dsRNA) assay using agarose gel electrophoresis (AGE) and the recently established fragmented and primer-ligated dsRNA sequencing (FLDS) protocol. By AGE, five virus-infected strains were detected. Then, full-length genomic sequences of 12 novel RNA viruses were revealed by FLDS, which were related to the families Mitoviridae, Narnaviridae, and Endornaviridae, ill-defined groups of single-stranded RNA (ssRNA) viruses with similarity to the established families Virgaviridae and Phasmaviridae, and the proposed family "Ambiguiviridae." All the characterized viruses, except a potential phasmavirid with a negative-sense RNA genome, had positive-sense RNA genomes. One virus belonged to a previously established species within the family Mitoviridae, whereas the other 11 viruses represented new species or even new genera. These results show that the fungal pathogen R. microsporus harbors diverse RNA viruses and extend our understanding of the diversity of RNA viruses in the fungal order Mucorales, division Mucoromycota. Identifying RNA viruses from clinical isolates of R. microsporus may expand the repertoire of natural therapeutic agents for mucormycosis in the future.IMPORTANCEThe diversity of mycoviruses in fungal hosts in the division Mucoromycota has been underestimated, mainly within the species Rhizopus microsporus. Only five positive-sense RNA genomes had previously been discovered in this species. Because current sequencing methods poorly complete the termini of genomes, we used fragmented and primer-ligated double-stranded RNA sequencing to acquire the full-length genomes. Eleven novel mycoviruses were detected in this study, including the first negative-sense RNA genome reported in R. microsporus. Our findings extend the understanding of the viral diversity in clinical strains of Mucoromycota, may provide insights into the pathogenesis and ecology of this fungus, and may offer therapeutic options.

DOI： 10.1128/msphere.00345-24

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Rice sheath blight, caused by the soil-borne fungus Rhizoctonia solani (teleomorph: Thanatephorus cucumeris, Basidiomycota), is one of the most devastating phytopathogenic fungal diseases and causes yield loss. Here, we report on a very high prevalence (100%) of potential virus-associated double-stranded RNA (dsRNA) elements for a collection of 39 fungal strains of R. solani from the rice sheath blight samples from at least four major rice-growing areas in the Philippines and a reference isolate from the International Rice Research Institute, showing different colony phenotypes. Their dsRNA profiles suggested the presence of multiple viral infections among these Philippine R. solani populations. Using next-generation sequencing, the viral sequences of the three representative R. solani strains (Ilo-Rs-6, Tar-Rs-3, and Tar-Rs-5) from different rice-growing areas revealed the presence of at least 36 viruses or virus-like agents, with the Tar-Rs-3 strain harboring the largest number of viruses (at least 20 in total). These mycoviruses or their candidates are believed to have single-stranded RNA or dsRNA genomes and they belong to or are associated with the orders Martellivirales, Hepelivirales, Durnavirales, Cryppavirales, Ourlivirales, and Ghabrivirales based on their coding-complete RNA-dependent RNA polymerase sequences. The complete genome sequences of two novel RNA viruses belonging to the proposed family Phlegiviridae and family Mitoviridae were determined.

DOI： 10.3390/v16071152

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The yadokari/yadonushi nature is a recently discovered virus lifestyle; "yadokari" refers to the ability of capsidless positive-sense (+) RNA viruses (yadokariviruses) to utilize the capsids of phylogenetically distant double-stranded RNA (dsRNA) viruses possibly as the replication site, while "yadonushi" refers to the ability of dsRNA viruses to provide capsids to yadokariviruses. This virus-virus interaction, however, has been only studied with limited pathosystems. Here, we established a new study model with a capsidless (+)RNA yadokarivirus YkV3 (family Yadokariviridae) and its capsid donor RnMBV3 (family Megabirnaviridae) in the original host fungus Rosellinia necatrix and a model filamentous fungal host Cryphonectria parasitica. YkV3 has a simple genome structure with one open reading frame of 4305 nucleotides encoding a single polyprotein with an RNA-dependent RNA polymerase and a 2A-like self-cleavage peptide domain. Reverse genetics of YkV3 in R. necatrix showed that YkV3 tolerates a nucleotide substitution in the extreme 5'-terminus. The insertion of two termination codons immediately downstream of the 2A-like cleavage site abolished YkV3 viability, suggesting the importance of the C-terminal portion of the polyprotein of unknown function. Transfection of RnMBV3 and YkV3 into an RNA silencing-deficient mutant Δdcl2 of C. parasitica showed the replication competency of both viruses. Comparison between the wild-type and Δdcl2 strains of C. parasitica in virus accumulation suggested that RnMBV3 and YkV3 are susceptible to RNA silencing in C. parasitica. Taken together, we have established a platform to further explore the yadokari/yadonushi nature using genetically manipulable host fungal and virus strains.

DOI： 10.1016/j.virusres.2023.199155

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High-throughput sequencing technologies have greatly expanded the RNA virome in general and have led to an exponential increase in new fungal viruses, also known as mycoviruses. Mycoviruses are omnipresent in fungi and usually induce symptomless infections. Some mycoviruses infecting fungi pathogenic to plants, insects, and mammals are known to modify host virulence positively and negatively and attract particular interests. In addition, fungal viruses continue to provide intriguing research materials and themes that lead to discoveries of peculiar viruses as infectious entities and insights into virus evolution and diversity. In this review, we outline the diversity and neolifestyle of recently discovered fungal RNA viruses, and phenotypic alterations induced by them. Furthermore, we discuss recent advances in research regarding the fungal antiviral defense and viral counterdefense, which are closely associated with host phenotype alterations. We hope that this article will enhance understanding of the interesting and growing fungal virology field.

DOI： 10.1016/j.mib.2023.102337

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Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

DOI： 10.1007/s00705-023-05797-4

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Other Link： https://link.springer.com/article/10.1007/s00705-023-05797-4/fulltext.html

Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

Abstract

Here, we report the discovery and complete genome sequence of a novel virus, designated as “Phytophthora heveae alphaendornavirus 1” (PhAEV1), from a single isolate of the plant pathogenic oomycete Phytophthora heveae (kingdom Stramenipila) isolated from a tropical evergreen lowland rainforest in northern Vietnam. PhAEV1 was detected by both cellulose affinity chromatography of dsRNA and high-throughput sequencing of total RNA, and its presence and sequence were confirmed by RT-PCR and Sanger sequencing. The PhAEV1 genome, 12,820 nucleotides (nt) in length, was predicted to encode a single large polyprotein with the catalytic core domain of viral (superfamily 1) RNA helicase (HEL, amino acid [aa] positions 1,287-1,531), glycosyltransferase (GT, aa positions ca. 2,800-3,125), and RNA-directed RNA polymerase (RdRp, aa positions 3,875-4,112). PhAEV1 is the most similar to Phytophthora cactorum alphaendornavirus 3, sharing 39.4% and 39.1% nt and aa sequence identity, respectively. In addition to the first 5′-terminal AUG codon, three additional in-frame methionine codons were found in close proximity (nt 14-16, 96-98, and 176-178), suggesting potential additional translation initiation sites. Conserved RdRp motifs (A-E) similar to those detected in related endornaviruses were identified in PhAEV1, as well as in several previously described alphaendornaviruses from other Phytophthora species in which these motifs had not been identified previously. Phylogenetic analysis showed that PhAEV1 clusters with members of the genus Alphaendornavirus in the family Endornaviridae and is basal to two other alphaendornaviruses described from another oomycete, Phytophthora cactorum. PhAEV1 is the first virus reported in P. heveae.

DOI： 10.1007/s00705-023-05786-7

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Publishing type：Research paper (scientific journal)   Publisher：Microbiology Society  

The taxonomy of viruses is developed and overseen by the International Committee on Taxonomy of Viruses (ICTV), which scrutinizes, approves and ratifies taxonomic proposals, and maintains a list of virus taxa with approved names (https://ictv.global). The ICTV has approximately 180 members who vote by simple majority. Taxon-specific Study Groups established by the ICTV have a combined membership of over 600 scientists from the wider virology community; they provide comprehensive expertise across the range of known viruses and are major contributors to the creation and evaluation of taxonomic proposals. Proposals can be submitted by anyone and will be considered by the ICTV irrespective of Study Group support. Thus, virus taxonomy is developed from within the virology community and realized by a democratic decision-making process. The ICTV upholds the distinction between a virus or replicating genetic element as a physical entity and the taxon category to which it is assigned. This is reflected by the nomenclature of the virus species taxon, which is now mandated by the ICTV to be in a binomial format (genus + species epithet) and is typographically distinct from the names of viruses. Classification of viruses below the rank of species (such as, genotypes or strains) is not within the remit of the ICTV. This article, authored by the ICTV Executive Committee, explains the principles of virus taxonomy and the organization, function, processes and resources of the ICTV, with the aim of encouraging greater understanding and interaction among the wider virology community.

DOI： 10.1099/jgv.0.001840

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Publishing type：Research paper (scientific journal)   Publisher：Research Square Platform {LLC}  

<jats:title>Abstract</jats:title>
<jats:p><jats:italic>Agrobacterium tumefaciens</jats:italic> (<jats:italic>Rhizobium radiobacter</jats:italic>) is used for the transient expression of foreign genes by the agroinfiltration method, but the introduction of foreign genes often induces transcriptional and/or post-transcriptional gene silencing (TGS and/or PTGS). In this study, we characterized the structural features of T-DNA that induce TGS during agroinfiltration. When <jats:italic>A. tumefaciens</jats:italic>cells harboring an empty T-DNA plasmid containing the cauliflower mosaic virus (CaMV) 35S promoter were infiltrated into the leaves<jats:italic> </jats:italic>of <jats:italic>Nicotiana benthamiana</jats:italic> line 16c with a GFP gene over-expressed under the control of the same promoter, no small interfering RNAs (siRNAs) were derived from the GFP sequence. However, siRNAs derived from the CaMV 35S promoter were detected, indicating that TGS against the GFP gene was induced. When the GFP gene was inserted into the T-DNA plasmid, PTGS against the GFP gene was induced whereas TGS against the CaMV 35S promoter was suppressed. In other words, depending on the combination of promoter and coding sequences on T-DNA and the host nuclear genome, either or both TGS and/or PTGS could be induced by agroinfiltration. We also showed the importance of terminator sequences in T-DNA for gene silencing and the possible involvement of three siRNA-producing Dicers in the induction of TGS. These results are valuable for controlling gene expression by agroinfiltration.</jats:p>

DOI： 10.21203/rs.3.rs-2796710/v1

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Publishing type：Research paper (scientific journal)   Publisher：MDPI AG  

Many aphid-borne viruses are important pathogens that affect wheat crops worldwide. An aphid-transmitted closterovirus named wheat yellow leaf virus (WYLV) was found to have infected wheat plants in Japan in the 1970s; however, since then, its viral genome sequence and occurrence in the field have not been investigated. We observed yellowing leaves in the 2018/2019 winter wheat-growing season in an experimental field in Japan where WYLV was detected five decades ago. A virome analysis of those yellow leaf samples lead to the discovery of a closterovirus together with a luteovirus (barley yellow dwarf virus PAV variant IIIa). The complete genomic sequence of this closterovirus, named wheat closterovirus 1 isolate WL19a (WhCV1-WL19a), consisted of 15,452 nucleotides harboring nine open reading frames. Additionally, we identified another WhCV1 isolate, WL20, in a wheat sample from the winter wheat-growing season of 2019/2020. A transmission test indicated that WhCV1-WL20 was able to form typical filamentous particles and transmissible by oat bird-cherry aphid (Rhopalosiphum pad). Sequence and phylogenetic analyses showed that WhCV1 was distantly related to members of the genus Closterovirus (family Closteroviridae), suggesting that the virus represents a novel species in the genus. Furthermore, the characterization of WhCV1-WL19a-derived small RNAs using high-throughput sequencing revealed highly abundant 22-nt-class small RNAs potentially derived from the 3′-terminal end of the WhCV1 negative-strand genomic RNA, indicating that this terminal end of the WhCV1 genome is likely particularly targeted for the synthesis of viral small RNAs in wheat plants. Our results provide further knowledge on closterovirus diversity and pathogenicity and suggest that the impact of WhCV1 on wheat production warrants further investigations.

DOI： 10.3390/pathogens12030358

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Publishing type：Research paper (scientific journal)   Publisher：Public Library of Science (PLoS)  

A universal taxonomy of viruses is essential for a comprehensive view of the virus world and for communicating the complicated evolutionary relationships among viruses. However, there are major differences in the conceptualisation and approaches to virus classification and nomenclature among virologists, clinicians, agronomists, and other interested parties. Here, we provide recommendations to guide the construction of a coherent and comprehensive virus taxonomy, based on expert scientific consensus. Firstly, assignments of viruses should be congruent with the best attainable reconstruction of their evolutionary histories, i.e., taxa should be monophyletic. This fundamental principle for classification of viruses is currently included in the International Committee on Taxonomy of Viruses (ICTV) code only for the rank of species. Secondly, phenotypic and ecological properties of viruses may inform, but not override, evolutionary relatedness in the placement of ranks. Thirdly, alternative classifications that consider phenotypic attributes, such as being vector-borne (e.g., “arboviruses”), infecting a certain type of host (e.g., “mycoviruses,” “bacteriophages”) or displaying specific pathogenicity (e.g., “human immunodeficiency viruses”), may serve important clinical and regulatory purposes but often create polyphyletic categories that do not reflect evolutionary relationships. Nevertheless, such classifications ought to be maintained if they serve the needs of specific communities or play a practical clinical or regulatory role. However, they should not be considered or called taxonomies. Finally, while an evolution-based framework enables viruses discovered by metagenomics to be incorporated into the ICTV taxonomy, there are essential requirements for quality control of the sequence data used for these assignments. Combined, these four principles will enable future development and expansion of virus taxonomy as the true evolutionary diversity of viruses becomes apparent.

DOI： 10.1371/journal.pbio.3001922

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

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DOI： 10.1371/journal.ppat.1011162

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Authorship：Corresponding author   Publishing type：Research paper (scientific journal)   Publisher：Microbiology Society  

The family Yadokariviridae, with the genera Alphayadokarivirus and Betayadokarivirus, includes capsidless non-segmented positive-sense (+) RNA viruses that hijack capsids from phylogenetically distant double-stranded RNA viruses. Yadokarivirids likely replicate inside the hijacked heterocapsids using their own RNA-directed RNA polymerase, mimicking dsRNA viruses despite their phylogenetic placement in a (+) RNA virus lineage. Yadokarivirids can have negative or positive impacts on their host fungi, through interactions with the capsid donor dsRNA viruses. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) report on the family Yadokariviridae, which is available at ictv.global/report/yadokariviridae.

DOI： 10.1099/jgv.0.001826

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Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.virusres.2022.198996

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Publishing type：Research paper (scientific journal)   Publisher：Microbiology Society  

Spinareoviridae is a large family of icosahedral viruses that are usually regarded as non-enveloped with segmented (9–12 linear segments) dsRNA genomes of 23–29 kbp. Spinareovirids have a broad host range, infecting animals, fungi and plants. Some have important pathogenic potential for humans (e.g. Colorado tick fever virus), livestock (e.g. avian orthoreoviruses), fish (e.g. aquareoviruses) and plants (e.g. rice ragged stunt virus and rice black streaked dwarf virus). This is a summary of the ICTV Report on the family Spinareoviridae, which is available at ictv.global/report/spinareoviridae.

DOI： 10.1099/jgv.0.001781

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Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

DOI： 10.1007/s00705-022-05516-5

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Other Link： https://link.springer.com/article/10.1007/s00705-022-05516-5/fulltext.html

Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

Mycoviruses are widely distributed across the kingdom Fungi, including ascomycetous yeast strains of the class Saccharomycetes. Geotrichum candidum is an important fungal pathogen belonging to Saccharomycetes and has a diverse host range. Here, we report the characterization of four new classical totiviruses from two distinct Geotrichum candidum strains from Pakistan. The four identified viruses were tentatively named "Geotrichum candidum totivirus 1, 2, 3a, and 3b" (GcTV1-3b). The complete dsRNA genomes of the identified totiviruses are 4621, 4592, 4576, and 4576 bp in length, respectively. All totivirus genomes have two open reading frames, encoding a capsid protein (CP) and an RNA-dependent RNA polymerase (RdRP), respectively. The downstream RdRP domain is assumed to be expressed as a CP-RdRP fusion product via -1 frameshifting mediated by a heptameric slippery site. Sequence comparisons and phylogenetic analysis showed that each of the discovered viruses belongs to a new species of the genus Totivirus in the family Totiviridae, with GcTV1 and GcTV3 (a and b strains) clustering in one subgroup and GcTV2 in another subgroup.

DOI： 10.1007/s00705-022-05611-7

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Publishing type：Research paper (scientific journal)   Publisher：Microbiology Society  

Sedoreoviridae is a large family of icosahedral viruses that are usually regarded as non-enveloped with segmented (10–12 linear segments) dsRNA genomes of 18–26 kbp. Sedoreovirids have a broad host range, infecting mammals, birds, crustaceans, arthropods, algae and plants. Some of them have important pathogenic potential for humans (e.g. rotavirus A), livestock (e.g. bluetongue virus) and plants (e.g. rice dwarf virus). This is a summary of the ICTV Report on the family Sedoreoviridae, which is available at ictv.global/report/sedoreoviridae.

DOI： 10.1099/jgv.0.001782

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

A novel dsRNA virus (Cryphonectria carpinicola fusagravirus 1, CcFGV1), isolated from a Japanese strain (JS13) of Cryphonectria carpinicola, was thoroughly characterized. The biological comparison of a set of isogenic CcFGV1-infected and -free (JS13VF) strains indicated asymptomatic infection by CcFGV1. The sequence analysis showed that the virus has a two open reading frame (ORF) genome of 9.6 kbp with the RNA-directed RNA polymerase domain encoded by ORF2. The N-terminal sequencing and peptide mass fingerprinting showed an N-terminally processed or degraded product (150 kDa) of the 5'-proximal ORF1-encoded protein (1462 amino acids) to make up the CcFGV1 spherical particles of ~40 nm in diameter. Interestingly, a portion of CcFGV1 dsRNA co-fractionated with a host protein of 70 kDa. The purified CcFGV1 particles were used to transfect protoplasts of JS13VF as well as the standard strain of an experimental model filamentous fungal host Cryphonectria parasitica. CcFGV1 was confirmed to be associated with asymptomatic infection of both fungi. RNA silencing was shown to target the virus in C. parasitica, resulting in reduced CcFGV1 accumulation by comparing the CcFGV1 content between RNA silencing-competent and -deficient strains. These results indicate the transfectability of spherical particles of a fusagravirus associated with asymptomatic infection.

DOI： 10.3390/v14081722

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Authorship：Corresponding author   Publishing type：Research paper (scientific journal)   Publisher：Frontiers Media SA  

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

DOI： 10.3389/fcimb.2022.913619

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Language：English   Publishing type：Research paper (scientific journal)  

The family Partitiviridae has five genera, among which is the genus Deltapartitivirus. We report here the complete genome sequence of a deltapartitivirus from red clover, termed "red clover cryptic virus 3" (RCCV3). RCCV3 has a bisegmented double-stranded (ds) RNA genome. dsRNA1 and dsRNA2 are 1580 and 1589 nucleotides (nt) in length and are predicted to encode an RNA-directed RNA polymerase (RdRP) and a capsid protein (CP), respectively. The RCCV3 RdRP shares the highest sequence identity with the RdRP of a previously reported deltapartitivirus, Medicago sativa deltapartitivirus 1 (MsDPV1) (76.5%), while the RCCV3 CP shows 50% sequence identity to the CP of MsDPV1. RdRP- and CP-based phylogenetic trees place RCCV3 into a clade of deltapartitiviruses. The sequence and phylogenetic analyses clearly indicate that RCCV3 represents a new species in the genus Deltapartitivirus. RCCV3 was detectable in all three tested cultivars of red clover.

DOI： 10.1007/s00705-022-05372-3

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Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

DOI： 10.1007/s00705-021-05323-4

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Authorship：Corresponding author   Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

Abstract

An increasing number of plant viruses and viroids have been reported from all over the world due largely to metavirogenomics approaches with technological innovation. Herein, the official changes of virus taxonomy, including the establishment of megataxonomy and amendments of the codes of virus classification and nomenclature, recently made by the International Committee on Taxonomy of Viruses were summarized. The continued efforts of the plant virology community of Japan to index all plant viruses and viroids occurring in Japan, which represent 407 viruses, including 303 virus species and 104 unclassified viruses, and 25 viroids, including 20 species and 5 unclassified viroids, as of October 2021, were also introduced. These viruses and viroids are collectively classified into 81 genera within 26 families of 3 kingdoms (Shotokuvirae, Orthornavirae, Pararnavirae) across 2 realms (Monodnaviria and Riboviria). This review also overviewed how Japan’s plant virus/viroid studies have contributed to advance virus/viroid taxonomy.

DOI： 10.1007/s10327-022-01051-y

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Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

DOI： 10.1007/s00705-021-05304-7

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.virusres.2021.198606

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An extensive screening survey was conducted on Pakistani filamentous fungal isolates for the identification of viral infections. A total of 396 fungal samples were screened, of which 36 isolates were found double-stranded (ds) RNA positive with an overall frequency of 9% when analysed by a classical dsRNA isolation method. One of 36 dsRNA-positive strains, strain SP1 of a plant pathogenic fungus Fusarium mangiferae, was subjected to virome analysis. Next-generation sequencing and subsequent completion of the entire genome sequencing by a classical Sanger sequencing method showed the SP1 strain to be co-infected by 11 distinct viruses, at least seven of which should be described as new taxa at the species level according to the ICTV (International Committee on Taxonomy of Viruses) species demarcation criteria. The newly identified F. mangiferae viruses (FmVs) include two partitivirids, one betapartitivirus (FmPV1) and one gammapartitivirus (FmPV2); six mitovirids, three unuamitovirus (FmMV2, FmMV4, FmMV6), one duamitovirus (FmMV5), and two unclassified mitovirids (FmMV1, FmMV3); and three botourmiavirids, two magoulivirus (FmBOV1, FmBOV3) and one scleroulivirus (FmBOV2). The number of coinfecting viruses is among the largest ones of fungal coinfections. Their molecular features are thoroughly described here. This represents the first large virus survey in the Indian sub-continent.

DOI： 10.1099/jgv.0.001690

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：MDPI AG  

Partitiviruses are one of the most prevalent double-stranded RNA viruses that have been identified mostly in filamentous fungi and plants. Partitiviruses generally infect host fungi asymptomatically but infrequently exert significant effect(s) on morphology and virulence, thus being considered a potential source of biological control agents against pathogenic fungi. In this study, we performed a screening for mycoviruses of a collection of Thai isolates of rice fungal pathogen Rhizoctonia oryzae-sativae, a causal agent of rice aggregated sheath spot disease. As a result, 36% of tested isolates carried potentially viral double-stranded RNAs with sizes ranging from 2 to 3 kbp. By conventional cDNA library construction and RNA-seq, we determined six new alphapartitiviruses that infected three isolates: tentatively named Rhizoctonia oryzae-sativae partitivirus 1 to 6 (RosPV1-6). Furthermore, RT-PCR detection of each virus revealed their omnipresent nature in different R. oryzae-sativae isolates. Although virus-curing of basidiomycetous fungi is generally difficult, our repeated attempts successfully obtained virus-free (for RosPV1, RosPV2, and uncharacterized partitiviruses), isogenic strain of R. oryzae-sativae TSS190442. The virus-cured strain showed slightly faster colony growth on the synthetic media and severe symptom development on the rice sheath compared to its virus-infected counterpart. Overall, this study shed light on the distribution of partitiviruses in R. oryzae-sativae in a paddy environment and exemplified a virus-curing protocol that may be applicable for other basidiomycetous fungi.

DOI： 10.3390/v13112269

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Cryphonectria is a fungal genus associated with economically significant disease of trees. Herein we characterized a novel double-stranded RNA virus from the fungal species Cryphonectria naterciae, a species unexplored as a virus host. De novo assembly of RNA-seq data and Sanger sequencing of RACE (rapid amplification of cDNA ends) clones gave the complete, non-segmented genome (10,164 bp) of the virus termed Cryphonectria naterciae fusagravirus (CnFGV1) that was phylogenetically placed within the previously proposed viral family Fusagraviridae. Of 31 fieldcollected strains of C. naterciae, 40% tested CnFGV1-positive. Cocultivation resulted in within-species transmission of CnFGV1 to virus-free strains of C. naterciae. Comparison of the mycelium phenotype and the growth rate of CnFGV1-infected and virus-free isogenic strains revealed frequent sectoring and growth reduction in C. naterciae upon virus infection. Co-culturing also led to cross-species transmission of CnFGV1 to Cryphonectria carpinicola and Cryphonectria radicalis, but not to Cryphonectria parasitica. The virus-infected C. naterciae and the experimentally infected Cryphonectria spp. readily transmitted CnFGV1 through asexual spores to the next generation. CnFGV1 strongly reduced conidiation and in some cases vegetative growth of C. carpinicola, which is involved in the European hornbeam disease. This study is the first report of a fusagravirus in the family Cryphonectriaceae and lays the groundwork for assessing a hypovirulence effect of CnFGV1 against the hornbeam decline in Europe.

DOI： 10.3390/jof7100861

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

DOI： 10.1007/s00705-021-05156-1

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

DOI： 10.1007/s00705-021-05176-x

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

The ascomycete Cryphonectria parasitica causes destructive chestnut blight. Biological control of the fungus by virus infection (hypovirulence) has been shown to be an effective control strategy against chestnut blight in Europe. To provide biocontrol effects, viruses must be able to induce hypovirulence and spread efficiently in chestnut trees. Field studies using living trees to date have focused on a selected family of viruses called hypoviruses, especially prototypic hypovirus CHV1, but there are now known to be many other viruses that infect C. parasitica. Here, we tested seven different viruses for their hypovirulence induction, biocontrol potential, and transmission properties between two vegetatively compatible but molecularly distinguishable fungal strains in trees. The test included cytosolically and mitochondrially replicating viruses with positive-sense single-stranded RNA or double-stranded RNA genomes. The seven viruses showed different in planta behaviors and were classified into four groups. Group I, including CHV1, had great biocontrol potential and could protect trees by efficiently spreading and converting virulent to hypovirulent cankers in the trees. Group II could induce high levels of hypovirulence but showed much smaller biocontrol potential, likely because of inefficient virus transmission. Group III showed poor performance in hypovirulence induction and biocontrol, while efficiently being transmitted in the infected trees. Group IV could induce hypovirulence and spread efficiently but showed poor biocontrol potential. Nuclear and mitochondrial genotyping of fungal isolates obtained from the treated cankers confirmed virus transmission between the two fungal strains in most isolates. These results are discussed in view of dynamic interactions in the tripartite pathosystem. IMPORTANCE The ascomycete Cryphonectria parasitica causes destructive chestnut blight, which is controllable by hypovirulence-conferring viruses infecting the fungus. The tripartite chestnut/C. parasitica/virus pathosystem involves the dynamic interactions of their genetic elements, i.e., virus transmission and lateral transfer of nuclear and mitochondrial genomes between fungal strains via anastomosis occurring in trees. Here, we tested diverse RNA viruses for their hypovirulence induction, biocontrol potential, and transmission properties between two vegetatively compatible but molecularly distinguishable fungal strains in live chestnut trees. The tested viruses, which are different in genome type (single-stranded or double-stranded RNA) and organization, replication site (cytosol or mitochondria), virus form (encapsidated or capsidless) and/or symptomatology, have been unexplored in the aforementioned aspects under controlled conditions. This study showed intriguing different in-tree behaviors of the seven viruses and suggested that to exert significant biocontrol effects, viruses must be able to induce hypovirulence and spread efficiently in the fungus infecting the chestnut trees.

DOI： 10.1128/JVI.01962-20

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

Cryphonectria nitschkei chrysovirus 1 (CnCV1), was described earlier from an ascomycetous fungus, Cryphonectria nitschkei strain OB5/11, collected in Japan; its partial sequence was reported a decade ago. Complete sequencing of the four genomic dsRNA segments revealed molecular features similar to but distinct from previously reported members of the family Chrysoviridae. Unique features include the presence of a mini-cistron preceding the major large open reading frame in each genomic segment. Common features include the presence of CAA repeats in the 5′-untranslated regions and conserved terminal sequences. CnCV1-OB5/11 could be laterally transferred to C. nitschkei and its relatives C. radicalis and C. naterciae via coculturing, virion transfection and protoplast fusion, but not to fungal species other than the three species mentioned above, even within the genus Cryphonectria, suggesting a very narrow host range. Phenotypic comparison of a few sets of CnCV1-infected and -free isogenic strains showed symptomless infection in new hosts.

DOI： 10.1016/j.virol.2020.11.011

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Previously, we have reported the ability of a symptomless hypovirus Cryphonectria hypovirus 4 (CHV4) of the chestnut blight fungus to facilitate stable infection by a co-infecting mycoreovirus 2 (MyRV2)—likely through the inhibitory effect of CHV4 on RNA silencing (Aulia et al., Virology, 2019). In this study, the N-terminal portion of the CHV4 polyprotein, termed p24, is identified as an autocatalytic protease capable of suppressing host antiviral RNA silencing. Using a bacterial expression system, CHV4 p24 is shown to cleave autocatalytically at the di-glycine peptide (Gly214-Gly215) of the polyprotein through its protease activity. Transgenic expression of CHV4 p24 in Cryphonectria parasitica suppresses the induction of one of the key genes of the antiviral RNA silencing, dicer-like 2, and stabilizes the infection of RNA silencing-susceptible virus MyRV2. This study shows functional similarity between CHV4 p24 and its homolog p29, encoded by the symptomatic prototype hypovirus CHV1.

DOI： 10.3390/biology10020100

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Dicing of double-stranded RNA (dsRNA) into small RNA is an essential process to trigger transcriptional and post-transcriptional gene silencing. Using cell-free extracts of the model filamentous fungus Neurospora crassa, we successfully detected the dicing activity of one of two N. crassa Dicers NcDCL2. The predominant 23-nucleotide (nt) cleavage product was always detected from 30-nt to 130-nt dsRNA substrates, and additional products of approximately 18 to 28 nt were occasionally produced. The enzymatic properties of NcDCL2 are different from those of insect and plant small interfering RNA (siRNA)-producing Dicers, Drosophila melanogaster Dicer-2 and Arabidopsis thaliana DCL3 and DCL4 (AtDCL3 and AtDCL4). Whereas AtDCL3 and AtDCL4 preferentially cleave short and long dsRNAs, respectively, NcDCL2 cleaved both short and long dsRNAs. These results suggest that N. crassa has a single siRNA-producing Dicer NcDCL2, which is a prototype of plant siRNA-producing Dicers with distinct functions in diverse RNA silencing pathways. The dicing assay reported here is convenient to detect and biochemically characterize the dicing activities of both plant and fungal Dicers, and is likely applicable to other organisms.

DOI： 10.1016/j.fgb.2020.103488

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Two isolates of Rosellinia necatrix (Rn118-8 and Rn480) have previously obtained from diseased avocado trees in commercial orchards of the coastal area in southern Spain. Rn118-8 and Rn480 have weak virulence on avocado plants, and are infected by R. necatrix hypovirus 2 (RnHV2). In this work, the possible biological effects of the hypovirus on R. necatrix were tested. First, RnHV2 was transmitted from each of Rn118-8 and Rn480 to a highly virulent, RnHV2-free isolate of R. necatrix (Rn400) through hyphal anastomosis, using zinc compounds which attenuate the mycelial incompatibility reactions and allow for horizontal virus transfer between vegetatively incompatible fungal strains. Next, we carried out an analysis of growth rate in vitro and a virulence test of these newly infected strains in avocado plants. We obtained five strains of Rn400 infected by RnHV2 after horizontal transmission, and showed some of them to have lower colony growth in vitro and lower virulence on avocado plants compared with virus-free Rn400. These results suggest that R. necatrix isolates infected by RnHV2 could be used as novel virocontrol agents to combat avocado white root rot.

DOI： 10.1016/j.funbio.2020.10.007

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Yellow mosaic disease in winter wheat is usually attributed to the infection by bymoviruses or furoviruses; however, there is still limited information on whether other viral agents are also associated with this disease. To investigate the wheat viromes associated with yellow mosaic disease, we carried out de novo RNA sequencing (RNA-seq) analyses of symptomatic and asymptomatic wheat-leaf samples obtained from a field in Hokkaido, Japan, in 2018 and 2019. The analyses revealed the infection by a novel betaflexivirus, which tentatively named wheat virus Q (WVQ), together with wheat yellow mosaic virus (WYMV, a bymovirus) and northern cereal mosaic virus (a cytorhabdovirus). Basic local alignment search tool (BLAST) analyses showed that the WVQ strains (of which there are at least three) were related to the members of the genus Foveavirus in the subfamily Quinvirinae (family Betaflexiviridae). In the phylogenetic tree, they form a clade distant from that of the foveaviruses, suggesting that WVQ is a member of a novel genus in the Quinvirinae. Laboratory tests confirmed that WVQ, like WYMV, is potentially transmitted through the soil to wheat plants. WVQ was also found to infect rye plants grown in the same field. Moreover, WVQ-derived small interfering RNAs accumulated in the infected wheat plants, indicating that WVQ infection induces antiviral RNA silencing responses. Given its common coexistence with WYMV, the impact of WVQ infection on yellow mosaic disease in the field warrants detailed investigation.

DOI： 10.3389/fmicb.2021.715545

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

The genus Polymycovirus of the family Polymycoviridae accommodates fungal RNA viruses with different genomic segment numbers (four, five, or eight). It is suggested that four members form no true capsids and one forms filamentous virus particles enclosing double-stranded RNA (dsRNA). In both cases, viral dsRNA is associated with a viral protein termed “proline-alanine-serine-rich protein” (PASrp). These forms are assumed to be the infectious entity. However, the detailed molecular characteristics of PASrps remain unclear. Here, we identified a novel five-segmented polymycovirus, Penicillium janthinellum polymycovirus 1 (PjPmV1), and characterized its purified fraction form in detail. The PjPmV1 had five dsRNA segments associated with PASrp. Density gradient ultracentrifugation of the PASrp-associated PjPmV1 dsRNA revealed its uneven structure and a broad fractionation profile distinct from that of typical encapsidated viruses. Moreover, PjPmV1-PASrp interacted in vitro with various nucleic acids in a sequence-non-specific manner. These PjPmV1 features are discussed in view of the diversification of genomic segment numbers of the genus Polymycovirus.

DOI： 10.3389/fmicb.2020.592789

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Many Mediteranean isolates of Rosellinia necatrix, causing root rot in avocado, have previously been screened for viruses. Among them is isolate Rn459 that has been shown to be infected by at least three viruses such as Rosellinia necatrix partitivirus 10 (RnPV10), Rosellinia necatrix hypovirus 2 (RnHV2), Rosellinia necatrix fusagravirus 1 (RnFGV1), and Rosellinia necatrix virga-like virus (RnVLV). Here, we attempted to eliminate the viruses by hyphal tip cultures to examine their effect on colony growth in vitro and virulence on avocado plants. The obtained fungal strain termed, Rn459_PV10F/VLVF, which was confirmed to be cured of RnPV10 and RnVLV, but still retaining RnHV2, manifested a phenotype different from the original Rn459. Colony growth comparison showed that Rn459_PV10F/VLVF grew faster than the original Rn459 isolate and the virulence on avocado plants of this Rn459_PV10F/VLVF strain was higher than the original Rn459 strain. These combined results suggest that RnPV10 and RnVLV, alone or together, contribute to confer hypovirulence on the R. necatrix isolates.

DOI： 10.1007/s10658-020-02058-x

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Publishing type：Research paper (scientific journal)   Publisher：Elsevier {BV}  

DOI： 10.1016/j.crviro.2020.100001

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

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

DOI： 10.3389/fmicb.2020.01064

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Aphids (order Hemiptera) are important insect pests of crops and are also vectors of many plant viruses. However, little is known about aphid-infecting viruses, particularly their diversity and relationship to plant viruses. To investigate the aphid viromes, we performed deep sequencing analyses of the aphid transcriptomes from infested barley plants in a field in Japan. We discovered virus-like sequences related to nege/kita-, flavi-, tombus-, phenui-, mononega-, narna-, chryso-, partiti-, and luteoviruses. Using RT-PCR and sequence analyses, we determined almost complete sequences of seven nege/kitavirus-like virus genomes; one of which was a variant of the Wuhan house centipede virus (WHCV-1). The other six seem to belong to four novel viruses distantly related to Wuhan insect virus 9 (WhIV-9) or Hubei nege-like virus 4 (HVLV-4). We designated the four viruses as barley aphid RNA virus 1 to 4 (BARV-1 to -4). Moreover, some nege/kitavirus-like sequences were found by searches on the transcriptome shotgun assembly (TSA) libraries of arthropods and plants. Phylogenetic analyses showed that BARV-1 forms a clade with WHCV-1 and HVLV-4, whereas BARV-2 to -4 clustered with WhIV-9 and an aphid virus, Aphis glycines virus 3. Both virus groups (tentatively designated as Centivirus and Aphiglyvirus, respectively), together with arthropod virus-like TSAs, fill the phylogenetic gaps between the negeviruses and kitaviruses lineages. We also characterized the flavi/jingmen-like and tombus-like virus sequences as well as other RNA viruses, including six putative novel viruses, designated as barley aphid RNA viruses 5 to 10. Interestingly, we also discovered that some aphid-associated viruses, including nege/kita-like viruses, were present in different aphid species, raising a speculation that these viruses might be distributed across different aphid species with plants being the reservoirs. This study provides novel information on the diversity and spread of nege/kitavirus-related viruses and other RNA viruses that are associated with aphids.

DOI： 10.3389/fmicb.2020.00509

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DOI： 10.3389/fcimb.2019.00456

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Mycoviruses are a diverse group that includes ssRNA, dsRNA, and ssDNA viruses, with or without a protein capsid, as well as with a complex envelope. Most mycoviruses are transmitted by cytoplasmic interchange and are thought to lack an extracellular phase in their infection cycle. Structural analysis has focused on dsRNA mycoviruses, which usually package their genome in a 120-subunit T = 1 icosahedral capsid, with a capsid protein (CP) dimer as the asymmetric unit. The atomic structure is available for four dsRNA mycovirus from different families: Saccharomyces cerevisiae virus L-A (ScV-L-A), Penicillium chrysogenum virus (PcV), Penicillium stoloniferum virus F (PsV-F), and Rosellinia necatrix quadrivirus 1 (RnQV1). Their capsids show structural variations of the same framework, with asymmetric or symmetric CP dimers respectively for ScV-L-A and PsV-F, dimers of similar domains of a single CP for PcV, or of two different proteins for RnQV1. The CP dimer is the building block, and assembly proceeds through dimers of dimers or pentamers of dimers, in which the genome is packed as ssRNA by interaction with CP and/or viral polymerase. These capsids remain structurally undisturbed throughout the viral cycle. The T = 1 capsid participates in RNA synthesis, organizing the viral polymerase (1–2 copies) and a single loosely packaged genome segment. It also acts as a molecular sieve, to allow the passage of viral transcripts and nucleotides, but to prevent triggering of host defense mechanisms. Due to the close mycovirus-host relationship, CP evolved to allocate peptide insertions with enzyme activity, as reflected in a rough outer capsid surface.

DOI： 10.1016/bs.aivir.2020.08.001

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Given that approximately 10 virus particles exist on Earth and all of them are parasitic in living organisms, it is not hard to imagine how virus infection might affect the physiology of hosts and their ecosystems. However, traditional virology research tends to focus on viral pathogenicity or the individual pathogenic viruses; hence, the significance of viruses and viral-mediated processes in the global ecosystem has been poorly understood. To identify the previously unrecognized “raison d'etre of viruses” in nature, we established a research community, designated as the ‘Neo-virology’ consortium. In this consortium, we define a virus as a component of the global ecosystem and our aim is to elucidate its key roles in host organisms, that is, the intra-host ecosystem. 31

DOI： 10.1016/j.virusres.2019.197751

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Megabirnaviridae is a family of non-enveloped spherical viruses with dsRNA genomes of two linear segments, each of 7.2-8.9 kbp, comprising 16.1 kbp in total. The genus Megabirnavirus includes the species Rosellinia necatrix megabirnavirus 1, the exemplar isolate of which infects the white root rot fungus (Rosellinia necatrix) to which it confers hypovirulence. Megabirnaviruses are characterized by their bisegmented genome with large 5'-untranslated regions (1.6 kb) upstream of both 5'-proximal coding strand ORFs, and large protrusions on the particle surface. This is a summary of the ICTV Report on the family Megabirnaviridae, which is available at ictv.global/report/megabirnaviridae.This Profile is dedicated to the memory of our valued colleague Professor Said A. Ghabrial.

DOI： 10.1099/jgv.0.001297

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Three ourmia-like viruses, designated Pyricularia oryzae ourmia-like virus (PoOLV) 1 to 3, were identified in a wheat-infecting isolate of P. oryzae. The sizes of the full-length PoOLV1-3 genomes were determined to be 2,528, 1,671, and 2,557 nt. Interestingly, we also found two abundant single-stranded RNAs sharing their 5’ terminal 25 and 255 nt with PoOLV1 RNA and PoOLV3 RNA, respectively. The PoOLV1- and PoOLV3-associated RNAs (ARNA1 and ARNA3) were 639 and 514 nt in length, and possessed one and two small ORFs, respectively. In the field isolates of P. oryzae, PoOLVs and ARNAs were detectable at varying levels, and the levels of PoOLV1 and ARNA1 as well as those of PoOLV3 and ARNA3, were tightly correlated. In addition, gene silencing of PoOLV1 and PoOLV3 resulted in a reduction of ARNA1 and ARNA3, respectively. There results indicated that replication of ARNA1 and ARNA3 was associated with PoOLV1 and PoOLV3, respectively.

DOI： 10.1016/j.virol.2019.05.015

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Grifola frondosa (Maitake mushroom) is an important cultivated mushroom due to its medicinal and nutrient values. In this study, we isolated and characterized a novel partitivirus (named Grifola frondosa partitivirus 1, GfPV1) infecting a standard G. frondosa strain Gf-N2. This virus has a two-segmented dsRNA genome (dsRNA1 and dsRNA2) with nucleotide lengths of 2.3 and 2.2 kbp, respectively. The coding strand of dsRNA1 and dsRNA2 segments carries single open reading frame encoding RNA-dependent RNA polymerase (RdRp) and a coat protein (CP), respectively. BLAST searches and phylogenetic analyses showed that GfPV1 is most closely related to a betapartitivirus, Lentinula edodes partitivirus 1 (RdRp <70% and CP <60% amino acid sequence identities), but the sequence divergence suggests that GfPV1 is classifiable as a new member of the genus Betapartitivirus, family Partitiviridae. The presence of GfPV1 does not affect colony morphology and fruiting body development of G. frondosa. This is the first report investigating the effects of a mycovirus infection on the colony morphology and fruiting body development of G. frondosa. Interestingly, GfPV1 accumulations markedly decreased along with the fruiting body maturation stages, suggesting the inhibition of virus multiplication during sexual phase of the G. frondosa life cycle.

DOI： 10.1016/j.myc.2019.01.005

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Publishing type：Research paper (scientific journal)   Publisher：Elsevier {BV}  

Field-collected US strain C18 of Cryphonectria parasitica, the chestnut blight fungus, was earlier reported to be infected by a double-stranded RNA virus, mycoreovirus 2 (MyRV2). Next-generation sequencing has revealed co-infection of C18 by a positive-strand RNA virus, hypovirus 4 (CHV4). The current molecular and genetic analyses showed interesting commensal interactions between the two viruses. CHV4 facilitated the stable infection and enhanced vertical transmission of MyRV2, which was readily lost during subculturing and showed reduced vertical transmission in single infections. Deletion of a key antiviral RNA silencing gene, dcl2, in isolate C18 increased stability of MyRV2 in single infections. The ability of CHV4 to facilitate stable infection with MyRV2 appears to be associated with the inhibitory effect of CHV4 on RNA silencing via compromising the induction of transcriptional up-regulation of dcl2. These results suggest that natural infection of isolate C18 by MyRV2 in the field was facilitated by CHV4 co-infection.

DOI： 10.1016/j.virol.2019.05.004

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There is still limited information on the diversity of (−)ssRNA viruses that infect fungi. Here, we have discovered two novel (−)ssRNA mycoviruses in the shiitake mushroom (Lentinula edodes). The first virus has a monopartite RNA genome and relates to that of mymonaviruses (Mononegavirales), especially to Hubei rhabdo-like virus 4 from arthropods and thus designated as Lentinula edodes negative-strand RNA virus 1. The second virus has a putative bipartite RNA genome and is related to the recently discovered bipartite or tripartite phenui-like viruses (Bunyavirales) associated with plants and ticks, and designated as Lentinula edodes negative-strand RNA virus 2 (LeNSRV2). LeNSRV2 is likely the first segmented (−)ssRNA virus known to infect fungi. Its smaller RNA segment encodes a putative nucleocapsid and a plant MP-like protein using a potential ambisense coding strategy. These findings enhance our understanding of the diversity, evolution and spread of (−)ssRNA viruses in fungi.

DOI： 10.1016/j.virol.2019.05.008

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The family Totiviridae currently contains five genera Totivirus, Victorivirus, Leishmavirus, Trichomonasvirus, and Giardiavirus. Members in this family generally have a set of two-open reading frame (ORF) elements in their genome with the 5’-proximalORF (ORF1) encoding a capsid protein (CP) and the 3’-proximal one (ORF2) for RNA-dependent RNA polymerase (RdRp). How the downstream open reading frames (ORFs) are expressed is genus-specific. All victoriviruses characterized thus far appear to use the stop/restart translation mechanism, allowing for the expression of two separate protein products from bicitronic genome-sized viral mRNA, while the totiviruses use a -1 ribosomal frame-shifting that leads to a fusion product of CP and RdRp. We report the biological and molecular characterization of a novel victorivirus termed Alternaria alternata victorivirus 1 (AalVV1) isolated from Alternaria alternata in Pakistan. The phylogenetic and molecular analyses showed AalVV1 to be distinct from previously reported victoriviruses. AalVV1 appears to have a sequence signature required for the -1 frame-shifting at the ORF1/2 junction region, rather than a stop/restart key mediator. By contrast, SDS–polyacrylamide gel electrophoresis and peptide mass fingerprinting analyses of purified virion preparations suggested the expression of two protein products, not a CP-RdRp fusion product. How these proteins are expressed is discussed in this study. Possible effects of infection by this virus were tested in two fungal species: A. alternata and RNA silencing proficient and deficient strains of Cryphonectria parasitica, a model filamentous fungus. AalVV1 showed symptomless infection in all of these fungal strains, even in the RNA silencing deficient C. parasitica strain.

DOI： 10.3390/v11060577

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier {BV}  

Mycoviruses ubiquitously infect a wide range of fungal hosts in the world. The current study reports a novel double stranded RNA (dsRNA) virus, termed Alternaria alternata botybirnavirus 1 (AaBbV1), infecting a Pakistani strain, 4a, of a phytopathogenic ascomycetous fungus Alternaria alternata. A combined approach of next generation and conventional terminal end sequencing of the viral genome revealed that the virus is a distinct member of the genus Botybirnavirus. This virus comprised of two segments (dsRNA1 and dsRNA2) of sizes 6127 bp and 5860 bp respectively. The dsRNA1-encoded protein carrying the RNA-dependent RNA polymerase domain showed 61% identity to the counterpart of Botrytis porri botybirnavirus 1 and lower levels of amino acid similarity with those of other putative botybirnaviruses and the fungal dsRNA viruses such as members of the families Totiviridae, Chrysoviridae and Megabirnaviridae. The dsRNA2-encoded protein showed resemblance with corresponding proteins of botybirnaviruses. Electron microscopy showed AaBbV1 to form spherical particles of 40 nm in diameter. Biochemical analyses showed that two structural proteins encoded by dsRNA1 and dsRNA2 underwent processing to some extent during particle purification, resulting in the appearance of multiple smaller products. Phylogenetic analyses of structural proteins suggested that their coding region might have been duplicated once and maintained without recombination. Protoplast fusion technique allowed for the introduction of AaBbV1 into a virus free Japanese strain of A. alternata and demonstrated its symptomless infection by the virus. Interesting similarities and dissimilarities between AaBbV1 and other previously reported botybirnaviruses are also discussed.

DOI： 10.1016/j.virusres.2019.01.006

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DOI： 10.1016/j.virusres.2018.12.012

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DOI： 10.1016/j.virusres.2017.11.020

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DOI： 10.1111/nph.15412

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Publisher：American Society for Microbiology  

DOI： 10.1128/JVI.01503-18

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier {BV}  

Many fungal viruses or mycoviruses have multi-segmented, rather than single-segmented, genomes. This multi-segment nature is frequently possessed by double-stranded RNA viruses, which include members of the Chrysoviridae, Quadriviridae, Megabirnaviridae, Partitiviridae, and Reoviridae families, and unassigned groups. Their genome segments are often packaged separately with the exception of mycoreoviruses, which are multi-segmented but mono-particulate viruses. These multi-segmented fungal dsRNA viruses, as exemplified by reoviruses, have been extensively studied among structural biologists, and contributed to discoveries of novel virion structures. Multi-component systems, interactions of viruses with subviral agents such as satellite and defective RNAs as typified by the yeast killer, and the rule-breaking neo-virus lifestyle exhibited by a capsidless single-stranded RNA virus hosted in an unrelated double-stranded RNA virus are also discussed. Fungal multi-segmented viruses and multicomponent virus systems would continue to provide virologists with interesting future challenges.

DOI： 10.1016/j.coviro.2018.07.009

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DOI： 10.1099/jgv.0.001152

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DOI： 10.3390/v10110584

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DOI： 10.3390/v10090481

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Microbiology Society  

The Hypoviridae, comprising one genus, Hypovirus, is a family of capsidless viruses with positive-sense, ssRNA genomes of 9.1–12.7 kb that possess either a single large ORF or two ORFs. The ORFs appear to be translated from genomic RNA by non-canonical mechanisms, i.e. internal ribosome entry site-mediated and stop/restart translation. Hypoviruses have been detected in ascomycetous or basidiomycetous filamentous fungi, and are considered to be replicated in host Golgi-derived, lipid vesicles that contain their dsRNA as a replicative form. Some hypoviruses induce hypovirulence to host fungi, while others do not. This is a summary of the current ICTV report on the taxonomy of the Hypoviridae, which is available at www.ictv.global/report/hypoviridae.

DOI： 10.1099/jgv.0.001055

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Blackwell Publishing Ltd  

To reveal mycovirus diversity, we conducted a search of as-yet-unexplored Mediterranean isolates of the phytopathogenic ascomycete Rosellinia necatrix for virus infections. Of seventy-nine, eleven fungal isolates tested RNA virus-positive, with many showing coinfections, indicating a virus incidence of 14%, which is slightly lower than that (approximately 20%) previously reported for extensive surveys of over 1000 Japanese R. necatrix isolates. All viral sequences were fully or partially characterized by Sanger and next-generation sequencing. These sequences appear to represent isolates of various new species spanning at least 6 established or previously proposed families such as Partiti-, Hypo-, Megabirna-, Yado-kari-, Fusagra- and Fusarividae, as well as a newly proposed family, Megatotiviridae. This observation greatly expands the diversity of R. necatrix viruses, because no hypo-, fusagra- or megatotiviruses were previously reported from R. necatrix. The sequence analyses showed a rare horizontal gene transfer event of the 2A-like protease domain between a dsRNA (phlegivirus) and a positive-sense, single-stranded RNA virus (hypovirus). Moreover, many of the newly detected viruses showed the closest relation to viruses reported from fungi other than R. necatrix, such as Fusarium spp., which are sympatric to R. necatrix. These combined results imply horizontal virus transfer between these soil-inhabitant fungi.

DOI： 10.1111/1462-2920.14065

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NLM (Medline)  

DOI： 10.1093/nar/gky078

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Society for Microbiology  

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

DOI： 10.1128/mBio.02350-17

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Small RNA (sRNA)-mediated gene silencing phenomena, exemplified by RNA interference (RNAi), require a unique class of proteins called Argonautes (AGOs). An AGO protein typically forms a protein-sRNA complex that contributes to gene silencing using the loaded sRNA as a specificity determinant. Here, we show that MoAGO2, one of the three AGO genes in the fungus Pyricularia oryzae (Magnaporthe oryzae) interferes with RNAi. Gene knockout (KO) studies revealed that MoAGO1 and MoAGO3 additively or redundantly played roles in hairpin RNA- and retrotransposon (MAGGY)-triggered RNAi while, surprisingly, the KO mutants of MoAGO2 (Δmoago2) showed elevated levels of gene silencing. Consistently, transcript levels of MAGGY and mycoviruses were drastically reduced in Δmoago2, supporting the idea that MoAGO2 impeded RNAi against the parasitic elements. Deep sequencing analysis revealed that repeat- and mycovirus-derived small interfering RNAs were mainly associated with MoAGO2 and MoAGO3, and their populations were very similar based on their size distribution patterns and positional base preference. Site-directed mutagenesis studies indicated that sRNA binding but not slicer activity of MoAGO2 was essential for the ability to diminish the efficacy of RNAi. Overall, these results suggest a possible interplay between distinct sRNA-mediated gene regulation pathways through a competition for sRNA.

DOI： 10.1093/nar/gkx1301

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Language：English   Publisher：Elsevier B.V.  

Recent studies illustrate that fungi as virus hosts provides a unique platform for hunting viruses and exploring virus/virus and virus/host interactions. Such studies have revealed a number of as-yet-unreported viruses and virus/virus interactions. Among them is a unique intimate relationship between a (+)ssRNA virus, yado-kari virus (YkV1) and an unrelated dsRNA virus, yado-nushi virus (YnV1). YkV1 dsRNA, a replicated form of YkV1, and RNA-dependent RNA polymerase, are trans-encapsidated by the capsid protein of YnV1. While YnV1 can complete its replication cycle, YkV1 relies on YnV1 for its viability. We previously proposed a model in which YkV1 diverts YnV1 capsids as the replication sites. YkV1 is neither satellite virus nor satellite RNA, because YkV1 appears to encode functional RdRp and enhances YnV1 accumulation. This represents a unique mutualistic virus/virus interplay and similar relations in other virus/host fungus systems are detectable. We propose to establish the family Yadokariviridae that accommodates YkV1 and recently discovered viruses phylogenetically related to YkV1. This article overviews what is known and unknown about the YkV1/YnV1 interactions. Also discussed are the YnV1 Phytoreo_S7 and YkV1 2A-like domains that may have been captured via horizontal transfer during the course of evolution and are conserved across extant diverse RNA viruses. Lastly, evolutionary scenarios are envisioned for YkV1 and YnV1.

DOI： 10.1016/j.virusres.2017.11.006

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Microbiology Society  

The Partitiviridae is a family of small, isometric, non-enveloped viruses with bisegmented double-stranded (ds) RNA genomes of 3-4.8 kbp. The two genome segments are individually encapsidated. The family has five genera, with characteristic hosts for members of each genus: either plants or fungi for genera Alphapartitivirus and Betapartitivirus, fungi for genus Gammapartitivirus, plants for genus Deltapartitivirus and protozoa for genus Cryspovirus. Partitiviruses are transmitted intracellularly via seeds (plants), oocysts (protozoa) or hyphal anastomosis, cell division and sporogenesis (fungi)
there are no known natural vectors. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of the Partitiviridae, which is available at www.ictv.global/report/partitiviridae.

DOI： 10.1099/jgv.0.000985

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Language：English   Publishing type：Part of collection (book)   Publisher：Academic Press Inc.  

Plant-associated fungi are infected by viruses at the incidence rates from a few % to over 90%. Multiple viruses often coinfect fungal hosts, and occasionally alter their phenotypes, but most of the infections are asymptomatic. Phenotypic alterations are grouped into two types: harmful or beneficial to the host fungi. Harmful interactions between viruses and hosts include hypovirulence and/or debilitation that are documented in a number of phytopathogenic fungi, exemplified by the chestnut blight, white root rot, and rapeseed rot fungi. Beneficial interactions are observed in a limited number of plant endophytic and pathogenic fungi where heat tolerance and virulence are enhanced, respectively. Coinfections of fungi provided a platform for discoveries of interesting virus/virus interactions that include synergistic, as in the case for those in plants, and unique antagonistic and mutualistic interactions between unrelated RNA viruses. Also discussed here are coinfection-induced genome rearrangements and frequently observed coinfections by the simplest positive-strand RNA virus, the mitoviruses.

DOI： 10.1016/bs.aivir.2017.10.003

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PUBLIC LIBRARY SCIENCE  

Unlike their counterparts in bacterial and higher eukaryotic hosts, most fungal viruses are transmitted intracellularly and lack an extracellular phase. Here we determined the cryo-EM structure at 3.7 angstrom resolution of Rosellinia necatrix quadrivirus 1 (RnQV1), a fungal double-stranded (ds) RNA virus. RnQV1, the type species of the family Quadriviridae, has a multipartite genome consisting of four monocistronic segments. Whereas most dsRNA virus capsids are based on dimers of a single protein, the similar to 450-angstrom-diameter, T = 1 RnQV1 capsid is built of P2 and P4 protein heterodimers, each with more than 1000 residues. Despite a lack of sequence similarity between the two proteins, they have a similar a-helical domain, the structural signature shared with the lineage of the dsRNA bluetongue virus-like viruses. Domain insertions in P2 and P4 preferential sites provide additional functions at the capsid outer surface, probably related to enzyme activity. The P2 insertion has a fold similar to that of gelsolin and profilin, two actin-binding proteins with a function in cytoskeleton metabolism, whereas the P4 insertion suggests protease activity involved in cleavage of the P2 383-residue C-terminal region, absent in the mature viral particle. Our results indicate that the intimate virus-fungus partnership has altered the capsid genome-protective and/or receptor-binding functions. Fungal virus evolution has tended to allocate enzyme activities to the virus capsid outer surface.

DOI： 10.1371/journal.ppat.1006755

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Orchid fleck virus (OFV) represents a rhabdovirus with a unique bipartite genome. OFV genetic diversity at the whole genome level has not been described. Using the partial genome sequence of RNA1, we have determined that several OFV isolates derived from orchids in Japan belong to two genetically distant subgroups: subgroup I, the members of which are distributed worldwide but previously not known in Asia, and subgroup II, which is commonly distributed in Japan. However, complete genome sequence analysis of a novel Japanese subgroup I isolate revealed that although its RNA1 sequence differs considerably from those of subgroup II isolates, its RNA2 sequence is almost identical to them. Based on phylogenetic and recombination analyses, the genome reassortment events were predicted to occur between OFV subgroups including other unseen strains. Our data show that genome reassortment contributes to the genetic diversities of the bipartite rhabdoviruses and its occurrence may be geographically constrained.

DOI： 10.1016/j.virol.2017.04.027

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Taylor and Francis Inc.  

Reactive oxygen species (ROS), including superoxide anion (O2 −), hydrogen peroxide (H2O2), and hydroxyl radical, act as signaling molecules to transduce biotic and abiotic stimuli into stress adaptations in plants. A respiratory burst oxidase homolog B of Nicotiana benthamiana (NbRBOHB) is responsible for O2 − production to inhibit pathogen infection during plant innate immunity. RBOH-derived O2 − can be immediately converted into H2O2 by the action of superoxide dismutase. Interestingly, we recently showed that red clover necrotic mosaic virus (RCNMV), a plant positive-strand RNA [(+)RNA] virus, hijacks the host's ROS-generating machinery during infection. An RCNMV replication protein associates with NbRBOHB and triggers intracellular ROS bursts. These bursts are required for robust viral RNA replication. However, what types of ROS are required for viral replication is currently unknown. Here, we found that RCNMV replication was sensitive to an O2 − scavenger but insensitive to an H2O2 scavenger. Interestingly, replication of another plant (+)RNA virus, brome mosaic virus, was sensitive to both types of scavengers. These results indicate a virus-specific pattern requirement of O2 − and H2O2 for (+)RNA virus replication and suggest a conserved nature of the roles of ROS in (+)RNA virus replication.

DOI： 10.1080/15592324.2017.1338223

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC MICROBIOLOGY  

Most double-stranded RNA (dsRNA) viruses are transcribed and replicated in a specialized icosahedral capsid with a T = 1 lattice consisting of 60 asymmetric capsid protein (CP) dimers. These capsids help to organize the viral genome and replicative complex( es). They also act as molecular sieves that isolate the virus genome from host defense mechanisms and allow the passage of nucleotides and viral transcripts. Rosellinia necatrix quadrivirus 1 (RnQV1), the type species of the family Quadriviridae, is a dsRNA fungal virus with a multipartite genome consisting of four monocistronic segments (segments 1 to 4). dsRNA-2 and dsRNA-4 encode two CPs (P2 and P4, respectively), which coassemble into similar to 450-angstrom-diameter capsids. We used three-dimensional cryo-electron microscopy combined with complementary biophysical techniques to determine the structures of RnQV1 virion strains W1075 and W1118. RnQV1 has a quadripartite genome, and the capsid is based on a single-shelled T = 1 lattice built of P2-P4 dimers. Whereas the RnQV1-W1118 capsid is built of full-length CP, P2 and P4 of RnQV1-W1075 are cleaved into several polypeptides, maintaining the capsid structural organization. RnQV1 heterodimers have a quaternary organization similar to that of homodimers of reoviruses and other dsRNA mycoviruses. The RnQV1 capsid is the first T = 1 capsid with a heterodimer as an asymmetric unit reported to date and follows the architectural principle for dsRNA viruses that a 120-subunit capsid is a conserved assembly that supports dsRNA replication and organization.
IMPORTANCE
Given their importance to health, members of the family Reoviridae are the basis of most structural and functional studies and provide much of our knowledge of dsRNA viruses. Analysis of bacterial, protozoal, and fungal dsRNA viruses has improved our understanding of their structure, function, and evolution, as well. Here, we studied a dsRNA virus that infects the fungus Rosellinia necatrix, an ascomycete that is pathogenic to a wide range of plants. Using three-dimensional cryo-electron microscopy and analytical ultracentrifugation analysis, we determined the structure and stoichiometry of Rosellinia necatrix quadrivirus 1 (RnQV1). The RnQV1 capsid is a T = 1 capsid with 60 heterodimers as the asymmetric units. The large amount of genetic information used by RnQV1 to construct a simple T = 1 capsid is probably related to the numerous virus-host and virus-virus interactions that it must face in its life cycle, which lacks an extracellular phase.

DOI： 10.1128/JVI.01013-16

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier B.V.  

The identification of mycoviruses contributes greatly to understanding of the diversity and evolutionary aspects of viruses. Powdery mildew fungi are important and widely studied obligate phytopathogenic agents, but there has been no report on mycoviruses infecting these fungi. In this study, we used a deep sequencing approach to analyze the double-stranded RNA (dsRNA) segments isolated from field-collected samples of powdery mildew fungus-infected red clover plants in Japan. Database searches identified the presence of at least ten totivirus (genus Totivirus)-like sequences, termed red clover powdery mildew-associated totiviruses (RPaTVs). The majority of these sequences shared moderate amino acid sequence identity with each other (&lt
44%) and with other known totiviruses (&lt
59%). Nine of these identified sequences (RPaTV1a, 1b and 2–8) resembled the genome of the prototype totivirus, Saccharomyces cerevisiae virus-L-A (ScV-L-A) in that they contained two overlapping open reading frames (ORFs) encoding a putative coat protein (CP) and an RNA dependent RNA polymerase (RdRp), while one sequence (RPaTV9) showed similarity to another totivirus, Ustilago maydis virus H1 (UmV-H1) that encodes a single polyprotein (CP-RdRp fusion). Similar to yeast totiviruses, each ScV-L-A-like RPaTV contains a –1 ribosomal frameshift site downstream of a predicted pseudoknot structure in the overlapping region of these ORFs, suggesting that the RdRp is translated as a CP-RdRp fusion. Moreover, several ScV-L-A-like sequences were also found by searches of the transcriptome shotgun assembly (TSA) libraries from rust fungi, plants and insects. Phylogenetic analyses show that nine ScV-L-A-like RPaTVs along with ScV-L-A-like sequences derived from TSA libraries are clustered with most established members of the genus Totivirus, while one RPaTV forms a new distinct clade with UmV-H1, possibly establishing an additional genus in the family. Taken together, our results indicate the presence of diverse, novel totiviruses in the powdery mildew fungus populations infecting red clover plants in the field.

DOI： 10.1016/j.virusres.2016.05.011

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DOI： 10.1016/j.virusres.2016.05.022

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

A new virus termed Rosellinia necatrix megabirnavirus 2 (RnMBV2) was molecularly and biologically characterized. RnMBV2 was originally harbored in isolate W8 of R. necatrix co-infected with the previously reported virus Rosellinia necatrix partitivirus 1 (RnPV1). RnMBV2 has molecular features similar and different from precedent megabirnaviruses, Rosellinia necatrix megabirnavirus 1 (RnMBV1) and Sclerotinia sclerotiorum megabirnavirus 1 (SsMBV1). The two genomic segments of RnMBV2 (9.0-kbp dsRNA1 and 8.0-kbp dsRNA2) each possess two open reading frames (ORF1 and 2 on dsRNA1 and ORF3 and 4 on dsRNA2), with a well conserved 5'-long untranslated region (UTR) of 1.7-1.8 kb between the segments, and relatively short 3'-UTR. The RnMBV2 dsRNA1-coded capsid protein (CP) and RNA-dependent RNA polymerase (RdRp) show higher sequence identity to those of SsMBV1 than to those of RnMBV1, whereas the RnMBV2 ORF3-coded protein is more closely related to the counterpart of RnMBV1. No significant amino acid sequence similarity was detected among ORF4-coded sequences of the three megabirnaviruses. Virion transfection and co-culturing allowed for single and double infection of mycelial incompatible isolates W37 and W97 by RnMBV2 and/or RnPV1. Their comparative analyses showed RnMBV2 to be able to confer hypovirulence with the aid of a co-infecting RnPV1, while the individual viruses exhibited asymptomatic infections. Interestingly, RnPV1 accumulation appeared to be increased in co-infected fungal strain with two segments of RnMBV2 relative to singly infected fungal strains. Furthermore, the dispensability of RnMBV2 dsRNA2 was demonstrated to be similar to that of the other two megabirnaviruses. (C) 2015 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.virusres.2015.12.009

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

The family Partitiviridae comprises of five genera with bi-segmented dsRNA genomes that accommodate members infecting plants, fungi or protists. All partitiviruses with only a few exceptions cause asymptomatic infections. We report the characterization of a novel betapartitivirus termed Rosellinia necatrix partitivirus 6 (RnPV6) from a field isolate of a plant pathogenic fungus, white root rot fungus. RnPV6 has typical partitivirus features: dsRNA1 and dsRNA2 are 2462 and 2499 bps in length encoding RNA dependent RNA polymerase and capsid protein. Purified particles are spherical with a diameter of 30 nm. Taking advantage of infectivity as virions, RnPV6 was introduced into a model filamentous fungal host, chestnut blight fungus to investigate virus/host interactions. Unlike other partitiviruses tested previously, RnPV6 induced profound phenotypic alterations with symptoms characterized by a reduced growth rate and enhanced pigmentation and was tolerant to host RNA silencing. In addition, a variety of defective RNAs derived from dsRNA1 appear after virion transfection. These sub-viral RNAs were shown to interfere with RnPV6 replication, at least for that of cognate segment dsRNA1. Presence of these sub-viral elements resulted in reduced symptom expression by RnPV6, suggesting their nature as defective-interfering RNAs. The features of RnPV6 are similar to but distinct from those of a previously reported alphapartitivirus, Rosellinia necatrix partitivirus 2 that is susceptible to RNA silencing. (C) 2015 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.virusres.2015.10.017

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Language：English   Publisher：ELSEVIER SCIENCE BV  

A transmissible disease of the plant pathogenic fungus Helminthosporium victoriae, the causal agent of Victoria blight of oats, was reported more than 50 years ago. Diseased, but not normal, isolates, of H. victoriae contain two distinct viruses designated according to their sedimentation values as victorivirus Helminthosporium victoriae virus 190S (HvV190S) and chrysovirus Helminthosporium victoriae 145S (HvV145S). Although a viral etiology of the disease was previously proposed, conclusive evidence was lacking. Here we present unequivocal evidence based on transfecting virus-free H. victoriae protoplasts with purified virus particles showing that HvV190S is essential for disease development. Furthermore, we show an expansion of the host range of HvV190S to include Cryphonectria parasitica and we also show similarity in a subset of phenotypic traits between HvV190S-infected RNA silencing deficient mutant (Delta dcl-2) of C. parasitica and a strain of H. victoriae. In virulence assays on detached American chestnut branches and Red Delicious apple fruits, HvV190S-infected C parasitica strain Delta dcl-2 was markedly less virulent than wild type and virus-free Delta dcl-2 C parasitica strains. Furthermore, the hypovirulent HvV190S-infected C parasitica Delta dcl-2 strain exhibited strong antifungal activity in dual culture with the plant pathogenic fungus Sclerotinia sclerotiorum. No such inhibitory activity was observed in comparable dual cultures with wild type and virus-free Delta dcl-2 C. parasitica strains. The discovery that infection with HvV190S induced a hypovirulent phenotype in a heterologous plant pathogenic host is very significant since it might be possible to convert other economically important plant pathogenic fungi to hypovirulence using HvV190S. (C) 2015 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.virusres.2016.05.010

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

The identification of mycoviruses contributes greatly to understanding of the diversity and evolutionary aspects of viruses. Powdery mildew fungi are important and widely studied obligate phytopathogenic agents, but there has been no report on mycoviruses infecting these fungi. In this study, we used a deep sequencing approach to analyze the double-stranded RNA (dsRNA) segments isolated from field collected samples of powdery mildew fungus-infected red clover plants in Japan. Database searches identified the presence of at least ten totivirus (genus Totivirus)-like sequences, termed red clover powdery mildew-associated totiviruses (RPaTVs). The majority of these sequences shared moderate amino acid sequence identity with each other (&lt;44%) and with other known totiviruses (&lt;59%). Nine of these identified sequences (RPaTV1a, 1b and 2-8) resembled the genome of the prototype totivirus, Saccharomyces cerevisiae virus-L-A (ScV-L-A) in that they contained two overlapping open reading frames (ORFs) encoding a putative coat protein (CP) and an RNA dependent RNA polymerase (RdRp), while one sequence (RPaTV9) showed similarity to another totivirus, Ustilago maydis virus H1 (UmV-H1) that encodes a single polyprotein (CP-RdRp fusion). Similar to yeast totiviruses, each ScV-L-A-like RPaTV contains a-1 ribosomal frameshift site downstream of a predicted pseudoknot structure in the overlapping region of these ORFs, suggesting that the RdRp is translated as a CP-RdRp fusion. Moreover, several ScV-L-A-like sequences were also found by searches of the transcriptome shotgun assembly (TSA) libraries from rust fungi, plants and insects. Phylogenetic analyses show that nine ScV-L-A-like RPaTVs along with ScV-L-A-like sequences derived from TSA libraries are clustered with most established members of the genus Totivirus, while one RPaTV forms a new distinct Glade with UmV-H1, possibly establishing an additional genus in the family. Taken together, our results indicate the presence of diverse, novel totiviruses in the powdery mildew fungus populations infecting red clover plants in the field. (C) 2015 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.virusres.2015.11.015

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

A transmissible disease of the plant pathogenic fungus Helminthosporium victoriae, the causal agent of Victoria blight of oats, was reported more than 50 years ago. Diseased, but not normal, isolates, of H. victoriae contain two distinct viruses designated according to their sedimentation values as victorivirus Helminthosporium victoriae virus 190S (HvV190S) and chrysovirus Helminthosporium victoriae 145S (HvV145S). Although a viral etiology of the disease was previously proposed, conclusive evidence was lacking. Here we present unequivocal evidence based on transfecting virus-free H. victoriae protoplasts with purified virus particles showing that HvV190S is essential for disease development. Furthermore, we show an expansion of the host range of HvV190S to include Cryphonectria parasitica and we also show similarity in a subset of phenotypic traits between HvV190S-infected RNA silencing deficient mutant (Delta dcl-2) of C parasitica and a strain of H. victoriae. In virulence assays on detached American chestnut branches and Red Delicious apple fruits, HvV190S-infected C parasitica strain Delta dcl-2 was markedly less virulent than wild type and virus-free Delta dcl-2 C parasitica strains. Furthermore, the hypovirulent HvV190S-infected C parasitica Delta dcl-2 strain exhibited strong antifungal activity in dual culture with the plant pathogenic fungus Sclerotinia sclerotiorum. No such inhibitory activity was observed in-comparable dual cultures with wild type and virus-free Delta dcl-2 C. parasitica strains. The discovery that infection with HvV190S induced a hypovirulent phenotype in a heterologous plant pathogenic host is very significant since it might be possible to convert other economically important plant pathogenic fungi to hypovirulence using HvV190S. (C) 2015 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.virusres.2015.12.011

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

Viral reactivations following hematopoietic stem cell transplantation are thought to result from the breakdown of both cell-mediated and humoral immunity. As a result, many viruses could be reactivated individually or simultaneously. Using a multiplex polymerase chain reaction (PCR), we prospectively examined many kinds of viral DNAs at a time in 105 patients who underwent allogeneic hematopoietic stem cell transplantation. In total, 591 whole blood samples were collected weekly from pre- to 42 days post-transplantation and the following 13 viruses were tested; herpes simplex virus 1 (HSV-1), HSV-2, varicella-zoster virus (VZV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), human herpes virus 6 (HHV-6), HHV-7, HHV-8, adenovirus, BK virus (BKV), JC virus (JCV), parvovirus B19, and hepatitis B virus (HBV). Several viral DNAs were detected in 12 patients before hematopoietic stem cell transplantation. The detection rate gradually increased after transplantation and peaked at 21 days. The most frequently detected virus was HHV-6 (n=63; 60.0%), followed by EBV (n=11; 10.5%), CMV (n=11; 10.5%), and HHV-7 (n=9; 8.6%). Adenovirus and HBV were each detected in one patient (1.0%). Detection of HHV-6 DNA was significantly more common among patients undergoing cord blood transplantation or with steroid treatment. EBV DNA tended to be more common in patients treated with anti-thymocyte globulin. Multiplex PCR was useful for detecting many viral reactivations after hematopoietic stem cell transplantation, simultaneously. Cord blood transplantation, steroid treatment, or anti-thymocyte globulin use was confirmed to be risk factors after transplantation. J. Med. Virol. 87:1427-1435, 2015. (c) 2015 Wiley Periodicals, Inc.

DOI： 10.1002/jmv.24161

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC GENERAL MICROBIOLOGY  

Rosellinia necatrix megabirnavirus 1 (RnMBV1) W779 is a bi-segmented dsRNA virus and a strain of the type species Rosellinia necatrix megabirnavirus 1 of the family Megabirnaviridae. RnMBV1 causes severe reduction of both mycelial growth of Rosellinia necatrix in synthetic medium and fungal virulence to plant hosts, and thus has strong potential for virocontrol (biological control using viruses) of white rot. The structure of RnMBV1 was examined by cryo-electron microscopy and three-dimensional reconstruction at 15.7 angstrom resolution. The diameter of the RnMBV1 capsid was 520 angstrom, and the capsid was composed of 60 asymmetrical dimers in the T=1 (so-called T=2) lattice that is well conserved among dsRNA viruses. However, RnMBV1 has putatively 120 large protrusions with a width of similar to 45 angstrom and a height of similar to 50 angstrom on the virus surface, making it distinguishable from the other dsRNA viruses.

DOI： 10.1099/vir.0.000182

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS INC  

Eukaryotes employ RNA silencing as an innate defense system against invading viruses. Dicer proteins play the most crucial role in initiating this antiviral pathway as they recognize and process incoming viral nucleic acids into small interfering RNAs. Generally, 2 successive infection stages constitute viral infection in plants. First, the virus multiplies in initially infected cells or organs after viral transmission and then the virus subsequently spreads systemically through the vasculature to distal plant tissues or organs. Thus, antiviral silencing in plants must cope with both local and systemic invasion of viruses. In a recent study using 2 sets of different experiments, we clearly demonstrated the differential requirement for Dicer-like 4 (DCL4) and DCL2 proteins in the inhibition of intracellular and systemic infection by potato virus X in Arabidopsis thaliana. Taken together with the results of other studies, here we further discuss the functional specificity of DCL proteins in the antiviral silencing pathway.

DOI： 10.1080/15592324.2015.1039214

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER WIEN  

Cymbidium chlorotic mosaic virus (CyCMV), isolated from a spring orchid (Cymbidium goeringii), was characterized molecularly. CyCMV isometric virions comprise a single, positive-strand RNA genome of 4,083 nucleotides and 30-kDa coat protein. The virus genome contains five overlapping open reading frames with a genomic organization similar to that of sobemoviruses. BLAST searches and phylogenetic analysis revealed that CyCMV is most closely related to papaya lethal yellowing virus, a proposed dicot-infecting sobemovirus (58.8 % nucleotide sequence identity), but has a relatively distant relationship to monocot-infecting sobemoviruses, with only modest sequence identities. This suggests that CyCMV is a new monocot-infecting member of the floating genus Sobemovirus.

DOI： 10.1007/s00705-015-2460-9

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Language：English   Publisher：FRONTIERS RESEARCH FOUNDATION  

DOI： 10.3389/fmicb.2015.00470

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Language：English   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

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 vieus assembly, function, and evolution. (C) 2015 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.virol.2015.02.034

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

The chestnut blight fungus, Cryphonectria parasitica, is an important plant pathogenic ascomycete. The fungus hosts a wide range of viruses and now has been established as a model filamentous fungus for studying virus/host and virus/virus interactions. This is based on the development of methods for artificial virus introduction and elimination, host genome manipulability, available host genome sequence with annotations, host mutant strains, and molecular tools. Molecular tools include subcellular distribution markers, gene expression reporters, and vectors with regulatable promoters that have been long available for unicellular organisms, cultured cells, individuals of animals and plants, and certain filamentous fungi. A comparison with other filamentous fungi such as Neurospora crassa has been made to establish clear advantages and disadvantages of C parasitica as a virus host. In addition, a few recent studies on RNA silencing vs. viruses in this fungus are introduced. (C) 2014 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.virol.2014.09.024

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

Members of the plant Dicer-like (DCL) protein family are the critical components of the RNA-silencing pathway that mediates innate antiviral defence. The distinct antiviral role of each individual DCL protein has been established with mostly based on observations of aerial parts of plants. Thus, although the roots are closely associated with the life cycle of many plant viruses, little is known about the antiviral activities of DCL proteins in roots. We observed that antiviral silencing strongly inhibits potato virus X (PVX) replication in roots of some susceptible Solanaceae species. Silencing of the DCL4 homolog in Nicotiana benthamiana partially elevated PVX replication levels in roots. In Arabidopsis thaliana, which was originally considered a non-host plant of PVX, high levels of PVX accumulation in inoculated leaves were achieved by inactivation of DCL4, while in the upper leaves and roots, it required the additional inactivation of DCL2. In transgenic A.thaliana carrying the PVX amplicon with a green fluorescent protein (GFP) gene insertion in the chromosome (AMP243 line), absence of DCL4 enabled high levels of PVX-GFP accumulation in various aerial organs but not in the roots, suggesting that DCL4 is critical for intracellular antiviral silencing in shoots but not in roots, where it can be functionally compensated by other DCL proteins. Together, the high level of functional redundancies among DCL proteins may contribute to the potent antiviral activities against PVX replication in roots.
Significance StatementThis study demonstrates the differential contributions of DCL proteins between leaves and roots.

DOI： 10.1111/tpj.12770

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Endogenous non-retroviral RNA like sequences (NRVSs) have been discovered in the genome of a wide range of eukaryotes. These are considered as fossil RNA viral elements integrated into host genomes by as-yet-known mechanisms, and in many cases, those fossils are estimated to be millions-of-years-old. It is likely that the number of NRVS records will increase rapidly due to the growing availability of whole-genome sequences for many kinds of eukaryotes. Discovery of the novel NRVSs and understanding of their phylogenetic relationship with modern viral relatives provide important information on deep evolutionary history of RNA virus-host interactions. In this chapter, therefore, the common strategies for the identification and characterization of endogenous NRVSs from plants, insects, and fungi are described.

DOI： 10.1007/978-1-4939-1743-3_7

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Language：English   Publisher：ELSEVIER SCIENCE BV  

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. (C) 2014 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.virusres.2014.04.007

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：FRONTIERS RESEARCH FOUNDATION  

Here we report a biological and molecular characterization of a novel positive-sense RNA virus isolated from a field isolate (NW10) of a filamentous phytopathogenic fungus, the white root rot fungus that is designated as Rosellinia necatrix fusarivirus 1 (RnFV1). A recently developed technology using zinc ions allowed us to transfer RnFV1 to two mycelially incompatible Rosellinia necatrix strains. A biological comparison of the virus-free and -recipient isogenic fungal strains suggested that RnFV1 infects latently and thus has no potential as a virocontrol agent. The virus has an undivided positive-sense RNA genome of 6286 nucleotides excluding a poly (A) tail. The genome possesses two non-overlapping open reading frames (ORFs): a large ORF1 that encodes polypeptides with RNA replication functions and a smaller ORF2 that encodes polypeptides of unknown function. A lack of coat protein genes was suggested by the failure of virus particles from infected mycelia. No evidence was obtained by Northern analysis or classical 5'-RACE for the presence of subgenomic RNA for the downstream ORE Sequence similarities were found in amino-acid sequence between RnFV1 putative proteins and counterparts of a previously reported mycovirus, Fusarium graminearum virus 1 (FgV1). Interestingly, several related sequences were detected by BLAST searches of independent transcriptome assembly databases one of which probably represents an entire virus genome. Phylogenetic analysis based on the conserved RNA-dependent RNA polymerase showed that RnFV1, FgV1, and these similar sequences are grouped in a cluster distinct from distantly related hypoviruses. It is proposed that a new taxonomic family termed Fusariviridae be created to include RnFV1 and FgV1.

DOI： 10.3389/fmicb.2014.00360

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC GENERAL MICROBIOLOGY  

Rosellinia necatrix megabirnavirus 1 (RnMBV1) with a bipartite dsRNA genome (dsRNA1 and dsRNA2) confers hypovirulence to its natural host, the white root rot fungus, and is thus regarded as a potential virocontrol (biocontrol) agent. Each segment has two large ORFs: ORF1 and partially overlapping ORF2 on dsRNA1 encode the major capsid protein (CP) and RNA-dependent RNA polymerase (RdRp), whilst ORF3 and ORF4 on dsRNA2 encode polypeptides with unknown functions. Here, we report the biological and molecular characterization of this virus in the chestnut blight fungus, Cryphonectria parasitica, a filamentous fungus that has been used as a model for mycovirus research. Transfection with purified RnMBV1 particles into an RNA-silencing-defective strain (Delta dcl-2) of C. parasitica and subsequent anastomosis with the WT strain (EP155) resulted in stable persistent infection in both host strains. However, accumulation levels in the two strains were different, being similar to 20-fold higher in Delta dcl-2 than in EP155. Intriguingly, whilst RnMBV1 reduced both virulence and growth rate in Delta dcl-2, it attenuated virulence without affecting significantly other traits in EP155. Western blot analysis using antiserum against recombinant proteins encoded by either ORF1 or ORF2 demonstrated the presence of a 250 kDa protein in purified virion preparations, suggesting that RdRp is expressed as a CP fusion product via a - 1 frameshift. Antiserum against the ORF3-encoded protein allowed the detection of 150, 30 and 23 kDa polypeptides specifically in RnMBV1-infected mycelia. Some properties of an RnMBV1 mutant with genome rearrangements, which occurred after transfection of Delta dcl-2 and EP155, were also presented. This study provides an additional example of C. parasitica serving as a versatile, heterologous fungus for exploring virus host interactions and virus gene expression strategies.

DOI： 10.1099/vir.0.058164-0

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

The transcriptional strategy of orchid fleck virus (OFV), which has a two-segmented negative-strand RNA genome and resembles plant nucleorhabdoviruses, remains unexplored. In this study, the transcripts of six genes encoded by OFV RNA1 and RNA2 in the poly(A)-enriched RNA fraction from infected plants were molecularly characterized. All of the OFV mRNAs were initiated at a start sequence 3'-UU-5' with one to three non-viral adenine nucleotides which were added at the 5' end of each mRNA, whereas their 3' termini ended with a 5'-AUUUAAA(U/G)AAAA(A)n-3' sequence. We also identified the presence of polyadenylated short transcripts derived from the 3'-terminal leader regions of both genomic and antigenomic strands, providing the first example of plus- and minus-strand leader RNAs in a segmented minus-strand RNA virus. The similarity in the transcriptional strategy between this bipartite OFV and monopartite rhabdoviruses, especially nucleorhabdoviruses (family Rhabdoviridae) is additional support for their close relationship. (C) 2014 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.virol.2014.01.007

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Rosellinia necatrix megabirnavirus 1 (RnMBV1) is a bi-segmented double-stranded RNA mycovirus that reduces the virulence of the fungal plant pathogen R. necatrix. We isolated strains of RnMBV1 with genome rearrangements (RnMBV1-RS1) that retained dsRNA1, encoding capsid protein (ORF1) and RNA-dependent RNA polymerase (ORF2), and had a newly emerged segment named dsRNAS1, but with loss of dsRNA2, which contains two ORFs of unknown function. Analyses of two variants of dsRNAS1 revealed that they both originated from dsRNA1 by deletion of ORFI and partial tandem duplication of ORF2, retaining a much shorter 5' untranslated region (UTR). R. necatrix transfected with RnMBV-RS1 virions showed maintenance of virulence on host plants compared with infection with RnMBV1. This suggests that dsRNAS1 is able to be transcribed and packaged, as well as suggesting that dsRNA2, while dispensable for virus replication, is required to reduce the virulence of R. necatrix. (C) 2013 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.virol.2013.12.002

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER WIEN  

The complete genomic sequence of Habenaria mosaic virus (HaMV), which infects terrestrial orchids (Habenaria radiata), has been determined. The genome is composed of 9,499 nucleotides excluding the 3'-terminal poly(A) tail, encoding a large polyprotein of 3,054 amino acids with the genomic features typical of a potyvirus. Putative proteolytic cleavage sites were identified by sequence comparison to those of known potyviruses. The HaMV polyprotein showed 58 % amino acid sequence identity to that encoded by the most closely related potyvirus, tobacco vein banding mosaic virus. Phylogenetic analysis of the polyprotein amino acid sequence and its coding sequences confirmed that HaMV formed a cluster with the chilli veinal mottle virus group, most of which infect solanaceous plants. These results suggest that HaMV is a distinct member of the genus Potyvirus.

DOI： 10.1007/s00705-013-1784-6

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Language：Japanese   Publisher：The Japanese Society for Virology  

Most of reported fungal viruses (mycoviruses) have double-stranded RNA (dsRNA) genomes. This may reflect the simple, easy method for mycovirus hunting that entails detection of dsRNAs as a sign of viral infections. There are an increasing number of screens of various fungi, particularly phytopathogenic fungi for viruses pathogenic to host fungi or able to confer hypovirulence to them. This bases on an attractive research field of biological control of fungal plant diseases using viruses (virocontrol), mainly targeting important phytopathogenic fungi. While isolated viruses usually induce asymptomatic symptoms, they show a considerably high level of diversity. As of 2014, fungal dsRNA viruses are classified into six families: Reoviridae, Totiviridae, Chrysoviridae, Partitiviridae, Megabirnaviridae and Quadriviridae. These exclude unassigned mycoviruses which will definitely be placed into distinct families and/or genera. In this review article, dsRNA viruses isolated from the kingdom Fungi including as-yet-unclassified taxa are overviewed. Some recent achievements in the related field are briefly introduced as well.

DOI： 10.2222/jsv.64.225

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DOI： 10.2222/jsv.64.11

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER WIEN  

Nyamanini virus (NYMV) and Midway virus (MIDWV) are unclassified tick-borne agents that infect land birds and seabirds, respectively. The recent molecular characterization of both viruses confirmed their already known close serological relationship and revealed them to be nonsegmented, single- and negative-stranded RNA viruses that are clearly related to, but quite distinct from, members of the order Mononegavirales (bornaviruses, filoviruses, paramyxoviruses, and rhabdoviruses). A third agent, soybean cyst nematode virus 1 (SbCNV-1, previously named soybean cyst nematode nyavirus), was recently found to be an additional member of this new virus group. Here, we review the current knowledge about all three viruses and propose classifying them as members of a new mononegaviral family, Nyamiviridae.

DOI： 10.1007/s00705-013-1674-y

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Orchid fleck virus (OFV) has a unique two-segmented negative-sense RNA genome that resembles that of plant nucleorhabdoviruses. In infected plant cells, OFV and nucleorhabdoviruses induce an intranuclear electron-lucent viroplasm that is believed to be the site for virus replication. In this study, we investigated the molecular mechanism by which OFV viroplasms are produced in vivo. Among OFV-encoded proteins, the nucleocapsid protein (N) and the putative phosphoprotein (P) were present in nuclear fractions of OFV-infected Nicotiana benthamiana plants. Transient coexpression of N and P, in the absence of virus infection, was shown to be sufficient for formation of an intranuclear viroplasm-like structure in plant cells. When expressed independently as a fluorescent protein fusion product in uninfected plant cells, N protein accumulated throughout the cell, while P protein accumulated in the nucleus. However, the N protein, when coexpressed with P, was recruited to a subnuclear region to induce a large viroplasm-like focus. Deletion and substitution mutagenesis demonstrated that the P protein contains a nuclear localization signal (NLS). Artificial nuclear targeting of the N-protein mutant was insufficient for formation of viroplasm-like structures in the absence of P. A bimolecular fluorescence complementation assay confirmed interactions between the N and P proteins within subnuclear viroplasm-like foci and interactions of two of the N. benthamiana importin-α homologues with the P protein but not with the N protein. Taken together, our results suggest that viroplasm formation by OFV requires nuclear accumulation of both the N and P proteins, which is mediated by P-NLS, unlike nucleorhabdovirus viroplasm utilizing the NLS on protein N. © 2013, American Society for Microbiology.

DOI： 10.1128/JVI.00270-13

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC MICROBIOLOGY  

A novel victorivirus, termed Rosellinia necatrix victorivirus 1 (RnVV1), was isolated from a plant pathogenic ascomycete, white root rot fungus Rosellinia necatrix, coinfected with a partitivirus. The virus was molecularly and biologically characterized using the natural and experimental hosts (chestnut blight fungus, Cryphonectria parasitica). RnVV1 was shown to have typical molecular victorivirus attributes, including a monopartite double-stranded RNA genome with two open reading frames (ORFs) encoding capsid protein (CP) and RNA-dependent RNA polymerase (RdRp), a UAAUG pentamer presumed to facilitate the coupled termination/reinitiation for translation of the two ORFs, a spherical particle structure similar to 40 nm in diameter, and moderate levels of CP and RdRp sequence identity (34 to 58%) to those of members of the genus Victorivirus within the family Totiviridae. A reproducible transfection system with purified RnVV1 virions was developed for the two distinct fungal hosts. Transfection assay with purified RnVV1 virions combined with virus elimination by hyphal tipping showed that the effects of RnVV1 on the phenotype of the natural host were negligible. Interestingly, comparison of the RNA silencing-competent (standard strain EP155) and -defective (Delta dcl-2) strains of C. parasitica infected with RnVV1 showed that RNA silencing acted against the virus to repress its replication, which was restored by coinfection with hypovirus or transgenic expression of an RNA silencing suppressor, hypovirus p29. Phenotypic changes were observed in the Delta dcl-2 strain but not in EP155. This is the first reported study on the host range expansion of a Totiviridae member that is targeted by RNA silencing.

DOI： 10.1128/JVI.00557-13

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We report the biological and molecular characterisation of a second quadrivirus strain termed Rosellinia necatrix quadrivirus 1 strain W1118 (RnQV1-W1118) from the ascomycete white root rot fungus. Commonalities with the first quadrivirus (RnQV1-W1075) include its quadripartite genome structure, spherical particle morphology, sequence heterogeneity in the extreme terminal end, 72-82% sequence identity between the corresponding proteins, and its ability to cause a latent infection. Distinguishing features include different conserved terminal sequences and the degree of susceptibility of two major capsid proteins to proteolytic degradation, which is thought to occur during virion purification. Identification of this second quadrivirus strain strengthens our earlier proposal that "Rosellinia necatrix quadrivirus 1" should be considered the type species of a novel family, "Quadriviridae".

DOI： 10.1007/s00705-012-1580-8

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC BIOSCIENCE BIOENGINEERING JAPAN  

A new multichannel microdevice flow system with stainless steel flow chamber was used for architecture visualization, development monitoring and structural quantification of GFP-labeled Pseudomonas aeruginosa PAO1 live biofilms. Direct in situ investigations using confocal laser scanning microscopy (CLSM) at 72 h revealed structural pattern differences as a result of nutrient concentration gradients. When grown in LB medium, round, dispersed cellular aggregates were formed whereas in 1/3-diluted LB medium, biofilms were mostly flat and compact. However, COMSTAT analyses showed no considerable differences in biomass and thickness between the two LB concentrations. Characterization of time-dependent development of biofilms grown in 1/3-diluted LB medium showed full maturation of colonies by 120 h reaching maximum biomass at 17.1 mu m(3)/mu m(2) and average thickness at 44.4 mu m. Consequent thinning and formation of openings through interior in colonies occurred by 168 h. These results suggest that the new system tested allowed a fast and thick biofilm development on the surface of the stainless steel flow chamber. These findings may provide better estimates of biofilm activity and systematic evaluation of the effects of different parameters on biofilm morphology and development in industrial and biomedical systems. (C) 2012, The Society for Biotechnology, Japan. All rights reserved.

DOI： 10.1016/j.jbiosc.2012.09.018

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A novel mycovirus termed Rosellinia necatrix partitivirus 2 (RnPV2), isolated from a phytopathogenic fungus, Rosellinina necatrix strain W57, was molecularly and biologically characterized in both natural and experimental host fungi. Three double-stranded RNA (dsRNA) segments, dsRNA1, dsRNA2, and defective interfering dsRNA1 (DI-dsRNA1), whose sizes were approximately 2.0, 1.8, and 1.7 kbp, respectively, were detected in W57. While the dsRNA2 sequence, encoding the coat protein, was reported previously, dsRNA1 and DI-dsRNA1 were shown to encode competent and defective (truncated) RNA-dependent RNA polymerase, respectively. Artificial introduction of RnPV2 into an RNA silencing-defective, Dicer-like 2 knockout mutant (Delta dcl-2) of a nonnatural host, Cryphonectria parasitica (chestnut blight fungus), resulted in successful infection by the DI-dsRNA1-carrying and -free RnPV2. The DI-dsRNA1-free RnPV2 strain was characterized by a higher ratio of accumulation of the intact dsRNA1 to dsRNA2, enhanced replication and severer symptom expression, compared with the DI-carrying strain. These findings confirmed the nature of DI-dsRNA1 as a DI-RNA. Both viral strains replicated to higher levels in a Delta dcl-2 mutant than in a wild-type C. parasitica fungal strain (EP155) and induced severe symptoms in the Delta dcl-2 mutant but subtle symptoms in EP155, indicating that the host RNA silencing targets the partitivirus. No obvious phenotypic effects of infection by either virus strain were detected in the natural host fungus. These combined results represent the first example of a partitivirus with DI-RNA that alters viral symptom induction in a host-dependent manner.

DOI： 10.1128/JVI.02835-12

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Fungal viruses comprise two groups: a major group of five families with double-stranded RNA genomes and a minor group with positive-sense single-stranded (ss)RNA genomes. Although many fungal viruses have been identified, no negative-stranded (-)ssRNA mycoviruses have been reported. Here we present two lines of evidence suggesting the presence of (-)ssRNA viruses in filamentous fungi based on an exhaustive search using extant (-)ssRNA viruses as queries. This revealed (-)ssRNA virus L protein-like sequences in the genome of a phytopathogenic obligate ascomycete, Erysiphe pisi. A similar search for (-)ssRNA viruses in fungal transcriptome shotgun assembly libraries demonstrated that two independent libraries from Sclerotinia homoeocarpa, another phytopathogenic ascomycete, contained several sequences considered to correspond to the entire mononegavirus L gene and likely originating from an infecting (-)ssRNA virus. These results provide strong evidence for both ancient and extant (-)ssRNA virus infections in fungi. © 2012 Elsevier Inc.

DOI： 10.1016/j.virol.2012.10.002

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Rosellinia necatrix is a filamentous ascomycete that is pathogenic to a wide range of perennial plants worldwide. An extensive search for double-stranded RNA of a large collection of field isolates led to the detection of a variety of viruses. Since the first identification of a reovirus in this fungus in 2002, several novel viruses have been molecularly characterized that include members of at least five virus families. While some cause phenotypic alterations, many others show latent infections. Viruses attenuating the virulence of a host fungus to its plant hosts attract much attention as agents for virocontrol (biological control using viruses) of the fungus, one of which is currently being tested in experimental fields. Like the Cryphonectria parasitica/viruses, the R. necatrix/viruses have emerged as an amenable system for studying virus/host and virus/virus interactions. Several techniques have recently been developed that enhance the investigation of virus etiology, replication, and symptom induction in this mycovirus/fungal host system.

DOI： 10.1016/B978-0-12-394315-6.00007-6

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Here we report the biological and molecular attributes of a novel dsRNA virus isolated from Rosellinia necatrix, a filamentous phytopathogenic fungus. The virus, termed Rosellinia necatrix quadrivirus 1 (RnQV1), forms rigid spherical particles approximately 45 nm in diameter in infected mycelia. The particles contain 4 dsRNA segments, dsRNA1 to dsRNA4, with a size range of 4.9 to 3.7 kbp, each possessing a single large ORF. A comparison of the virus-infected and -cured isogenic fungal strains suggested that RnQV1 infection has no appreciable phenotypic effects. Phylogenetic analysis using the dsRNA3-encoded RdRp sequence revealed that RnQV1 is more distantly related to quadripartite chrysoviruses than to monopartite totiviruses, and is placed in a distinct group from other mycoviruses. No significant sequence similarities were evident between known proteins and RnQV1 structural proteins shown to be encoded by dsRNA2 or dsRNA4. These suggest that RnQV1 is a novel latent virus, belonging to a new family. (C) 2012 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.virol.2012.01.013

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Language：English   Publisher：FRONTIERS RESEARCH FOUNDATION  

The family Reoviridae is one of the largest virus families with genomes composed of 9-12 double-stranded RNA segments. It includes members infecting organisms from protists to humans. It is well known that reovirus genomes are prone to various types of genome alterations including intragenic rearrangement and reassortment under laboratory and natural conditions. Recently distinct genetic alterations were reported for members of the genus Mycoreovirus, Mycoreovirus 1 (MyRV1), and MyRV3 with 11 (S1-S11) and 12 genome segments (S1-S12), respectively. While MyRV3 S8 is lost during subculturing of infected host fungal strains, MyRV1 rearrangements undergo alterations spontaneously and inducibly. The inducible MyRV1 rearrangements are different from any other previous examples of reovirus rearrangements in their dependence on an unrelated virus factor, a multifunctional protein, p29, encoded by a distinct virus Cryphonectria parasitica hypovirus 1 (CHV1). A total of 5 MyRV1 variants with genome rearranged segments (S1-S3, S6 and S10) are generated in the background of a single viral strain in the presence of CHV1 p29 supplied either transgenically or by coinfection. MyRV1 S4 and S10 are rearranged, albeit very infrequently, in a CHV1 p29 independent fashion. A variant of MyRV1 with substantial deletions in both S4 and S10, generated through a combined reassortment and rearrangement approach, shows comparable replication levels to the wild-type MyRV1. In vivo and in vitro interactions of CHV1 p29 and MyRV1VP9 are implicated in the induction of MyRV1 rearrangements. However, the mechanism underlying p29-mediated rearrangements remains largely unknown. MyRV1 S4 rearrangements spontaneously occurred independently of CHV1 p29. In the absence of reverse genetics systems for mycoreoviruses, molecular and biological characterization of these MyRV1 and MyRV3 variants contribute to functional analyses of the protein products encoded by those rearranged segments.

DOI： 10.3389/fmicb.2012.00186

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC GENERAL MICROBIOLOGY  

Mycoreovirus 1 (MyRV1), a member of the family Reoviridae possessing a genome consisting of 11 dsRNA segments (S1-S11), infects the chestnut blight fungus and reduces its virulence (hypovirulence). Studies have previously demonstrated reproducible induction of intragenic rearrangements of MyRV1 S6 (S6L: almost full-length duplication) and S10 (S10ss: internal deletion of three-quarters of the ORF), mediated by the multifunctional protein p29 encoded by the prototype hypovirus, Cryphonectria hypovirus 1 (CHV1) strain EP713, of the family Hypoviridae with ssRNA genomes. The current study showed that CHV1 p29 also induced rearrangements of the three largest MyRV1 segments, S1, S2 and S3, which encode structural proteins. These rearranged segments involved in-frame extensions of almost two-thirds of the ORFs (S1L, S2L and S3L, respectively), which is rare for a reovirus rearrangement. MyRV1 variants carrying S1L, S2L or S3L always contained S10ss (MyRV1/S1L+S10ss2, MyRV1/S2L+S10ss2 or MyRV1/S3L+S10ss2). Levels of mRNAs for the rearranged and co-existing unaltered genome segments in fungal colonies infected with each of the MyRV1 variants appeared to be comparable to those for the corresponding normal segments in wild-type MyRV1-infected colonies, suggesting that the rearranged segments were fully competent for packaging and transcription. Protein products of the rearranged segments were detectable in fungal colonies infected with S2L MyRV1/S2L+S10ss2 and S3L MyRV1/S3L+S10ss2, whilst S1L-encoded protein remained undetectable. S1L, S2L and S3L were associated with enhancement of the aerial hyphae growth rate. This study has provided additional examples of MyRV1 intragenic rearrangements induced by p29, and suggests that normal S1, S2 and S3 are required for the symptoms caused by MyRV1.

DOI： 10.1099/vir.0.031138-0

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Non-retroviral RNA virus sequences (NRVSs) have been found in the chromosomes of vertebrates and fungi, but not plants. Here we report similarly endogenized NRVSs derived from plus-, negative-, and double-stranded RNA viruses in plant chromosomes. These sequences were found by searching public genomic sequence databases, and, importantly, most NRVSs were subsequently detected by direct molecular analyses of plant DNAs. The most widespread NRVSs were related to the coat protein (CP) genes of the family Partitiviridae which have bisegmented dsRNA genomes, and included plant- and fungus-infecting members. The CP of a novel fungal virus (Rosellinia necatrix partitivirus 2, RnPV2) had the greatest sequence similarity to Arabidopsis thaliana ILR2, which is thought to regulate the activities of the phytohormone auxin, indole-3-acetic acid (IAA). Furthermore, partitivirus CP-like sequences much more closely related to plant partitiviruses than to RnPV2 were identified in a wide range of plant species. In addition, the nucleocapsid protein genes of cytorhabdoviruses and varicosaviruses were found in species of over 9 plant families, including Brassicaceae and Solanaceae. A replicase-like sequence of a betaflexivirus was identified in the cucumber genome. The pattern of occurrence of NRVSs and the phylogenetic analyses of NRVSs and related viruses indicate that multiple independent integrations into many plant lineages may have occurred. For example, one of the NRVSs was retained in Ar. thaliana but not in Ar. lyrata or other related Camelina species, whereas another NRVS displayed the reverse pattern. Our study has shown that single- and double-stranded RNA viral sequences are widespread in plant genomes, and shows the potential of genome integrated NRVSs to contribute to resolve unclear phylogenetic relationships of plant species. © 2011 Chiba et al.

DOI： 10.1371/journal.ppat.1002146

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Rice dwarf virus (RDV), with 12 double-stranded RNA (dsRNA) genome segments (S1 to S12), replicates in and is transmitted by vector insects. The RDV-plant host-vector insect system allows us to examine the evolution, adaptation, and population genetics of a plant virus. We compared the effects of long-term maintenance of RDV on population structures in its two hosts. The maintenance of RDV in rice plants for several years resulted in gradual accumulation of nonsense mutations in S2 and S10, absence of expression of the encoded proteins, and complete loss of transmissibility. RDV maintained in cultured insect cells for 6 years retained an intact protein-encoding genome. Thus, the structural P2 protein encoded by S2 and the nonstructural Pns10 protein encoded by S10 of RDV are subject to different selective pressures in the two hosts, and mutations accumulating in the host plant are detrimental in vector insects. However, one round of propagation in insect cells or individuals purged the populations of RDV that had accumulated deleterious mutations in host plants, with exclusive survival of fully competent RDV. Our results suggest that during the course of evolution, an ancestral form of RDV, of insect virus origin, might have acquired the ability to replicate in a host plant, given its reproducible mutations in the host plant that abolish vector transmissibility and viability in nature.

DOI： 10.1128/JVI.02147-10

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Language：Japanese   Publishing type：Research paper (scientific journal)  

Viruses are widespread in all major groups of fungi. The transmission of fungal viruses occurs intracellularly during cell division, sporogenesis, and cell fusion. They apparently lack an extracellular route for infection. Recent searches of the collections of field fungal isolates have detected an increasing number of novel viruses and lead to discoveries of novel genome organizations, expression strategies and virion structures. Those findings enhanced our understanding of virus diversity and evolution. The majority of fungal viruses have dsRNA genomes packaged in spherical particles, while ssRNA mycoviruses, possessing or lacking the ability to form particles, have increasingly been reported. This review article discusses the current status of mycovirus studies and virocontrol (biocontrol) of phytopathogenic fungi using viruses that infect them and reduce their virulence. Selected examples of virocontrol-associated systems include the chestnut/chestnut blight/hypovirus and fruit trees/white root rot fungus/mycoviruses. Natural dissemination and artificial introduction of hypovirulent fungal strains efficiently contributed to virocontrol of chestnut blight in European forests. Attempts to control white root rot with hypovirulence-conferring mycoviruses are now being made in Japan.

DOI： 10.2222/jsv.60.163

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

With the global spread of the pandemic H1N1 and the ongoing pandemic potential of the H5N1 subtype, the influenza virus represents one of the most alarming viruses spreading worldwide. The influenza virus sialidase is an effective drug target, and a number of inhibitors are clinically effective against the virus (zanamivir, oseltamivir, peramivir). Here we report structural and biochemical studies of the human cytosolic sialidase Neu2 with influenza virus sialidase-targeting drugs and related compounds.

DOI： 10.1021/jm100078r

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Rearrangements of two segments, S6 and S10, of Mycoreovirus 1 (MyRV1), a member of the family Reoviridae, were previously shown to be induced at a high rate by the multifunctional protein p29 encoded by a distinct ssRNA virus, the prototype hypovirus CHV1-EP713 (Sun and Suzuki, RNA 14, 2557-2571, 2008). Here we report the occurrence of rearrangements of MyRV1 S4. albeit at a very low frequency, in the absence of CHV1 p29, resulting in internal 80-90% deletions of the open reading frame (ORF) in S4. Comparative analyses of fungal strains infected by wild-type MyRV1 and its variants carrying rearrangements of S4, S4 Plus S10 and S10 indicated that S4-encoded VP4, like VP10, is non-essential for virus replication but required for efficient vertical transmission and symptom expression caused by MyRV1. This is the first example of a reovirus variant that carries deletions of over 75% of the ORFs in two genome segments and is still replication-competent. (C) 2009 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.virol.2009.11.035

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The Arabidopsis genome encodes six members of microRNA395 (miR395) family previously determined to regulate the expression of ATP sulfurylase (APS) and the sulfate transporter SULTR2
1. However, the mRNA targets for the individual miR395 family members and the biological consequences produced by target gene regulation of each miR395 remain to be identified. In this study, a transgenic approach was employed to determine the mRNA targets for each miR395 family member as well as the role each member plays in plant growth under abiotic stress conditions. Over-expression of miR395c or miR395e retarded and accelerated, respectively, the seed germination of Arabidopsis under high salt or dehydration stress conditions. Despite a single nucleotide difference between miR395c and miR395e, the cleavage of mRNA targets,APS1,APS3, APS4 and SULTR2
1, was not same in miR395c- and miR395e-overexpressing plants. These results demonstrate that a given miRNA family containing a single nucleotide difference can guide the cleavage of various mRNA targets, thereby acting as a positive or negative regulator of seed germination under stress. © Springer-Verlag 2010.

DOI： 10.1007/s00425-010-1267-x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC MICROBIOLOGY  

White root rot, caused by the ascomycete Rosellinia necatrix, is a devastating disease worldwide, particularly in fruit trees in Japan. Here we report on the biological and molecular properties of a novel bipartite double-stranded RNA (dsRNA) virus encompassing dsRNA-1 (8,931 bp) and dsRNA-2 (7,180 bp), which was isolated from a field strain of R. necatrix, W779. Besides the strictly conserved 5&apos; (24 nt) and 3&apos; (8 nt) terminal sequences, both segments show high levels of sequence similarity in the long 5&apos; untranslated region of approximately 1.6 kbp. dsRNA-1 and -2 each possess two open reading frames (ORFs) named ORF1 to -4. Although the protein encoded by 3&apos;-proximal ORF2 on dsRNA-1 shows sequence identities of 22 to 32% with RNA-dependent RNA polymerases from members of the families Totiviridae and Chrysoviridae, the remaining three virus-encoded proteins lack sequence similarities with any reported mycovirus proteins. Phylogenetic analysis showed that the W779 virus belongs to a separate clade distinct from those of other known mycoviruses. Purified virions similar to 50 nm in diameter consisted of dsRNA-1 and -2 and a single major capsid protein of 135 kDa, which was shown by peptide mass fingerprinting to be encoded by dsRNA-1 ORF1. We developed a transfection protocol using purified virions to show that the virus was responsible for reduction of virulence and mycelial growth in several host strains. These combined results indicate that the W779 virus is a novel bipartite dsRNA virus with potential for biological control (virocontrol), named Rosellinia necatrix megabirna-virus 1 (RnMBV1), that possibly belongs to a new virus family.

DOI： 10.1128/JVI.01830-09

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：MOSBY-ELSEVIER  

Objective To assess the etiology, prognosis, and appropriate treatment of hemophagocytic lymphohistiocytosis (HLH) in neonates.
Study design We collected information on neonates in whom HLH was diagnosed between 1997 and 2007 from participating members of the Japanese Society of Pediatric Hematology.
Results HLH was diagnosed in 20 patients within 4 weeks after birth. Of the diagnostic criteria for HLH-2004, the incidence of fever was quite low in preterm infants, and hypertriglyceridemia and neutropenia were uncommon. Familial HLH (n = 6) or severe combined immunodeficiency-associated HLH (n = 1) was diagnosed in 7 patients, and 2 of them have survived. Herpes simplex virus-associated HLH was diagnosed in 6 patients, and 2 of them have survived. The overall survival rate for the 20 patients was 40%.
Conclusions HLH is rare in neonates and has a poor prognosis. Early diagnosis and immediate treatment are required when considering the possibility of herpes simplex virus-associated or familial HLH. (J Pediatr 2009; 155:235-8).

DOI： 10.1016/j.jpeds.2009.02.050

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

Cryphonectria hypovirus 1 (CHV1), associated with the picorna-like superfamily, infects the chestnut blight fungus and attenuates the virulence of the host fungus. The genomic RNA of the virus has two continuous open reading frames, A and B, separated by the pentanucleotide UAAUG. We present here evidence suggesting that ORF B is translated from genome-sized virus mRNA by a coupled termination/reinitiation mechanism mediated by the pentamer. In the coupled translation, the overlapping UAA and AUG triplets serve as the stop codon of ORF A and the initiator of ORF B, respectively. This was established by the use of a luciferase assay with a basic construct containing the ORF A sequence and the firefly luciferase gene while retaining the pentamer between the two coding sequences. The proportion of ribosomes reinitiating translation after terminating was determined to be 2.54.4 by three independent assay systems in fungal and insect cells. Use of a series of mutant constructs identified two sequence elements, the pentamer and the p40 sequence, that affect the efficiency of coupled translation and virus replication. Together, these results provide the first example of coupled translation facilitated by the pentanucleotide UAAUG in the kingdom Fungi. The mechanism by which the preceding p40-coding sequence promotes reinitiation is discussed.

DOI： 10.1093/nar/gkp224

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL PUBLISHING, INC  

BK polyomavirus (BKV) is ubiquitous among humans, usually infecting them asymptomatically during childhood. BKV persists in renal tissue of individuals and their progeny are excreted in urine, particularly in immunocompromised patients. JC virus, another human polyomavirus, has been considered to be transmitted from parents to children during prolonged cohabitation. However, BKV has been supposed to be transmitted not only within but also outside the family. In the present study, to clarify this possibility, we analyzed phylogenetically 35 BKV which were excreted in the urine by Japanese children and adults undergoing stem cell transplantation. Subtypes I, III and IV were detected in 15, two and one children and in 15, one and one adults, respectively. Among 15 subtype I isolates from children, three, four and eight belonged to subgroups Ia, Ib-1 and Ic, respectively. All the three children from whom Ia was detected were less than 9 years old. In contrast in the adults, three subtype I belonged to Ib-1 and the other 12 to Ic. These findings may reflect the recent transmission of BKV Ia strains to Japanese children.

DOI： 10.1111/j.1348-0421.2009.00129.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

The karyotypes of nine strains including three transformants of the chestnut blight fungus Cryphonectria parasitica were analyzed by pulsed-field gel electrophoresis (PFGE) and cytology using a fluorescence microscope. Cytology of the mitotic metaphase showed n = 9 for both standard strain EP155 and field strain GH2 infected by Cryphonectria hypovirus 3. Chromosomes were morphologically characterized by size, heterochromatic segment, and constriction. PFGE resolved 5 or 6 chromosomal DNA bands ranging from 3.3 Mbp to 9.7 Mbp, but accurate determination of the chromosome number was hampered by clumping of some bands. Banding profiles in PFGE were similar among the strains except for GH2, in which a chromosome translocation was detected by Southern blot analysis. By integrating the data from cytology and PFGE, the genome size of C parasitica was estimated to be ca. 50 Mbp. This is the first report of a cytological karyotype in the order Diaporthales. (C) 2009 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.fgb.2009.01.005

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：COLD SPRING HARBOR LAB PRESS, PUBLICATIONS DEPT  

Mycoreovirus 1 (MyRV1), a member of the Reoviridae family possessing a genome consisting of 11 dsRNA segments (S1-S11), and the prototype hypovirus (CHV1-EP713) of the Hypoviridae family, which is closely related to the monopartite picorna-like superfamily with a ssRNA genome, infect the chestnut blight fungus and cause virulence attenuation and distinct phenotypic alterations in the host. Here, we present evidence for reproducible induction of intragenic rearrangements of MyRV1 S6 and S10, mediated by the multifunctional protein p29 encoded by CHV1. S6 and S10 underwent an almost full-length ORF duplication (S6L) and an internal deletion of three-fourths of the ORF (S10ss). No significant influence on symptom induction in the fungal host was associated with the S6L rearrangement. In contrast, S10-encoded VP10, while nonessential for MyRV1 replication, was shown to contribute to virulence reduction and reduced growth of aerial mycelia. Furthermore, p29 was found to copurify with MyRV1 genomic RNA and bind to VP9 in vitro and in vivo, suggesting direct interactions of p29 with the MyRV1 replication machinery. This study provides the first example of a viral factor involved in RNA genome rearrangements of a different virus and shows its usefulness as a probe into the mechanism of replication and symptom expression of a heterologous virus.

DOI： 10.1261/rna.1125408

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER TOKYO  

The type virus of the family Hypoviridae, Cryphonectria hypovirus 1 strain EP713 (CHV1-EP713), infects Cryphonectria parasitica, the filamentous causal fungus of chestnut blight, and reduces its virulence. This pathosystem serves as a model to study fungus-mycovirus interactions. We previously developed a genetic screening protocol for host factors associated with symptom induction by CHV1-EP713 and its mutants. In the procedure the standard field fungal isolate EP155 was transformed by cDNA from a mild hypovirus mutant Cys(72), launching virus infection, and mutagenized by random plasmid insertion with pHygR conferring hygromycin resistance. We now report an extension of the study to characterize different mutant strains, with different phenotypes than their parental strain TCys(72)-1. TCys(72)-1 is moderately reduced in pigmentation and sporulation compared to the uninfected wild-type strain EP155. Mutants sfb1, sfb2 and k202 were characterized biologically and molecularly in comparison to the previously isolated mutant (namA) and the parental strain. These mutants harbored one (sfb1) or more copies (sfb2 and k202) of the mutagenic plasmid, pHygR. The three mutants had similar biological attributes; that is, vegetative growth rate, conidiation and virulence (assay on apples) was reduced on potato dextrose agar media, relative to the parental strain and pigmentation was the same or slightly increased. Interestingly, viral dsRNA accumulation levels were apparently unaltered in these mutants. The screening method was efficient for mining fungal mutants with unusual hypovirus symptoms. Further, characterization of the mutants provides interesting insights into symptom induction by the hypovirus.

DOI： 10.1007/s10327-008-0121-5

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The prototype hypovirus CRV1-EP713 causes virulence attenuation and severe suppression of asexual sporulation and pigmentation in its host, the chestnut blight fungus, Cryphonectria parasitica. We identified a factor associated with symptom induction in C. parasitica using a transformation of C. parasitica strain EP155 with a full-length cDNA clone from a mild mutant virus strain, Cys(72). This was accomplished by using mutagenesis of the transformant fungal strain TCys(72)-1 by random integration of plasmid pHygR, conferring hygromycin resistance. The mutant, nam4 (after nami-gata, meaning wave shaped), showed an irregular fungal morphology with reduced conidiation and pigmentation while retaining similar levels of virulence and virus accumulation relative to TCys(72)-1- or Cys (72)-infected strain EP155. However, the colony morphology of virus-cured namA (VC-namA) was indistinguishable from those of EP155 and virus-cured TCys(72)-1 [VC-TCys(72)-1]. The phenotypic difference between VC-namA and VC-TCys(72)-1 was found only when these strains infected with the wild type or certain mutant CHV1-EP713 strains but not when infected with Mycoreoviras 1. Sequence analysis of inverse-PCR-amplified genomic DNA fragments and cDNA identified the insertion site of the mutagenic plasmid in exon 8 of the nam-1 gene. NAM-1, comprising 1,257 amino acids, shows sequence similarities to counterparts from other filamentous fungi and possesses the CorA domain that is conserved in a class of Mg2+ transporters from prokaryotes and eukaryotes. Complementation assays using the wild-type and mutant alleles and targeted disruption of nam-1 showed that nam-1 with an extension of the pHygR-derived sequence contributed to the altered phenotype in the namA mutant. The molecular mechanism underlying virus-specific fungal symptom modulation in VC-namA is discussed.

DOI： 10.1128/JVI.02015-07

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC GENERAL MICROBIOLOGY  

The type member Mycoreovirus 1 (MyRV-1) of a newly described genus, Mycoreovirus, isolated from a hypovirulent strain 9B21 of the chestnut blight fungus, has a genome composed of 11 dsRNA segments (S1-S11). All of the segments have single ORFs on their capped, positive-sense strands. Infection of insect cells by baculovirus recombinants carrying full-length cDNAs of S1-S1 1 resulted in overexpression of protein products of the expected sizes, based on their deduced amino acid sequences. This expression system was utilized to identify the S3-encoded protein (VP3) as a guanylyltransferase by an autoguanylylation assay, in which only VP3 was radiolabelled with [alpha-(32) P]GTP. A series of progressive N-terminal and C-terminal deletion mutants was made to localize the autoguanylylation-active site of VP3 to aa residues 133-667. Within this region, a sequence stretch (aa 170-250) with relatively high sequence similarity to homologues of two other mycoreoviruses and two coltivinuses was identified. Site-directed mutagenesis of conserved aa residues revealed that H233, H242, Y243, F244 and F246, but not K172 or K202, play critical roles in guanylyltransferase activities. Together with broader sequence alignments of 'turreted' reoviruses, these results supported the a/vxxHx(8)Hyf/lvf motif, originally noted for orthoreovirus and aquareoviruses, as an active site for guanylyltransferases of viruses within the Orthoreovirus, Aquareovirus, Cypovirus, Oryzavirus, Fijivirus, Coltivirus and Mycoreovirus genera, as well as for the proposed Dinovernavirus genus.

DOI： 10.1099/vir.0.82318-0

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Infection of the chestnut blight fungus, Cryphonectria parasitica, by the prototypic hypovirus Cryphonectria hypovirus 1-EP713 (CHV1-EP713) or by the type member, Mycoreovirus 1-Cp9B21 (MyRV1-Cp9B21), of a novel genus (Mycoreovirus) of the family Reoviridae results in hypovirulence, but with a different spectrum of phenotypic changes. The former virus depresses pigmentation and conidiation dramatically, whilst the latter virus has little effect on these processes. This study showed that double infection by the two viruses resulted in a phenotype similar to that of CHV1-EP713 singly infected colonies, but with further decreased levels of host conidiation and vegetative growth and increased levels of MyRV1-Cp9B21 genomic dsRNA accumulation (twofold) and vertical transmission (sixfold). In contrast, CHV1-EP713 RNA accumulation was not altered by MyRV1-Cp9B21 infection. It was also found that the papain-like cysteine protease p29, encoded by CHV1-EP713 ORF A, contributes to the phenotypic alterations and transactivation of MyRV1-Cp9B21 replication and transmission. Chromosomally expressed p29 was able to increase MyRV1-Cp9B21 vertical transmission by more than twofold and genomic RNA accumulation by 80 %. Transactivation was abolished by Cys -&gt; Gly mutations at p29 residues 70 and 72 located within the previously identified symptom-determinant domain required for suppression of host pigmentation and sporulation and p29-mediated in trans enhancement of homologous Delta p29 mutant virus RNA replication. Transactivation was not altered by Ser substitutions at the p29 protease catalytic residue Cys(162). These results indicated a link between p29-mediated enhancement of heterologous virus accumulation and transmission and p29-mediated host symptom expression. The role of p29 as a suppressor of RNA silencing is discussed.

DOI： 10.1099/vir0.82213-0

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Rice dwarf virus (RDV), a member of the family Reoviridae, has a 12-segmented dsRNA genome. Seven segments, designated S1, S2, S3, S5, S7, S8, and S9, encode structural proteins, while the remainder encode nonstructural proteins. One of the nonstructural proteins, Pns4, which is encoded by S4, was characterized. Pns4 was a phosphorylatable substrate in a phosphorylation assay in vivo
it associated with large cytoplasmic fibrils and formed novel minitubules in infected cultured cells of its leafhopper insect vector, as revealed by immunofluorescence and immunoelectron microscopy. Early in infection, Pns4 was detected at the periphery of the viroplasm, and it was then observed on amorphous or fibrillar inclusions, which were identified as bundles of minitubules, at later stages of infection. Since viroplasms are believed to be the site of RDV replication, the intracellular location of Pns4 suggests that this protein might be involved in the process of assembly of the RDV virion. © Springer-Verlag 2006.

DOI： 10.1007/s00705-006-0757-4

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC MICROBIOLOGY  

Various cytopathological structures, known as inclusion bodies, are formed upon infection of cultured leafhopper cells by Rice dwarf virus, a member of the family Reoviridae. These structures include tubules of approximately 85 nm in diameter which are composed of the nonstructural viral protein Pns10 and contain viral particles. Such tubular structures were produced in heterologous non-host insect cells that expressed PnsIO of the virus. These tubules, when associated with actin-based filopodia, were able to protrude from the surface of cells and to penetrate neighboring cells. A binding assay in vitro revealed the specific binding of Pns10 to actin. Infection of clusters of cells was readily apparent 5 days after inoculation at a low multiplicity of infection with the virus, even in the presence of neutralizing antibodies. However, treatment of host cells with drugs that inhibited the elongation of actin filaments abolished the extension of PnsIO tubules from the surface of cells, with a significant simultaneous decrease in the extent of infection of neighboring cells. These results together revealed a previously undescribed aspect of the intercellular spread of Rice dwarf virus, wherein the virus exploits tubules composed of a nonstructural viral protein and actin-based filopodia to move into neighboring cells.

DOI： 10.1128/JVI.00537-06

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Cytoplasmic inclusion bodies, known as viroplasms or viral factories, are assumed to be the sites of replication of members of the family Reoviridae. Immunocytochemical and biochemical analyses were carried out to characterize the poorly understood viroplasms of the phytoreovirus Rice dwarf virus (RDV). Within 6 h of inoculation of cells, viroplasms, namely discrete cytoplasmic inclusions, were formed that contained the non-structural proteins Pns6, Pns11 and Pns12 of RDV, which appeared to be the constituents of the inclusions. Formation of similar inclusions in non-host insect cells upon expression of Pns12 in a baculovirus system and the association of molecules of Pns12 in vitro suggested that the inclusions observed in RDV-infected cells were composed basically of Pns12. Core proteins P1, P3, P5 and P7 and core virus particles were identified in the interior region of the inclusions. In contrast accumulation of the outer capsid proteins P2, P8 and P9 and of intact virus particles was evident in the peripheral regions of the inclusions. These observations suggest that core particles were constructed inside the inclusions, whereas outer capsid proteins were assembled at the periphery of the inclusions. Viral inclusions were shown to be the sites of viral RNA synthesis by labelling infected cells with 5-bromouridine 5′-tri phosphate. The number of viroplasms decreased with time post-inoculation as their sizes increased, suggesting that inclusions might fuse with one another during the virus-propagation process. Our results are consistent with a model, proposed for vertebrate reoviruses, in which viroplasms play a pivotal role in virus assembly. © 2006 SGM.

DOI： 10.1099/vir.0.81425-0

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC GENERAL MICROBIOLOGY  

Mycoreovirus 1 (MYRV-1) is the type species of the newly described genus Mycoreovirus of the large virus family Reoviridae. The virus was isolated from a hypovirulent strain (9B21) of the chestnut blight fungus, Cryphonectria parasitica. A previous study showed that double-shelled particles introduced to fungal spheroplasts resulted in stably infected colonies. Of the 11 double-stranded RNA genomic segments (S1-S11), the three largest (S1-S3) were sequenced previously and shown to have moderate levels of similarity to the homologous segments of mammal-pathogenic coltiviruses (Eyach virus and Colorado tick fever virus) and another fungus-infecting reovirus, Mycoreovirus 3 of Rosellinia necatrix strain W370 (MYRV-3/RnW370). The sequences of the remaining segments (S4-S11) are reported here. All of the segments have single ORFs on their positive strands and the terminal sequences 5'-GAUCA----GCAGUCA-3' are conserved among currently and previously sequenced segments. Oligo-cap analysis showed that the positive strands of the genomic segments are capped, whereas the negative strands are not. Similarities among the four evolutionarily related viruses include low or moderate levels of amino acid sequence identity (14(.)7-34(.)2%) and isoelectric points among equivalent polypeptides, e.g. proteins encoded by segments S4 and S5 of the four viruses. Phylogenetic analysis indicated that MYRV-1/Cp9B21 is related more closely to MYRV-3/RnW370 than to the coltiviruses. An interesting dissimilarity is found in codon-choice pattern among the four viruses, i.e. MYRV-1/Cp9B21 segments have a lower frequency of [XYG + XYC] than corresponding segments of the other viruses, suggesting a possible adjustment of virus codon usage to their host environments.

DOI： 10.1099/vir.0.80293-0

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RNA viruses of filamentous fungi fall into two broad categories, those that contain double-stranded RNA (dsRNA) genomes in rigid particles and those that are more closely related to positive-sense, single-stranded RNA viruses with dsRNA replicative intermediates found within lipid vesicles. Effective infectivity systems have been described for the latter, using RNA transcripts, but not for the former. We report the characterization of a reovirus from Cryphonectria parasitica, the filamentous fungus that causes chestnut blight disease. The virus substantially reduces the virulence of the fungus and results in dramatically altered colony morphology, as well as changes in other associated fungal traits, relative to the virus-free isogenic strain. Virus particles from infected mycelium contained 11 segments of dsRNA and showed characteristics typical of the family Reoviridae. Sequences of the largest three segments revealed that the virus is closely related to the Coltivirus genus of animal pathogens, which includes the human pathogen Colorado tick fever virus. The introduction of purified virus particles into protoplasts from virus-free isolates of the fungus resulted in a newly infected mycelium with the same morphology and virus composition as the original virus-infected isolate. This represents the completion of Koch's postulates for a true dsRNA virus from a filamentous fungus and the description of a definitive fungal member of the family Reoviridae.

DOI： 10.1128/JVI.78.2.892-898.2004

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DOI： 10.1016/S0065-3527(04)63007-7

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC MICROBIOLOGY  

The prototypic hypovirus CHV1-EP713 attenuates virulence (hypovirulence) and alters several physiological processes of the chestnut blight fungus Cryphonectria parasitica. The papain-like protease, p29, and the highly basic protein, p40, derived, respectively, from the N-terminal and C-terminal portions of the CHV1-EP713-encoded open reading frame (ORF) A polyprotein, p69, both contribute to reduced pigmentation and sporulation. The p29 coding region was shown to suppress pigmentation and asexual sporulation in the absence of virus infection in transformed C. parasitica, whereas transformants containing the p40-coding domain exhibited a wild-type, untransformed phenotype. Deletion of either p29 or p40 from the viral genome also results in reduced accumulation of viral RNA. We now show that p29, but not p40, functions in trans to enhance genomic RNA accumulation and vertical transmission of p29 deletion mutant viruses. The frequency of virus transmission through conidia was found to decrease with reduced accumulation of viral genomic double-stranded RNA (dsRNA): from almost 100% for wild-type virus to similar to50% for Deltap29, and 10 to 20% for Deltap69. When expressed from a chromosomally integrated cDNA copy, p29 elevated viral dsRNA accumulation and transmission for Deltap29 mutant virus to the level shown by wild-type virus. Increased viral RNA accumulation levels were also observed for a Deltap69 mutant lacking almost the entire ORF A sequence. Such enhancements were not detected in transgenic fungal colonies expressing p40. Mutation of p29 residues Cys(70) or Cys(72), strictly conserved in hypovirus p29 and potyvirus HC-Pro, resulted in the loss of both p29-mediated suppressive activity in virus-free transgenic C. parasitica and in trans enhancement of RNA accumulation and transmission, suggesting a linkage between these functional activities. These results suggest that p29 is an enhancer of viral dsRNA accumulation and vertical virus transmission through asexual spores.

DOI： 10.1128/JVI.77.21.11697-11707.2003

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER PHYTOPATHOLOGICAL SOC  

Biolistic bombardment was used to successfully transform three phytopathogenic fungal species with an infectious cDNA clone of the prototypic hypovirus, CHV1-EP713, a genetic element responsible for the virulence attenuation (hypovirulence) of the chestnut blight fungus, Cryphonectria parasitica. The fungal species included two strains each of C. parasitica and Valsa ceratosperma, as well as one strain of Phomopsis G-type (teleomorph Diaporthe Nitschke); all are members of the order Diaporthales but classified into three different genera. A subset of transformants for each of the fungal species contained CHV1EP713 dsRNA derived from chromosomally integrated viral cDNA. As has been reported for CHV1-EP713 infection of the natural host C. parasitica, biolistic introduction of CHV1-EP713 into the new fungal hosts V. ceratosperma and Phomopsis G-type resulted in altered colony morphology and, more importantly, reduced virulence. These results suggest a potential for hypoviruses as biological control agents in plant-infecting fungal pathogens other than the chestnut blight fungus and closely related species. In addition, the particle delivery technique offers a convenient means of transmitting hypoviruses to potential host fungi that provides new avenues for fundamental mycovirus research and may have practical applications for conferring hypovirulence directly on infected plants in the field.

DOI： 10.1094/MPMI.2002.15.8.780

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The papain-like protease p29, derived from the N-terminal portion of the hypovirus CHV1-EP713-encoded open reading frame (ORF) A polyprotein, p69, was previously shown to contribute to reduced pigmentation and sporulation by the infected host, the chestnut blight fungus Cryphonectria parasitica, while being dispensable for virus replication and attenuation of fungal virulence (hypovirulence). We now report that deletion of the C-terminal portion of p69, which encodes the highly basic protein p40, resulted in replication-competent mutant viruses that were, however, significantly reduced in RNA accumulation. While the Deltap40 mutants retained the ability to confer hypovirulence, Deltap40-infected fungal strains produced more asexual spores than strains infected with either wild-type CHV1-EP713 or a Deltap29 mutant virus. As observed for Deltap29-infected colonies, pigment production was significantly increased in Deltap40-infected fungal strains relative to that in CHV1-EP713-infected strains. Virus-mediated suppression of laccase production was not affected by p40 deletion. A gain-of-function analysis was employed to map the p40 symptom determinant to the N-terminal domain, encompassing p69 amino acid residues Thr(288) to Arg(312). Evidence that the gain of function was due to the encoded protein rather than the corresponding RNA sequence element was provided by introducing frameshift mutations on either side of the activity determinant domain. Moreover, restoration of symptoms correlated with increased accumulation of viral RNA. These results suggest that p40 indirectly contributes to virus-mediated suppression of fungal pigmentation and conidiation by providing an accessory function in hypovirus RNA amplification. A possible role for p40 in facilitating ORF B expression and the relationship between hypovirus RNA accumulation and symptom expression are discussed.

DOI： 10.1128/JVI.76.15.7747-7759.2002

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© 2002 by CRC Press LLC. The concept of using a parasite of a parasite for disease control is appealing from both an intellectual and an ecological perspective. The phenomenon of transmissible hypovirulence, in which virulence of the chestnut blight fungus, Cryphonectria parasitica, is attenuated by double-stranded RNA viruses in the family Hypoviridae, is often cited as a classic example of this approach to biological disease control. Progress in the development of an infectious cDNA-based reverse genetics system for hypoviruses has provided the means for engineering these viral agents for both fundamental and practical applications, e.g., as a tool for probing signal transduction processes underlying fungal pathogenesis and for enhanced biocontrol potential. This chapter describes the basic techniques used for manipulating hypovirus genomes and provides specific examples of how they are being applied to identify virus-encoded determinants responsible for altering fungal host phenotype, to gain insights into dispensable and essential elements of viral replication, and to fine tune the interaction between a fungal pathogen and its plant host.

DOI： 10.1201/9781420039122

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC MICROBIOLOGY  

We have investigated whether hypoviruses, viral agents responsible for virulence attenuation (hypovirulence) of the chestnut blight fungus Cryphonectria parasitica, could serve as gene expression vectors, The infectious cDNA clone of the prototypic hypovirus CHV1-EP713 was modified to generate 20 different vector candidates. Although transient expression was achieved for a subset of vectors that contained the green fluorescent protein gene from Aequorea victoria, long-term expression (past day 8) was not observed for any vector construct. analysis of viral RNAs recovered from transfected fungal colonies revealed that the foreign genes were readily deleted from the replicating virus, although small portions of foreign sequences were retained by some vectors after months of replication, However, the results of vector viability and progeny characterization provided unexpected new insights into essential and dispensable elements of hypovirus replication. The N-terminal portion (codons 1 to 24) of the 5'-proximal open reading frame (ORF), ORF ii was found to be required for virus replication, while the remaining 598 codons of this ORF were completely dispensable. Substantial alterations were tolerated in the pentanucleotide UAAUG that contains the ORF A termination codon and the overlapping putative initiation codon of the second of the two hypovirus ORFs, ORF B, Replication competence was maintained following either a frameshift mutation that caused a two-codon extension of ORF A or a modification that produced a single-ORF genomic organization, These results are discussed in terms of determinants of hypovirus replication, the potential utility of hypoviruses as gene expression vectors, and possible mechanisms by which hypoviruses recognize and delete foreign sequences.

DOI： 10.1128/JVI.74.16.7568-7577.2000

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC  

Nucleotide sequences of RNAs 1 and 2 of a Japanese strain of soil-borne wheat mosaic virus (sBWMV), the type species of the genus Furovirus, and sorghum chlorotic spot virus (SCSV) were determined from cloned cDNA. The relationship among the Japanese and US strains of SBWMV, SCSV, oat golden stripe virus (OGSV), and recently proposed Chinese wheat mosaic and European wheat mosaic viruses (CWMV and EWMV) were examined at the nucleotide and amino acid levels. Pairwise comparisons of genome-encoded proteins among the six viruses showed that the US strains of SBWMV and CWMV were the most closely related pair in RNA 1 and the Japanese strains of SBWMV and EWMV were most closely related in RNA 2. SCSV was most distantly related to the other five viruses. Phylogenetic analysis indicated that there may have been an ancient reassortment between RNAs 1 and 2 of the four wheat-infecting viruses and OGSV, while SCSV was shown to have separated from the rest before the other five viruses diverged. The fact that CWMV and EWMV have almost identical biological properties as well as the sequence similarities to the two strains of SBWMV suggests that they be regarded as strains of SBWMV, considering that SBWMV consists of generically diverged strains. OGSV and SCSV are distinct in biological properties in addition to genetic divergence in the genus Furovirus. (C) 2000 Academic Press.

DOI： 10.1006/viro.2000.0251

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Hypovirus infection of the chestnut blight fungus Cryphonectria parasitica results in a spectrum of phenotypic changes that can include alterations in colony morphology and significant reductions in pigmentation, asexual sporulation, and virulence (hypovirulence). Deletion of 88% [Phe(25) to Pro(243)] of the virus-encoded papain-like protease, p29, in the context of an infectious cDNA clone of the prototypic hypovirus CHV1-EP713 (recombinant virus Ap29) partially relieved virus-mediated suppression of pigmentation and sporulation without altering the level of hypovirulence. We now report mapping of the p29 symptom determinant domain to a region extending from Phe(25) through Gln(73) by a gain-of-function analysis following progressive repair of the Δp29 deletion mutant. This domain was previously shown to share sequence similarity [including conserved cysteine residues Cys(38), Cys(48), Cys(70), and Cys(72)] with the N-terminal portion of the potyvirus-encoded helper component-proteinase (HC-Pro), a multifunctional protein implicated in aphid-mediated transmission, genome amplification, polyprotein processing, long-distance movement, and suppression of posttranscriptional silencing. Substitution of a glycine residue for either Cys(38) or Cys(48) resulted in no qualitative or quantitative changes in virus-mediated symptoms. Unexpectedly, mutation of Cys(70) resulted in a very severe phenotype that included significantly reduced mycelial growth and profoundly altered colony morphology. In contrast, substitution for Cys(72) resulted in a less severe symptom phenotype approaching that observed for Δp29. The finding that p29-mediated symptom expression is influenced by two cysteine residues that are conserved in the potyvirus-encoded HC-Pro raises the possibility that these related viral-papain-like proteases function in their respective fungal and plant hosts by impacting ancestrally related regulatory pathways.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER-VERLAG WIEN  

In vivo and in vitro phosphorylation and intracellular location of rice dwarf phytoreovirus Pns12, which is encoded by one of the twelve dsRNA genome segments, S12, and comprises 312 amino acids, was investigated. When [P-32]phosphoric acid was incorporated into RDV-infected leafhopper cultured cells, labelled Pns12 was immunoprecipitated from the cells by a monospecific anti-Pns12 polyclonal antibody. Recombinant Pns12 was purified from Spodoptera frugiperda cells infected with AcRS12, a baculovirus recombinant carrying a full-length cDNA of RDV S12. Purified Pns12 was also demonstrated to be phosphorylated in vitro by a kinase activity present in host (rice, barley, wheat, leafhopper) cells and non-host (tobacco, spinach, white clover, S. frugiperda, mosquito, mammals) cells as well. Immunocytochemical studies showed Pns12 accumulated in the cytoplasm of infected cells, and frequently localized in a slightly electron-dense patch. These results demonstrated that RDV Pns12 was a cytoplasmic nonstructural phosphoprotein.

DOI： 10.1007/s007050050593

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A transmission-defective (TD) isolate of rice dwarf phytoreovirus lacked the ability to infect cells when derived from the virus-free insect vector Nephotettix cincticeps. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified virus showed that among six structural proteins, the P2 outer capsid protein (encoded by genome segment S2) was absent from the TD isolate, whereas all six proteins were present in the transmission-competent (TC) isolate. P2 was not detected on immunoblots of rice plants infected with the TD isolate. Genome segment S2 and its transcript were detected in both TD and TC isolates. Sequence analysis of the S2 segment of the TD isolate revealed the presence of a termination codon due to a point mutation in the open reading frame, which might explain the absence of P2 in the TD isolate. These results demonstrate that the P2 protein is one of the factors essential for infection by the virus of vector cells and, thus, influences transmissibility by vector insects.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC MICROBIOLOGY  

Genomic segment S12 of rice dwarf virus and segment S9 of wound tumor virus, both members of the genus Phytoreovirus, have small out-of-phase overlapping open reading frames (ORFs). Western blot (immunoblot) analysis revealed that rice dwarf virus S12 mRNA specified translation products from the large ORF and two overlapping small ORFs both in rice plant hosts and in Spodoptera frugiperda insect cells. These results provide the first example of a tricistronic mRNA for a segmented double-stranded RNA virus. Similarly, wound tumor virus S9 mRNA was found to direct the synthesis of protein products from both the large ORF and small out-of-frame ORF in S. frugiperda cells. Results of site-specific and deletion mutagenesis studies were consistent with a leaky scanning translation mechanism for the synthesis of the small ORFs.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC JNL-COMP SUBSCRIPTIONS  

Rice dwarf phytoreovirus (RDV) mRNA synthesized from purified virion has a cap structure, m(7)GpppAm-, which suggests the presence of guanylyltransferase activity in the virion. We attempted to identify the enzyme involved in the cap formation by using a nucleoside triphosphate binding assay. Incubation of virion with [alpha-P-32]GTP resulted in labeling of an 89-kDa protein that had not previously been identified in purified virus preparations. Interestingly this protein also covalently bound UTP and ATP, which is not a property of the known guanylyltransferases. RDV particles catalyzed GTP-PPi, dGTP-PPi, ATP-PPi, and UTP-PPi exchange reactions. In SDS-polyacrylamide gel electrophoresis, the 89-kDa protein comigrated with the SS-coded protein, P5, which had been expressed by a baculovirus vector. Moreover, the labeled 89-kDa protein was precipitated by an antiserum against this recombinant RDV P5. Careful reinvestigation of purified virus particles by SDS-polyacrylamide gel electrophoresis and Western blotting analyses showed that they contained a small amount of P5 (&lt;0.5% of the total protein) within the core. These results may suggest that the minor core protein of RDV, which is coded by S5, is a candidate guanylyltransferase, although the biological significance of its ATP and UTP binding activities remains largely unknown. (C) 1996 Academic Press, Inc.

DOI： 10.1006/viro.1996.0273

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Language：Chinese   Publishing type：Research paper (scientific journal)  

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We have isolated a low temperature-induced maize gene, mlip15, via cross hydridization using rice lip19. The longest cDNA isolated comprised 1179 bp and coded for a 135 amino acid bZIP (basic region/leucine zipper) protein. The gene showed 61.4% and 68.9% identity with the rice gene at the DNA and amino acid sequence levels, respectively, and is distinct from other maize genes that code for bZIP proteins. The level of mlip15 transcript was positively regulated by low temperature in the same way as the lip19 transcript. The levels of the transcript were also strongly increased by salt stress and exogenous abscisic acid, and slightly increased by anaerobiosis, but were not affected by heat shock and drought. The mLIP15 protein and truncated derivatives, produced in rabbit reticulocyte lysates or in an Escherichia coli expression system, were able to bind to a fragment of the wheat histone H3 gene promoter. This binding was diminished by addition of a molar excess of the hexamer sequence 5'-ACGTCA-3' found in the promoter and of the G-box-like sequence, but not by the addition of the ocs sequence or a mutated hexamer sequence. The factor bound to a promoter region of the maize Adh1 gene, expression of which is also induced by low temperature. These results lead to the conclusion that mlip15 is a strong candidate for a low temperature-induced transcription factor in maize.

DOI： 10.1007/BF02423445

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Language：Japanese   Publisher：The Phytopathological Society of Japan (PSJ)  

DOI： 10.3186/jjphytopath.61.176

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DOI： 10.1006/smvy.1995.0011

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：KARGER  

The complete nucleotide sequence of rice dwarf phytoreovirus genome segment 2 (S2) was determined to be 3,512 nucleotides long with one open reading frame initiating at nucleotide 15 and terminating at nucleotide 3363. The encoded polypeptide was predicted to have 1,116 residues with a relative molecular weight of 123 kD. Comparison of S2 of two isolates showed they had identical lengths and 97 and 98.3% nucleotide and amino acid sequence identities, respectively. A search of the Swiss-Prot data base (R 22.0) failed to fmd any proteins with significant homology to the S2-encoded protein. Determination of the nucleotide sequence of the S2 has completed the sequence determination of the genome of rice dwarf virus. Homology searches made for proteins encoded by each of the genomic segments showed that the polypeptide encoded by S11 has similarity to histone H1 protein and VP6 of blue tongue virus, indicating it might possess nucleic acid binding properties.

DOI： 10.1159/000150348

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Peptides encoded by truncated (S1 and S2) or full-length (S3 to S11) cDNAs of 11 of the 12 rice dwarf phytoreovirus (RDV) genome segments were expressed in a baculovirus vector system. Antibodies raised against each of the expressed peptides were used as probes to detect the authentic proteins encoded by the RDV open reading frame. Polypeptides identified as gene products of S1 to S11 in both RDV-infected rice leaf and leafhopper (Nephotettix cincticeps) homogenates were the P1 minor core (170 kDa), P2 (130 kDa), P3 major core (110 kDa), Pns4 nonstructural (83 kDa), P5 (89 kDa), Pns6 nonstructural (56 kDa), P7 minor core (58 kDa), P8 outercapsid (43 kDa), Pns9 nonstructural (49 kDa), Pns10 nonstructural (35 kDa), and Pns11a nonstructural (23 kDa) proteins. These molecular masses were in accord with those obtained from previous in vitro translation analysis. The locations of P2 and P5 remain to be determined although both of these are assumed to be outer layer proteins. Quantitative detection showed that accumulation (per gram of total proteins) of the virus-coded proteins in rice leaves is much greater (more than 15 times) than that in leafhoppers and that the content of the individual proteins varies within a sample from rice or leafhopper and also varies among different samples. © 1994 Academic Press. All rights reserved.

DOI： 10.1006/viro.1994.1320

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER VERLAG  

We have isolated from rice suspension cells three non-sequence-related cDNAs the expression of which is markedly induced by low, non-freezing temperature. Here we further characterize one of the cDNA clones, lip19. Expression of lip19 is positively regulated by low temperature, but not affected by high (40-degrees-C) temperature. Sequencing and primer extension analyses showed that lip19 has a long (552 bp) 5' non-coding sequence followed by a single open reading frame specifying a protein of 148 amino acids. The deduced amino acid sequence of the protein, Lip19, shows at its amino-terminus a conserved basic region followed by a ''leucine-zipper'' domain. The reported sequence most similar to Lip19 is maize OCSBF-1, which is a bZip-type DNA binding protein. The possibility is suggested that Lip19 is a transcriptional factor that is positively controlled by low temperature.

DOI： 10.1007/BF00276876

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We have determined the complete nucleotide sequence of the largest segment S1 of rice dwarf phytoreovirus (RDV), a member of the family Reoviridae. S1 is 4423 nucleotides long with a segment-specific inverted repeat located adjacent to the conserved termini (5′GGCAAA- - -UGAU3′). A major open reading frame (bases 36 to 4367) on the S1 plus strand, which is preceded by a minicistron (bases 6 to 29), encodes the polypeptide (P1) consisting of 1444 amino acids with a Mr of 164, 142. The sense-strand transcript derived from the full-length S1 cDNA, the minicistron of which was abolished, directed the synthesis of a polypeptide of 170 kDa in addition to smaller polypeptides in wheat germ extracts, and the 170-kDa product comigrated with the minor core protein in SDS-polyacrylamide gel. Thus, P1 is assumed to be localized in the viral core particle. The consensus sequence element conserved in RNA-dependent RNA polymerase is observed in the P1 amino acid sequence predicted from the nucleotide sequence. Based on the dendrogram established from the sequence alignment around the polymerase module region, and sequence identity within the alignment, P1 of plant-infecting RDV was evolutionarily compared with VP1, λ3, and VP1 of three other animal-infecting members of the family, rota-, reo-, and bluetongue viruses. Consequently, RDV S1 was shown to be more closely related to the rotavirus gene segment 1, in terms of molecular evolution, than the animal-infecting members are to one another. © 1992.

DOI： 10.1016/0042-6822(92)91210-L

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Sequence analysis revealed that rice dwarf phytoreovirus segment S12 is 1066 nucleotides long with a small out-of-phase, overlapping open reading frame (ORF) as well as a major ORF. The large ORF (positions 42 to 980) encodes 312 amino acids, while the small one (bases 313 to 591) encodes 92 amino acids with an additional in-frame AUG codon (positions 337-339) 24 nucleotides downstream from the first one. Transcripts from a full-length cDNA directed the in vitro synthesis of three polypeptides of 33 (considered to be translated from the long ORF), 8, and 7 kDa. Alteration of each of the two ATG codons on the small ORF demonstrated their involvement in the generation of the 8- and 7-kDa polypeptides. Although it is still unknown whether these proteins are expressed in vivo, the small ORF is shown to be conserved in S9s of two other members of the genus Phytoreovirus, rice gall dwarf virus and wound tumor virus, suggesting its common, important function. © 1992.

DOI： 10.1016/0042-6822(92)90279-X

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPANESE SOC PLANT PHYSIOLOGISTS  

We have isolated cDNAs that correspond to three distinct low-temperature-induced mRNAs from rice cells using an effective, simple subtraction method. The corresponding genes were designated lip (gene encoding low-temperature-induced protein) -5, -9 and -19. The expression of these genes was slightly stimulated (lip5 and lip19) or unaffected (lip9) by abscisic acid, unlike the expression of the rab16A-D genes which are readily induced by either abscisic acid or high osmotic stress. Time-course analysis revealed that while the lip5 gene was induced 3 h after temperature down-shift, and the lip9 and lip19 genes were induced 6 h after such a shift.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC GENERAL MICROBIOLOGY  

The complete nucleotide sequence of rice dwarf phytoreovirus (RDV) genome segment S11 was determined. S11 is 1067 nucleotides long. There is an inverted repeat of 10 bp adjacent to the conserved 5'-terminal hexanucleotide (5' GGUAAA 3') and 3'-terminal tetranucleotide (5' UAGU 3') sequences. A single large open reading frame found in the plus strand of S11 begins with the first AUG codon (bases 6 to 8) and extends for 567 bases. Evolutionary relatedness between RDV S11 and wound tumour phytoreovirus S12 based on amino acid sequence similarity (25.8%) was found. In addition to the first AUG triplet, RDV S11 possesses a second in-phase AUG triplet (positions 30 to 32) nearby, which conforms to the Kozak consensus sequence. Two forms of the protein were identified by using an in vitro transcription and translation system in which a tailored full-length cDNA was the initial template. The abolition of the first AUG codon by site-directed mutagenesis resulted in disappearance of the larger translation product. These results strongly suggest that the two products are translated from the first and second AUG codons. Whether the two proteins are expressed in vivo is at present unclear.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER VERLAG  

The genes coding for d-ribulose-1,5-bisphosphate carboxylase (RuBPCase) from an iron-oxidizing bacterium, Thiobacillus ferrooxidans, were cloned into an Escherichia coli plasmid, pUC18. The recombinant plasmid, termed pTR11, contained a 4.0-kb PstI fragment including the entire coding regions for both large and small subunits of RuBPCase. Escherichia coli carrying pTR11 did not show any CO2-fixing activity. However, a derivative plasmid with an appropriate deletion, which was placed under the control of a tac promoter, conferred ribulose bisphosphate-dependent CO2-fixing activity on the host cell. Analysis of gel-filtration chromatography of the RuBPCase synthesized in E. coli revealed that it had a hexadecameric form like the native enzyme of T. ferrooxidans. © 1991 Springer-Verlag New York Inc.

DOI： 10.1007/BF02106210

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The 46K outer capsid protein encoded by RNA segment S8 and the 42K polypeptide, previously thought to be the segment S9-encoded structural protein, were isolated from a rice dwarf phytoreovirus purified preparation, and then analysed by peptide mapping and electroblot-ELlSA. Staphylococcus aureus V8 protease peptide mapping patterns of the 42K and 46K proteins were similar. Two monoclonal antibodies (MAbs), obtained after immunization with virus particles dissociated by 0.1% SDS, were each specific for both the 42K and 46K proteins. Furthermore, the MAbs bound common peptide fragments which were generated by digestion of the 42K and 46K proteins with V8 protease or proteinase K. These results strongly suggest that the 42K protein is not a gene product of S9 but a product overlapping with the 46K outer capsid protein. Whether the two proteins are functionally distinct remains to be determined.

DOI： 10.1099/0022-1317-72-9-2239

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Language：English   Publishing type：Research paper (scientific journal)  

The complete nucleotide sequence of rice dwarf phytoreovirus (RDV) genome segment S11 was determined. S11 is 1067 nucleotides long. There is an inverted repeat of 10 bp adjacent to the conserved 5'terminal hexanucleotide (5' GGUAAA 3') and 3'terminal tetranucleotide (5' UAGU 3') sequences. A single large open reading frame found in the plus strand of S11 begins with the first AUG codon (bases 6 to 8) and extends for 567 bases. Evolutionary relatedness between RDV S11 and wound tumour phytoreovirus S12 based on amino acid sequence similarity (25-8%) was found. In addition to the first AUG triplet, RDV S11 possesses a second in-phase AUG triplet (positions 30 to 32) nearby, which conforms to the Kozak consensus sequence. Two forms of the protein were identified by using an in vitro transcription and translation system in which a tailored full-length cDNA was the initial template. The abolition of the first AUG codon by site-directed mutagenesis resulted in disappearance of the larger translation product. These results strongly suggest that the two products are translated from the first and second AUG codons. Whether the two proteins are expressed in vivo is at present unclear.

DOI： 10.1099/0022-1317-72-9-2233

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS UNITED KINGDOM  

DOI： 10.1093/nar/18.4.1071

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：KARGER  

Cylindrical inclusion proteins (CIPs), amorphous inclusion proteins (AIPs) and virus particles were partially purified from Cucurbita maxima leaves infected with zucchini yellow mosaic virus (ZYMV), watermelon mosaic virus 2 (WMV2), and the watermelon mosaic virus 1 strain of papaya ringspot virus (PRSV-W). Antisera to these individual antigens, except AIPs of ZYMV and WMV2, were prepared and used to determine with electroblot-ELISA the serological differentiation indices (SDIs) between homologous and heterologous antigens. The average SDIs between ZYMV and WMV2 CIPs, between ZYMV and PRSV-W CIPs, and between WMV2 and PRSV-W CIPs were 1.5, 3.5, and 2.5, respectively. The SDIs between ZYMV and PRSV-W AIPs and between WMV2 and PRSV-W AIPs were 5 and 3, respectively. The average SDIs between ZYMV and WMV2 capsid proteins (CPs), between ZYMV and PRSV-W CPs, and between WMV2 and PRSV-W CPs were 3, 7, and 8, respectively. These data suggest that the serological relationship between ZYMV and WMV2 is much closer than that between PRSV-W and ZYMV or between PRSV-W and WMV2 and that antigenic determinants of CIP and AIP were conserved more than those of CP among the three potyviruses.

DOI： 10.1159/000150133

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The complete nucleotide sequences of genome segments S4, S5, and S6 of rice dwarf phytoreovirus (RDV) were determined. S4 and S5 consist of 2468 and 2570 base pairs, respectively, S5 thus being larger in size than S4, contrary to the situation suggested by their relative migration in a polyacrylamide gel. S6 is 1699 nucleotides long. The individual segments have segment-specific inverted repeats adjacent to the conserved terminal sequences (5′GGUAAA-UGAU3′ for S4, 5′GGCAAA-UGAU3′ for S5 and S6). S4, S5, and S6 each have single long open reading frames encoding 727, 801, and 509 amino acids, respectively. A low level of amino acid sequence homology was observed between RDV S4 and wound tumor virus (WTV) S4 (22.4%), and between RDV S6 and WTV S6 (20.2%). On the other hand, RDV S5 and WTV S5 show 52.0% amino acid sequence similarity, indicating that S5 is much more conserved than any other segments of RDV and WTV reported so far. Further comparative analyses indicate that the RDV segment shows a greater frequency of usage of codons XYG and XYC, and much less frequent usage of codon XYA than the equivalent WTV segment, this codon preference bias being more conspicuous than expected from the base contents. © 1990.

DOI： 10.1016/0042-6822(90)90313-G

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The primary structure of rice dwarf phytoreovirus (RDV) genome segment S3 was determined. RDV S3 consists of 3195 nucleotides. A 14-bp segment-specific inverted repeat is located immediately adjacent to the conserved terminal sequence (5′GGCAAA---UGAU3′). A single long open reading frame encoding for 1019 amino acids with an Mr of 114,289 is also identified. In order to investigate the localization of the predicted polypeptide, we determined the amino acid sequence of the 26-kDa peptide fragment obtained from the structural core protein digested by Staphylococcus aureus V8 protease. The sequence of the fragment was found in the translational product presumed from the nucleotide sequence of RDV S3, indicating that RDV S3 encodes the major structural core protein of 114 kDa. © 1990.

DOI： 10.1016/0042-6822(90)90314-H

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Language：English   Publisher：OXFORD UNIV PRESS UNITED KINGDOM  

DOI： 10.1093/nar/17.21.8858

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC GENERAL MICROBIOLOGY  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：The Phytopathological Society of Japan (PSJ)  

We examined the effects of concentration methods, extraction buffers, clarification reagents, and resuspension buffers on virus yield and purity by using SDS-polyacrylamide gel electrophoresis (SDS-PAGE), so as to establish optimal purification procedures of zucchini yellow mosaic virus (ZYMV), watermelon mosaic virus 2 (WMV2) and the watermelon mosaic virus 1 strain of papaya ringspot virus (PRSV-W). We then investigated the physicochemical properties of the three viruses. SDS-PAGE proved very useful to monitor virus purification in a small scale. The three viruses were purified with similar yields of 5-10 mg per 100 g of infected pumpkin leaves by the respective optimal methods. The procedures basically involved clarification with Triton X-100, differential centrifugation using a 20% sucrose pad, and 20-50% sucrose density gradient centrifugation. From WMV2 and PRSV-W purified preparations, capsid protein (CP) of 34,000 (34K) was predominantly detected in addition to minor degradation products of 27K to 33K, respectively. A breakdown product of 28K was, however, as detectabel as intact CP of 33K from ZYMV purified preparation. The RNAs of WMV2 and PRSV-W, which were a little larger than ZYMV RNA, were indistinguishable in size as evaluated with 1.7% agarose gel electrophoresis in denaturing conditions.

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Other Link： http://agriknowledge.affrc.go.jp/RN/2010450878

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Total pages：315p   Language：Japanese

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Total pages：4 v.   Language：English

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Authorship：Corresponding author  

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Publishing type：Book review, literature introduction, etc.  

DOI： 10.1007/s10327-017-0740-9

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Language：Japanese   Publisher：東京家政大学生活科学研究所  

東京家政大学生活科学研究所研究報告は、本研究所の本年度の活動成果を取りまとめたものです。本研究報告の内容の一部は、別途学会誌等に発表されることがありますのでご了承ください。

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Language：Japanese   Publisher：東京家政大学生活科学研究所  

東京家政大学生活科学研究所研究報告は、本研究所の本年度の活動成果を取りまとめたものです。本研究報告の内容の一部は、別途学会誌等に発表されることがありますのでご了承ください。

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Language：English   Publisher：Elsevier Ltd  

DOI： 10.1016/B978-0-12-382219-2.00492-0

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Language：Japanese   Publisher：The Phytopathological Society of Japan (PSJ)  

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Language：Japanese   Publisher：Japan Society for Bioscience, Biotechnology, and Agrochemistry  

DOI： 10.1271/kagakutoseibutsu1962.41.335

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