Cellular anatomy of the mouse primary motor cortex.

An essential step toward understanding brain function is to establish a structural framework with cellular resolution on which multi-scale datasets spanning molecules, cells, circuits and systems can be integrated and interpreted1. Here, as part of the collaborative Brain Initiative Cell Census Network (BICCN), we derive a comprehensive cell type-based anatomical description of one exemplar brain structure, the mouse primary motor cortex, upper limb area (MOp-ul). Using genetic and viral labelling, barcoded anatomy resolved by sequencing, single-neuron reconstruction, whole-brain imaging and cloud-based neuroinformatics tools, we delineated the MOp-ul in 3D and refined its sublaminar organization. We defined around two dozen projection neuron types in the MOp-ul and derived an input-output wiring diagram, which will facilitate future analyses of motor control circuitry across molecular, cellular and system levels. This work provides a roadmap towards a comprehensive cellular-resolution description of mammalian brain architecture.

Argonaute-independent, Dicer-dependent antiviral defense against RNA viruses.

Antiviral RNA interference (RNAi) is conserved from yeasts to mammals. Dicer recognizes and cleaves virus-derived double-stranded RNA (dsRNA) and/or structured single-stranded RNA (ssRNA) into small-interfering RNAs, which guide effector Argonaute to homologous viral RNAs for digestion and inhibit virus replication. Thus, Argonaute is believed to be essential for antiviral RNAi. Here, we show Argonaute-independent, Dicer-dependent antiviral defense against dsRNA viruses using Cryphonectria parasitica (chestnut blight fungus), which is a model filamentous ascomycetous fungus and hosts a variety of viruses. The fungus has two dicer-like genes (dcl1 and dcl2) and four argonaute-like genes (agl1 to agl4). We prepared a suite of single to quadruple agl knockout mutants with or without dcl disruption. We tested these mutants for antiviral activities against diverse dsRNA viruses and ssRNA viruses. Although both DCL2 and AGL2 worked as antiviral players against some RNA viruses, DCL2 without argonaute was sufficient to block the replication of other RNA viruses. Overall, these results indicate the existence of a Dicer-alone defense and different degrees of susceptibility to it among RNA viruses. We discuss what determines the great difference in susceptibility to the Dicer-only defense.

Replication of single viruses across the kingdoms, Fungi, Plantae, and Animalia.

It is extremely rare that a single virus crosses host barriers across multiple kingdoms. Based on phylogenetic and paleovirological analyses, it has previously been hypothesized that single members of the family Partitiviridae could cross multiple kingdoms. Partitiviridae accommodates members characterized by their simple bisegmented double-stranded RNA genome; asymptomatic infections of host organisms; the absence of an extracellular route for entry in nature; and collectively broad host range. Herein, we show the replicability of single fungal partitiviruses in three kingdoms of host organisms: Fungi, Plantae, and Animalia. Betapartitiviruses of the phytopathogenic fungusRosellinia necatrix could replicate in protoplasts of the carrot (Daucus carota), Nicotiana benthamiana and Nicotiana tabacum, in some cases reaching a level detectable by agarose gel electrophoresis. Moreover, betapartitiviruses showed more robust replication than the tested alphapartitiviruses. One of the fungal betapartitiviruses, RnPV18, could persistently and stably infect carrot plants regenerated from virion-transfected protoplasts. Both alpha- and betapartitiviruses, although with different host preference, could replicate in two insect cell lines derived from the fall armyworm Spodoptera frugiperda and the fruit fly Drosophila melanogaster. Our results indicate the replicability of single partitiviruses in members of three kingdoms and provide insights into virus adaptation, host jumping, and evolution.

Identification of a negative-strand RNA virus with natural plant and fungal hosts.

The presence of viruses that spread to both plant and fungal populations in nature has posed intriguingly scientific question. We found a negative-strand RNA virus related to members of the family Phenuiviridae, named Valsa mali negative-strand RNA virus 1 (VmNSRV1), which induced strong hypovirulence and was prevalent in a population of the phytopathogenic fungus of apple Valsa canker (Valsa mali) infecting apple orchards in the Shaanxi Province of China. Intriguingly, VmNSRV1 encodes a protein with a viral cell-to-cell movement function in plant tissue. Mechanical leaf inoculation showed that VmNSRV1 could systemically infect plants. Moreover, VmNSRV1 was detected in 24 out of 139 apple trees tested in orchards in Shaanxi Province. Fungal inoculation experiments showed that VmNSRV1 could be bidirectionally transmitted between apple plants and V. mali, and VmNSRV1 infection in plants reduced the development of fungal lesions on leaves. Additionally, the nucleocapsid protein encoded by VmNSRV1 is associated with and rearranged lipid droplets in both fungal and plant cells. VmNSRV1 represents a virus that has adapted and spread to both plant and fungal hosts and shuttles between these two organisms in nature (phyto-mycovirus) and is potential to be utilized for the biocontrol method against plant fungal diseases. This finding presents further insights into the virus evolution and adaptation encompassing both plant and fungal hosts.

The intriguing phenomenon of cross-kingdom infections of plant and insect viruses to fungi: Can other animal viruses also cross-infect fungi?

Fungi are highly widespread and commonly colonize multicellular organisms that live in natural environments. Notably, studies on viruses infecting plant-associated fungi have revealed the interesting phenomenon of the cross-kingdom transmission of viruses and viroids from plants to fungi. This implies that fungi, in addition to absorbing water, nutrients, and other molecules from the host, can acquire intracellular parasites that reside in the host. These findings further suggest that fungi can serve as suitable alternative hosts for certain plant viruses and viroids. Given the frequent coinfection of fungi and viruses in humans/animals, the question of whether fungi can also acquire animal viruses and serve as their hosts is very intriguing. In fact, the transmission of viruses from insects to fungi has been observed. Furthermore, the common release of animal viruses into the extracellular space (viral shedding) could potentially facilitate their acquisition by fungi. Investigations of the cross-infection of animal viruses in fungi may provide new insights into the epidemiology of viral diseases in humans and animals.

Decoding the RNA virome of the tree parasite Armillaria provides new insights into the viral community of soil-borne fungi.

The globally distributed basidiomycete genus Armillaria includes wood decomposers that can act as opportunistic parasites, causing deadly root rot on woody plants. To test whether RNA viruses are involved in this opportunistic behaviour, a large isolate collection of five Armillaria species collected over 40 years in Switzerland from trees, dead wood and soil was analysed. De novo assembly of RNA-Seq data revealed 21 viruses, 14 of which belong to putative new species. Two dsRNA viruses and an unclassified Tymovirales are formally described for the first time for Armillaria. One mitovirus occurred with a high prevalence of 71.1%, while all other viruses were much less prevalent (0.6%-16.9%). About half of all viruses were found only in one fungal species, others occurred in 2-6 fungal species. Co-infections of 2-7 viruses per isolate were not uncommon (34.9%), and most viruses persisted circulating within fungal populations for decades. Some viruses were related to viruses associated with other Armillaria species, supporting the hypothesis that virus transmission can occur between different fungal species. Although no specific correlation between viruses and the fungal trophic strategy was found, this study opens new insights into viral diversity hidden in the soil microbiome.

Identification of a novel member of the genus Laulavirus (family Phenuiviridae) from the entomopathogenic ascomycete fungus Cordyceps javanica.

The virus family Phenuiviridae (order Hareavirales, comprising segmented negative-sense single stranded RNA viruses) has highly diverse members that are known to infect animals, plants, protozoans, and fungi. In this study, we identified a novel phenuivirus infecting a strain of the entomopathogenic fungus Cordyceps javanica isolated from a small brown plant hopper (Laodelphax striatellus), and this virus was tentatively named "Cordyceps javanica negative-strand RNA virus 1" (CjNRSV1). The CjNRSV1 genome consists of three negative-sense single stranded RNA segments (RNA1-3) with lengths of 7252, 2401, and 1117 nt, respectively. The 3'- and 5'-terminal regions of the RNA1, 2, and 3 segments have identical sequences, and the termini of the RNA segments are complementary to each other, reflecting a common characteristic of viruses in the order Hareavirales. RNA1 encodes a large protein (∼274 kDa) containing a conserved domain for the bunyavirus RNA-dependent RNA polymerase (RdRP) superfamily, with 57-80% identity to the RdRP encoded by phenuiviruses in the genus Laulavirus. RNA2 encodes a protein (∼79 kDa) showing sequence similarity (47-63% identity) to the movement protein (MP, a plant viral cell-to-cell movement protein)-like protein (MP-L) encoded by RNA2 of laulaviruses. RNA3 encodes a protein (∼28 kDa) with a conserved domain of the phenuivirid nucleocapsid protein superfamily. Phylogenetic analysis using the RdRPs of various phenuiviruses and other unclassified phenuiviruses showed CjNRSV1 to be grouped with established members of the genus Laulavirus. Our results suggest that CjNRSV1 is a novel fungus-infecting member of the genus Laulavirus in the family Phenuiviridae.

Possible Biological Control of Ash Dieback Using the Mycoparasite Hymenoscyphus Fraxineus Mitovirus 2.

Invasive fungal diseases represent a major threat to forest ecosystems worldwide. As the application of fungicides is often unfeasible and not a sustainable solution, only a few other control options are available, including biological control. In this context, the use of parasitic mycoviruses as biocontrol agents of fungal pathogens has recently gained particular attention. Since the 1990s, the Asian fungus Hymenoscyphus fraxineus has been causing lethal ash dieback across Europe. In the present study, we investigated the biocontrol potential of the mitovirus Hymenoscyphus fraxineus mitovirus 2 (HfMV2) previously identified in Japanese populations of the pathogen. HfMV2 could be successfully introduced via co-culturing into 16 of 105 HfMV2-free isolates. Infection with HfMV2 had contrasting effects on fungal growth in vitro, from cryptic to detrimental or beneficial. Virus-infected H. fraxineus isolates whose growth was reduced by HfMV2 showed overall a lower virulence on ash (Fraxinus excelsior) saplings as compared with their isogenic HfMV2-free lines. The results suggest that mycoviruses exist in the native populations of H. fraxineus in Asia that have the potential for biological control of ash dieback in Europe. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

ICTV Virus Taxonomy Profile: Hadakaviridae 2023.

The family Hadakaviridae, including the genus Hadakavirus, accommodates capsidless viruses with a 10- or 11-segmented positive-sense (+) RNA genome. Currently known hosts are ascomycetous filamentous fungi. Although phylogenetically related to polymycovirids with a segmented double-stranded RNA genome and certain encapsidated picorna-like viruses, hadakavirids are distinct in their lack of a capsid ('hadaka' means naked in Japanese) and their consequent inability to be pelleted by conventional ultracentrifugation; they show ribonuclease susceptibility in host tissue homogenates. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Hadakaviridae, which is available at ictv.global/report/hadakaviridae.

ICTV Virus Taxonomy Profile: Yadokariviridae 2023.

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.

Identification of a novel dicistro-like virus associated with the roots of tomato plants.

Viruses belonging to the family Dicistroviridae have a monopartite positive-sense single-stranded RNA genome and infect a variety of arthropods. Using high-throughput sequencing, we detected a novel dicistro-like virus, tentatively named "tomato root-associated dicistro-like virus" (TRaDLV), in the roots of tomato plants showing yellow mosaic symptoms on the leaves. The diseased tomato plants were coinfected with multiple plant viruses, and TRaDLV was present in the roots but not in the leaves. The genome of TRaDLV is 8726 nucleotides in length, excluding the poly(A) tail, and contains two open reading frames (ORFs) separated by an intergenic region (IGR). The TRaDLV genome showed characteristics similar to those of dicistroviruses, including the presence of a 3C-like protease domain, repeated amino acid sequences representing multiple copies of viral genome-linked protein (VPg)-like sequences in the ORF1 polyprotein, and a series of stem-loop structures resembling an internal ribosome entry site in the IGR. Phylogenetic analysis revealed that TRaDLV clustered with unclassified dicistro-like viruses from invertebrates or identified in samples of plant-derived material. These findings indicate the existence of a novel dicistro-like virus that may associate with plant roots or a root-inhabiting organism.

Facilitative and synergistic interactions between fungal and plant viruses.

Plants and fungi are closely associated through parasitic or symbiotic relationships in which bidirectional exchanges of cellular contents occur. Recently, a plant virus was shown to be transmitted from a plant to a fungus, but it is unknown whether fungal viruses can also cross host barriers and spread to plants. In this study, we investigated the infectivity of Cryphonectria hypovirus 1 (CHV1, family Hypoviridae), a capsidless, positive-sense (+), single-stranded RNA (ssRNA) fungal virus in a model plant, Nicotiana tabacum CHV1 replicated in mechanically inoculated leaves but did not spread systemically, but coinoculation with an unrelated plant (+)ssRNA virus, tobacco mosaic virus (TMV, family Virgaviridae), or other plant RNA viruses, enabled CHV1 to systemically infect the plant. Likewise, CHV1 systemically infected transgenic plants expressing the TMV movement protein, and coinfection with TMV further enhanced CHV1 accumulation in these plants. Conversely, CHV1 infection increased TMV accumulation when TMV was introduced into a plant pathogenic fungus, Fusarium graminearum In the in planta F. graminearum inoculation experiment, we demonstrated that TMV infection of either the plant or the fungus enabled the horizontal transfer of CHV1 from the fungus to the plant, whereas CHV1 infection enhanced fungal acquisition of TMV. Our results demonstrate two-way facilitative interactions between the plant and fungal viruses that promote cross-kingdom virus infections and suggest the presence of plant-fungal-mediated routes for dissemination of fungal and plant viruses in nature.

ICTV Virus Taxonomy Profile: Mymonaviridae 2022.

Typical members of the family Mymonaviridae produce filamentous, enveloped virions containing a single molecule of linear, negative-sense RNA of about about 10 kb, but some may not produce any virions. The family includes several genera, some with multiple species. Mymonavirids usually infect filamentous fungi, but a few have been identified associated with insects, oomycetes or plants. At least one virus, Sclerotinia sclerotiorum negative-stranded RNA virus 1, induces hypovirulence in its fungal host. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Mymonaviridae, which is available at ictv.global/report/mymonaviridae.

ICTV Virus Taxonomy Profile: Rhabdoviridae 2022.

The family Rhabdoviridae comprises viruses with negative-sense (-) RNA genomes of 10-16 kb. Virions are typically enveloped with bullet-shaped or bacilliform morphology but can also be non-enveloped filaments. Rhabdoviruses infect plants or animals, including mammals, birds, reptiles, amphibians or fish, as well as arthropods, which serve as single hosts or act as biological vectors for transmission to animals or plants. Rhabdoviruses include important pathogens of humans, livestock, fish or agricultural crops. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Rhabdoviridae, which is available at ictv.global/report/rhabdoviridae.

A novel deltapartitivirus from red clover.

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.

Identification of novel totiviruses from the ascomycetous fungus Geotrichum candidum.

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.

A novel victorivirus from the phytopathogenic fungus Neofusicoccum parvum.

Neofusicoccum parvum is an important plant-pathogenic ascomycetous fungus that causes trunk diseases in a variety of plants. A limited number of reports on mycoviruses from this fungus are available. Here, we report the characterization of a novel victorivirus, Neofusicoccum parvum victorivirus 3 (NpVV3). An agarose gel dsRNA profile of a Pakistani strain of N. parvum, NFN, showed a band of ~5 kbp that was not detectable in Japanese strains of N. parvum. Taking a high-throughput and Sanger sequencing approach, the complete genome sequence of NpVV3 was determined to be 5226 bp in length with two open reading frames (ORF1 and ORF2) that encode a capsid protein (CP) and an RNA-dependent RNA polymerase (RdRP). The RdRP appears to be translated by a stop/restart mechanism facilitated by the junction sequence AUGucUGA, as is found in some other victoriviruses. BLASTp searches showed that NpVV3 CP and RdRP share the highest amino acid sequence identity (80.5% and 72.4%, respectively) with the corresponding proteins of NpVV1 isolated from a French strain of N. parvum. However, NpVV3 was found to be different from NpVV1 in its terminal sequences and the stop/restart facilitator sequence. NpVV3 particles ~35 nm in diameter were partially purified and used to infect an antiviral-RNA-silencing-deficient strain (∆dcl2) of an experimental ascomycetous fungal host, Cryphonectria parasitica. NpVV3 showed symptomless infection in the new host strain.

Dicer functions transcriptionally and posttranscriptionally in a multilayer antiviral defense.

In antiviral RNA interference (RNAi), Dicer plays a primary role in processing double-stranded RNA (dsRNA) molecules into small-interfering RNAs (siRNAs) that guide Argonaute effectors to posttranscriptional suppression of target viral genes. Here, we show a distinct role for Dicer in the siRNA-independent transcriptional induction of certain host genes upon viral infection in a filamentous fungus. Previous studies have shown that the two key players, dicer-like 2 (dcl2) and argonaute-like 2 (agl2), of antiviral RNAi in a phytopathogenic ascomycete, Cryphonectria parasitica, are highly transcriptionally induced upon infection with certain RNA mycoviruses, including the positive-stranded RNA hypovirus mutant lacking the RNAi suppressor (Cryphonectria hypovirus 1-Δp69, CHV1-Δp69). This induction is regulated by the Spt-Ada-Gcn5 acetyltransferase (SAGA) complex, a well-known transcriptional coactivator. The present study shows that diverse host genes, in addition to dcl2 and agl2, were up-regulated more than 10-fold by SAGA upon infection with CHV1-Δp69. Interestingly, DCL2, but not AGL2, was essential for SAGA-mediated global gene up-regulation. Moreover, deletion of certain virus-induced genes enhanced a CHV1-Δp69 symptom (growth rate) but not its accumulation. Constitutive, modest levels of dcl2 expression drastically reduced viral siRNA accumulation but were sufficient for full-scale up-regulation of host genes, suggesting that high induction of dcl2 and siRNA production are not essential for the transcriptional up-regulation function of DCL2. These data clearly demonstrate the dual functionality of DCL2: as a dsRNA-specific nuclease in posttranscriptional antiviral RNA silencing and as a key player in SAGA-mediated host gene induction, which independently represses viral replication and alleviates virus-induced symptom expression.

Symptomatic plant viroid infections in phytopathogenic fungi.

Viroids are pathogenic agents that have a small, circular noncoding RNA genome. They have been found only in plant species; therefore, their infectivity and pathogenicity in other organisms remain largely unexplored. In this study, we investigate whether plant viroids can replicate and induce symptoms in filamentous fungi. Seven plant viroids representing viroid groups that replicate in either the nucleus or chloroplast of plant cells were inoculated to three plant pathogenic fungi, Cryphonectria parasitica, Valsa mali, and Fusarium graminearum By transfection of fungal spheroplasts with viroid RNA transcripts, each of the three, hop stunt viroid (HSVd), iresine 1 viroid, and avocado sunblotch viroid, can stably replicate in at least one of those fungi. The viroids are horizontally transmitted through hyphal anastomosis and vertically through conidia. HSVd infection severely debilitates the growth of V. mali but not that of the other two fungi, while in F. graminearum and C. parasitica, with deletion of dicer-like genes, the primary components of the RNA-silencing pathway, HSVd accumulation increases. We further demonstrate that HSVd can be bidirectionally transferred between F. graminearum and plants during infection. The viroids also efficiently infect fungi and induce disease symptoms when the viroid RNAs are exogenously applied to the fungal mycelia. These findings enhance our understanding of viroid replication, host range, and pathogenicity, and of their potential spread to other organisms in nature.

Coat protein of Chinese wheat mosaic virus upregulates and interacts with cytosolic glyceraldehyde-3-phosphate dehydrogenase, a negative regulator of plant autophagy, to promote virus infection.

Autophagy is an intracellular degradation mechanism involved in antiviral defense, but the strategies employed by plant viruses to counteract autophagy-related defense remain unknown for the majority of the viruses. Herein, we describe how the Chinese wheat mosaic virus (CWMV, genus Furovirus) interferes with autophagy and enhances its infection in Nicotiana benthamiana. Yeast two-hybrid screening and in vivo/in vitro assays revealed that the 19 kDa coat protein (CP19K) of CWMV interacts with cytosolic glyceraldehyde-3-phosphate dehydrogenases (GAPCs), negative regulators of autophagy, which bind autophagy-related protein 3 (ATG3), a key factor in autophagy. CP19K also directly interacts with ATG3, possibly leading to the formation of a CP19K-GAPC-ATG3 complex. CP19K-GAPC interaction appeared to intensify CP19K-ATG3 binding. Moreover, CP19K expression upregulated GAPC gene transcripts and reduced autophagic activities. Accordingly, the silencing of GAPC genes in transgenic N. benthamiana reduced CWMV accumulation, whereas CP19K overexpression enhanced it. Overall, our results suggest that CWMV CP19K interferes with autophagy through the promotion and utilization of the GAPC role as a negative regulator of autophagy.