Nyamanini Virus Nucleoprotein and Phosphoprotein Organize Viral Inclusion Bodies That Associate with Host Biomolecular Condensates in the Nucleus.

Many mononegaviruses form inclusion bodies (IBs) in infected cells. However, little is known about nuclear IBs formed by mononegaviruses, since only a few lineages of animal-derived mononegaviruses replicate in the nucleus. In this study, we characterized the IBs formed by Nyamanini virus (NYMV), a unique tick-borne mononegavirus undergoing replication in the nucleus. We discovered that NYMV forms IBs, consisting of condensates and puncta of various sizes and morphologies, in the host nucleus. Likewise, we found that the expressions of NYMV nucleoprotein (N) and phosphoprotein (P) alone induce the formation of condensates and puncta in the nucleus, respectively, even though their morphologies are somewhat different from the IBs observed in the actual NYMV-infected cells. In addition, IB-like structures can be reconstructed by co-expressions of NYMV N and P, and localization analyses using a series of truncated mutants of P revealed that the C-terminal 27 amino acid residues of P are important for recruiting P to the condensates formed by N. Furthermore, we found that nuclear speckles, cellular biomolecular condensates, are reorganized and recruited to the IB-like structures formed by the co-expressions of N and P, as well as IBs formed in NYMV-infected cells. These features are unique among mononegaviruses, and our study has contributed to elucidating the replication mechanisms of nuclear-replicating mononegaviruses and the virus-host interactions.

2022 taxonomic update of phylum Negarnaviricota (Riboviria: Orthornavirae), including the large orders Bunyavirales and Mononegavirales.

In March 2022, following the annual International Committee on Taxonomy of Viruses (ICTV) ratification vote on newly proposed taxa, the phylum Negarnaviricota was amended and emended. The phylum was expanded by two new families (bunyaviral Discoviridae and Tulasviridae), 41 new genera, and 98 new species. Three hundred forty-nine species were renamed and/or moved. The accidentally misspelled names of seven species were corrected. This article presents the updated taxonomy of Negarnaviricota as now accepted by the ICTV.

ICTV Virus Taxonomy Profile: Nyamiviridae 2021.

Nyamiviridae is a family of viruses in the order Mononegavirales, with unsegmented (except for members of the genus Tapwovirus), negative-sense RNA genomes of 10-13 kb. Nyamviruses have a genome organisation and content similar to that of other mononegaviruses. Nyamiviridae includes several genera that form monophyletic clades on phylogenetic analysis of the RNA polymerase. Nyamiviruses have been found associated with diverse invertebrates as well as land- and seabirds. Members of the genera Nyavirus and Socyvirus produce enveloped, spherical virions. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Nyamiviridae, which is available at ictv.global/report/nyamiviridae.

Structures of the Mononegavirales Polymerases.

Mononegavirales, known as nonsegmented negative-sense (NNS) RNA viruses, are a class of pathogenic and sometimes deadly viruses that include rabies virus (RABV), human respiratory syncytial virus (HRSV), and Ebola virus (EBOV). Unfortunately, no effective vaccines and antiviral therapeutics against many Mononegavirales are currently available. Viral polymerases have been attractive and major antiviral therapeutic targets. Therefore, Mononegavirales polymerases have been extensively investigated for their structures and functions. Mononegavirales mimic RNA synthesis of their eukaryotic counterparts by utilizing multifunctional RNA polymerases to replicate entire viral genomes and transcribe viral mRNAs from individual viral genes as well as synthesize 5' methylated cap and 3' poly(A) tail of the transcribed viral mRNAs. The catalytic subunit large protein (L) and cofactor phosphoprotein (P) constitute the Mononegavirales polymerases. In this review, we discuss the shared and unique features of RNA synthesis, the monomeric multifunctional enzyme L, and the oligomeric multimodular adapter P of Mononegavirales We outline the structural analyses of the Mononegavirales polymerases since the first structure of the vesicular stomatitis virus (VSV) L protein determined in 2015 and highlight multiple high-resolution cryo-electron microscopy (cryo-EM) structures of the polymerases of Mononegavirales, namely, VSV, RABV, HRSV, human metapneumovirus (HMPV), and human parainfluenza virus (HPIV), that have been reported in recent months (2019 to 2020). We compare the structures of those polymerases grouped by virus family, illustrate the similarities and differences among those polymerases, and reveal the potential RNA synthesis mechanisms and models of highly conserved Mononegavirales We conclude by the discussion of remaining questions, evolutionary perspectives, and future directions.

2021 Taxonomic update of phylum Negarnaviricota (Riboviria: Orthornavirae), including the large orders Bunyavirales and Mononegavirales.

In March 2021, following the annual International Committee on Taxonomy of Viruses (ICTV) ratification vote on newly proposed taxa, the phylum Negarnaviricota was amended and emended. The phylum was expanded by four families (Aliusviridae, Crepuscuviridae, Myriaviridae, and Natareviridae), three subfamilies (Alpharhabdovirinae, Betarhabdovirinae, and Gammarhabdovirinae), 42 genera, and 200 species. Thirty-nine species were renamed and/or moved and seven species were abolished. This article presents the updated taxonomy of Negarnaviricota as now accepted by the ICTV.

Pneumoviral Phosphoprotein, a Multidomain Adaptor-Like Protein of Apparent Low Structural Complexity and High Conformational Versatility.

Mononegavirales phosphoproteins (P) are essential co-factors of the viral polymerase by serving as a linchpin between the catalytic subunit and the ribonucleoprotein template. They have highly diverged, but their overall architecture is conserved. They are multidomain proteins, which all possess an oligomerization domain that separates N- and C-terminal domains. Large intrinsically disordered regions constitute their hallmark. Here, we exemplify their structural features and interaction potential, based on the Pneumoviridae P proteins. These P proteins are rather small, and their oligomerization domain is the only part with a defined 3D structure, owing to a quaternary arrangement. All other parts are either flexible or form short-lived secondary structure elements that transiently associate with the rest of the protein. Pneumoviridae P proteins interact with several viral and cellular proteins that are essential for viral transcription and replication. The combination of intrinsic disorder and tetrameric organization enables them to structurally adapt to different partners and to act as adaptor-like platforms to bring the latter close in space. Transient structures are stabilized in complex with protein partners. This class of proteins gives an insight into the structural versatility of non-globular intrinsically disordered protein domains.

2020 taxonomic update for phylum Negarnaviricota (Riboviria: Orthornavirae), including the large orders Bunyavirales and Mononegavirales.

In March 2020, following the annual International Committee on Taxonomy of Viruses (ICTV) ratification vote on newly proposed taxa, the phylum Negarnaviricota was amended and emended. At the genus rank, 20 new genera were added, two were deleted, one was moved, and three were renamed. At the species rank, 160 species were added, four were deleted, ten were moved and renamed, and 30 species were renamed. This article presents the updated taxonomy of Negarnaviricota as now accepted by the ICTV.

Addicted to sugar: roles of glycans in the order Mononegavirales.

Glycosylation is a biologically important protein modification process by which a carbohydrate chain is enzymatically added to a protein at a specific amino acid residue. This process plays roles in many cellular functions, including intracellular trafficking, cell-cell signaling, protein folding and receptor binding. While glycosylation is a common host cell process, it is utilized by many pathogens as well. Protein glycosylation is widely employed by viruses for both host invasion and evasion of host immune responses. Thus better understanding of viral glycosylation functions has potential applications for improved antiviral therapeutic and vaccine development. Here, we summarize our current knowledge on the broad biological functions of glycans for the Mononegavirales, an order of enveloped negative-sense single-stranded RNA viruses of high medical importance that includes Ebola, rabies, measles and Nipah viruses. We discuss glycobiological findings by genera in alphabetical order within each of eight Mononegavirales families, namely, the bornaviruses, filoviruses, mymonaviruses, nyamiviruses, paramyxoviruses, pneumoviruses, rhabdoviruses and sunviruses.

Aedes Anphevirus: an Insect-Specific Virus Distributed Worldwide in Aedes aegypti Mosquitoes That Has Complex Interplays with Wolbachia and Dengue Virus Infection in Cells.

Insect-specific viruses (ISVs) of the yellow fever mosquito Aedes aegypti have been demonstrated to modulate transmission of arboviruses such as dengue virus (DENV) and West Nile virus by the mosquito. The diversity and composition of the virome of A. aegypti, however, remains poorly understood. In this study, we characterized Aedes anphevirus (AeAV), a negative-sense RNA virus from the order Mononegavirales AeAV identified from Aedes cell lines was infectious to both A. aegypti and Aedes albopictus cells but not to three mammalian cell lines. To understand the incidence and genetic diversity of AeAV, we assembled 17 coding-complete and two partial genomes of AeAV from available transcriptome sequencing (RNA-Seq) data. AeAV appears to transmit vertically and be present in laboratory colonies, wild-caught mosquitoes, and cell lines worldwide. Phylogenetic analysis of AeAV strains indicates that as the A. aegypti mosquito has expanded into the Americas and Asia-Pacific, AeAV has evolved into monophyletic African, American, and Asia-Pacific lineages. The endosymbiotic bacterium Wolbachia pipientis restricts positive-sense RNA viruses in A. aegypti Reanalysis of a small RNA library of A. aegypti cells coinfected with AeAV and Wolbachia produces an abundant RNA interference (RNAi) response consistent with persistent virus replication. We found Wolbachia enhances replication of AeAV compared to a tetracycline-cleared cell line, and AeAV modestly reduces DENV replication in vitro The results from our study improve understanding of the diversity and evolution of the virome of A. aegypti and adds to previous evidence that shows Wolbachia does not restrict a range of negative-strand RNA viruses.IMPORTANCE The mosquito Aedes aegypti transmits a number of arthropod-borne viruses (arboviruses), such as dengue virus and Zika virus. Mosquitoes also harbor insect-specific viruses that may affect replication of pathogenic arboviruses in their body. Currently, however, there are only a few insect-specific viruses described from A. aegypti in the literature. Here, we characterize a novel negative-strand virus, AeAV. Meta-analysis of A. aegypti samples showed that it is present in A. aegypti mosquitoes worldwide and is vertically transmitted. Wolbachia-transinfected mosquitoes are currently being used in biocontrol, as they effectively block transmission of several positive-sense RNA viruses in mosquitoes. Our results demonstrate that Wolbachia enhances the replication of AeAV and modestly reduces dengue virus replication in a cell line model. This study expands our understanding of the virome in A. aegypti as well as providing insight into the complexity of the Wolbachia virus restriction phenotype.

Taxonomy of the order Mononegavirales: update 2019.

In February 2019, following the annual taxon ratification vote, the order Mononegavirales was amended by the addition of four new subfamilies and 12 new genera and the creation of 28 novel species. This article presents the updated taxonomy of the order Mononegavirales as now accepted by the International Committee on Taxonomy of Viruses (ICTV).

Taxonomy of the order Mononegavirales: second update 2018.

In October 2018, the order Mononegavirales was amended by the establishment of three new families and three new genera, abolishment of two genera, and creation of 28 novel species. This article presents the updated taxonomy of the order Mononegavirales as now accepted by the International Committee on Taxonomy of Viruses (ICTV).

Taxonomy of the order Mononegavirales: update 2018.

In 2018, the order Mononegavirales was expanded by inclusion of 1 new genus and 12 novel species. This article presents the updated taxonomy of the order Mononegavirales as now accepted by the International Committee on Taxonomy of Viruses (ICTV) and summarizes additional taxonomic proposals that may affect the order in the near future.

Taxonomy of the order Mononegavirales: update 2017.

In 2017, the order Mononegavirales was expanded by the inclusion of a total of 69 novel species. Five new rhabdovirus genera and one new nyamivirus genus were established to harbor 41 of these species, whereas the remaining new species were assigned to already established genera. Furthermore, non-Latinized binomial species names replaced all paramyxovirus and pneumovirus species names, thereby accomplishing application of binomial species names throughout the entire order. This article presents the updated taxonomy of the order Mononegavirales as now accepted by the International Committee on Taxonomy of Viruses (ICTV).

Invertebrate RNA virus diversity from a taxonomic point of view.

Invertebrates are hosts to diverse RNA viruses that have all possible types of encapsidated genomes (positive, negative and ambisense single stranded RNA genomes, or a double stranded RNA genome). These viruses also differ markedly in virion morphology and genome structure. Invertebrate RNA viruses are present in three out of four currently recognized orders of RNA viruses: Mononegavirales, Nidovirales, and Picornavirales, and 10 out of 37 RNA virus families that have yet to be assigned to an order. This mini-review describes general properties of the taxonomic groups, which include invertebrate RNA viruses on the basis of their current classification by the International Committee on Taxonomy of Viruses (ICTV).

[Molecular basis for negative-strand RNA virus entry and neutralization by antibodies].

Mononegaviruses are non-segmented negative-strand RNA viruses, and include measles, mumps, Marburg, and Ebola viruses. Measles virus and mumps virus, members of the family Paramyxoviridae, are immunotropic and neurotropic, respectively. Marburg virus and Ebola virus, members of the family Filoviridae, cause highly lethal hemorrhagic fever. In this paper, I summarize the recent structural and functional studies on the viral glycoproteins (GPs) of these viruses, which have shed light on virus entry and the humoral response. The structural and functional analyses of the interaction between viral GPs and receptors/antibodies also illuminate directions toward therapeutics against the viruses.

Taxonomy of the order Mononegavirales: update 2016.

In 2016, the order Mononegavirales was emended through the addition of two new families (Mymonaviridae and Sunviridae), the elevation of the paramyxoviral subfamily Pneumovirinae to family status (Pneumoviridae), the addition of five free-floating genera (Anphevirus, Arlivirus, Chengtivirus, Crustavirus, and Wastrivirus), and several other changes at the genus and species levels. This article presents the updated taxonomy of the order Mononegavirales as now accepted by the International Committee on Taxonomy of Viruses (ICTV).

Virome analysis of Amblyomma americanum, Dermacentor variabilis, and Ixodes scapularis ticks reveals novel highly divergent vertebrate and invertebrate viruses.

UNLABELLED: A wide range of bacterial pathogens have been identified in ticks, yet the diversity of viruses in ticks is largely unexplored. In the United States, Amblyomma americanum, Dermacentor variabilis, and Ixodes scapularis are among the principal tick species associated with pathogen transmission. We used high-throughput sequencing to characterize the viromes of these tick species and identified the presence of Powassan virus and eight novel viruses. These included the most divergent nairovirus described to date, two new clades of tick-borne phleboviruses, a mononegavirus, and viruses with similarity to plant and insect viruses. Our analysis revealed that ticks are reservoirs for a wide range of viruses and suggests that discovery and characterization of tick-borne viruses will have implications for viral taxonomy and may provide insight into tick-transmitted diseases. IMPORTANCE: Ticks are implicated as vectors of a wide array of human and animal pathogens. To better understand the extent of tick-borne diseases, it is crucial to uncover the full range of microbial agents associated with ticks. Our current knowledge of the diversity of tick-associated viruses is limited, in part due to the lack of investigation of tick viromes. In this study, we examined the viromes of three tick species from the United States. We found that ticks are hosts to highly divergent viruses across several taxa, including ones previously associated with human disease. Our data underscore the diversity of tick-associated viruses and provide the foundation for further studies into viral etiology of tick-borne diseases.

Structure and assembly of a paramyxovirus matrix protein.

Many pleomorphic, lipid-enveloped viruses encode matrix proteins that direct their assembly and budding, but the mechanism of this process is unclear. We have combined X-ray crystallography and cryoelectron tomography to show that the matrix protein of Newcastle disease virus, a paramyxovirus and relative of measles virus, forms dimers that assemble into pseudotetrameric arrays that generate the membrane curvature necessary for virus budding. We show that the glycoproteins are anchored in the gaps between the matrix proteins and that the helical nucleocapsids are associated in register with the matrix arrays. About 90% of virions lack matrix arrays, suggesting that, in agreement with previous biological observations, the matrix protein needs to dissociate from the viral membrane during maturation, as is required for fusion and release of the nucleocapsid into the host's cytoplasm. Structure and sequence conservation imply that other paramyxovirus matrix proteins function similarly.

Reverse genetics identifies the product of open reading frame 4 as an essential particle assembly factor of Nyamanini virus.

We established a reverse genetics system for Nyamanini virus (NYMV) and recovered green fluorescent protein (GFP)-expressing virus from full-length cDNA. Using this technology, we assessed the functions of two poorly characterized viral genes. NYMV lacking open reading frame 2 (ORF2) could not be rescued, whereas virus lacking ORF4 was replication competent. ORF4-deficient NYMV readily established a persisting noncytolytic infection but failed to produce infectious viral particles, supporting the view that ORF4 represents an essential factor for NYMV particle assembly.

Discovery and Genomic Analysis of Three Novel Viruses in the Order Mononegavirales in Leafhoppers.

Leafhoppers are economically important pests and may serve as vectors for pathogenic viruses that cause substantial crop damage. In this study, using deep transcriptome sequencing, we identified three novel viruses within the order Mononegavirales, including two viruses belonging to the family Rhabdoviridae and one to the family Lispiviridae. The complete genome sequences were obtained via the rapid amplification of cDNA ends and tentatively named Recilia dorsalis rhabdovirus 1 (RdRV1, 14,251 nucleotides, nt), Nephotettix virescens rhabdovirus 1 (NvRV1, 13,726 nt), and Nephotettix virescens lispivirus 1 (NvLV1, 14,055 nt). The results of a phylogenetic analysis and sequence identity comparison suggest that RdRV1 and NvRV1 represent novel species within the family Rhabdoviridae, while NvLV1 is a new virus belonging to the family Lispiviridae. As negative-sense single-strand RNA viruses, RdRV1 and NvRV1 contain the conserved transcription termination signal and intergenic trinucleotides in the non-transcribed region. Intergenomic sequence and transcriptome profile analyses suggested that all these genes were co-transcriptionally expressed in these viral genomes, facilitated by specific intergenic trinucleotides and putative transcription initiation sequences.