Design and Execution of In Vitro Polymerase Assays for Measles Virus and Related Mononegaviruses.

Morbilliviruses such as measles virus (MeV) are responsible for major morbidity and mortality worldwide, despite the availability of an effective vaccine and global vaccination campaigns. MeV belongs to the mononegavirus order of viral pathogens that store their genetic information in non-segmented negative polarity RNA genomes. Genome replication and viral gene expression are carried out by a virus-encoded RNA-dependent RNA polymerase (RdRP) complex that has no immediate host cell analog. To better understand the organization and regulation of the viral RdRP and mechanistically characterize antiviral candidates, biochemical RdRP assays have been developed that employ purified recombinant polymerase complexes and synthetic RNA templates to monitor the initiation of RNA synthesis and RNA elongation in vitro. In this article, we will discuss strategies for the efficient expression and preparation of mononegavirus polymerase complexes, provide detailed protocols for the execution and optimization of RdRP assays, evaluate alternative options for the choice of template and detection system, and describe the application of the assay for the characterization of inhibitor candidates. Although MeV RdRP assays are the focus of this article, the general strategies and experimental approaches are readily transferable to related viruses in the mononegavirus order.

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.

Structure and function of negative-strand RNA virus polymerase complexes.

Viruses with negative-strand RNA genomes (NSVs) include many highly pathogenic and economically devastating disease-causing agents of humans, livestock, and plants-highlighted by recent Ebola and measles virus epidemics, and continuously circulating influenza virus. Because of their protein-coding orientation, NSVs face unique challenges for efficient gene expression and genome replication. To overcome these barriers, NSVs deliver a large and multifunctional RNA-dependent RNA polymerase into infected host cells. NSV-encoded polymerases contain all the enzymatic activities required for transcription and replication of their genome-including RNA synthesis and mRNA capping. Here, we review the structures and functions of NSV polymerases with a focus on key domains responsible for viral replication and gene expression. We highlight shared and unique features among polymerases of NSVs from the Mononegavirales, Bunyavirales, and Articulavirales orders.

Sendai Virus and a Unified Model of Mononegavirus RNA Synthesis.

Vesicular stomatitis virus (VSV), the founding member of the mononegavirus order (Mononegavirales), was found to be a negative strand RNA virus in the 1960s, and since then the number of such viruses has continually increased with no end in sight. Sendai virus (SeV) was noted soon afterwards due to an outbreak of newborn pneumonitis in Japan whose putative agent was passed in mice, and nowadays this mouse virus is mainly the bane of animal houses and immunologists. However, SeV was important in the study of this class of viruses because, like flu, it grows to high titers in embryonated chicken eggs, facilitating the biochemical characterization of its infection and that of its nucleocapsid, which is very close to that of measles virus (MeV). This review and opinion piece follow SeV as more is known about how various mononegaviruses express their genetic information and carry out their RNA synthesis, and proposes a unified model based on what all MNV have in common.

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.

Virome characterization of Cryphonectria parasitica isolates from Azerbaijan unveiled a new mymonavirus and a putative new RNA virus unrelated to described viral sequences.

Cryphonectria parasitica, the causal agent of chestnut blight is controlled in Europe through natural spread of Cryphonectria hypovirus 1 (CHV1), a mycovirus able to induce hypovirulence to the host. In recent years C. parasitica was reported infecting Azerbaijani population of chestnut, but the presence of CHV1 still needs to be confirmed. Aim of this work was to investigate fifty-five C. parasitica isolates collected in Azerbaijan to describe the associated viruses. Our work found i) the first negative-sense ssRNA virus known to infect C. parasitica naturally for which we propose the name Cryphonectria parasitica sclerotimonavirus 1 (CpSV1) and ii) an RNA sequence showing peculiar features suggesting a viral nature for which we propose the name Cryphonectria parasitica ambivirus 1 (CpaV1). The discovery of CpaV1 expands our knowledge of the RNA virosphere suggesting the existence of a new lineage that cannot presently be reliably associated to the monophyletic Riboviria.

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.

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).

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.

Chaperoning the Mononegavirales: Current Knowledge and Future Directions.

The order Mononegavirales harbors numerous viruses of significant relevance to human health, including both established and emerging infections. Currently, vaccines are only available for a small subset of these viruses, and antiviral therapies remain limited. Being obligate cellular parasites, viruses must utilize the cellular machinery for their replication and spread. Therefore, targeting cellular pathways used by viruses can provide novel therapeutic approaches. One of the key challenges confronted by both hosts and viruses alike is the successful folding and maturation of proteins. In cells, this task is faced by cellular molecular chaperones, a group of conserved and abundant proteins that oversee protein folding and help maintain protein homeostasis. In this review, we summarize the current knowledge of how the Mononegavirales interact with cellular chaperones, highlight key gaps in our knowledge, and discuss the potential of chaperone inhibitors as antivirals.

Viral regulation of host cell biology by hijacking of the nucleolar DNA-damage response.

Recent studies indicate that nucleoli play critical roles in the DNA-damage response (DDR) via interaction of DDR machinery including NBS1 with nucleolar Treacle protein, a key mediator of ribosomal RNA (rRNA) transcription and processing. Here, using proteomics, confocal and single molecule super-resolution imaging, and infection under biosafety level-4 containment, we show that this nucleolar DDR pathway is targeted by infectious pathogens. We find that the matrix proteins of Hendra virus and Nipah virus, highly pathogenic viruses of the Henipavirus genus in the order Mononegavirales, interact with Treacle and inhibit its function, thereby silencing rRNA biogenesis, consistent with mimicking NBS1-Treacle interaction during a DDR. Furthermore, inhibition of Treacle expression/function enhances henipavirus production. These data identify a mechanism for viral modulation of host cells by appropriating the nucleolar DDR and represent, to our knowledge, the first direct intranucleolar function for proteins of any mononegavirus.

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.

Implementation of Objective PASC-Derived Taxon Demarcation Criteria for Official Classification of Filoviruses.

The mononegaviral family Filoviridae has eight members assigned to three genera and seven species. Until now, genus and species demarcation were based on arbitrarily chosen filovirus genome sequence divergence values (≈50% for genera, ≈30% for species) and arbitrarily chosen phenotypic virus or virion characteristics. Here we report filovirus genome sequence-based taxon demarcation criteria using the publicly accessible PAirwise Sequencing Comparison (PASC) tool of the US National Center for Biotechnology Information (Bethesda, MD, USA). Comparison of all available filovirus genomes in GenBank using PASC revealed optimal genus demarcation at the 55-58% sequence diversity threshold range for genera and at the 23-36% sequence diversity threshold range for species. Because these thresholds do not change the current official filovirus classification, these values are now implemented as filovirus taxon demarcation criteria that may solely be used for filovirus classification in case additional data are absent. A near-complete, coding-complete, or complete filovirus genome sequence will now be required to allow official classification of any novel "filovirus." Classification of filoviruses into existing taxa or determining the need for novel taxa is now straightforward and could even become automated using a presented algorithm/flowchart rooted in RefSeq (type) sequences.

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).

New antiviral approaches for respiratory syncytial virus and other mononegaviruses: Inhibiting the RNA polymerase.

Worldwide, respiratory syncytial virus (RSV) causes severe disease in infants, the elderly, and immunocompromised people. No vaccine or effective antiviral treatment is available. RSV is a member of the non-segmented, negative-strand (NNS) group of RNA viruses and relies on its RNA-dependent RNA polymerase to transcribe and replicate its genome. Because of its essential nature and unique properties, the RSV polymerase has proven to be a good target for antiviral drugs, with one compound, ALS-8176, having already achieved clinical proof-of-concept efficacy in a human challenge study. In this article, we first provide an overview of the role of the RSV polymerase in viral mRNA transcription and genome replication. We then review past and current approaches to inhibiting the RSV polymerase, including use of nucleoside analogs and non-nucleoside inhibitors. Finally, we consider polymerase inhibitors that hold promise for treating infections with other NNS RNA viruses, including measles and Ebola.

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).

Reverse genetics of Mononegavirales: How they work, new vaccines, and new cancer therapeutics.

The order Mononegavirales includes five families: Bornaviridae, Filoviridae, Nyamaviridae, Paramyxoviridae, and Rhabdoviridae. The genome of these viruses is one molecule of negative-sense single strand RNA coding for five to ten genes in a conserved order. The RNA is not infectious until packaged by the nucleocapsid protein and transcribed by the polymerase and co-factors. Reverse genetics approaches have answered fundamental questions about the biology of Mononegavirales. The lack of icosahedral symmetry and modular organization in the genome of these viruses has facilitated engineering of viruses expressing fluorescent proteins, and these fluorescent proteins have provided important insights about the molecular and cellular basis of tissue tropism and pathogenesis. Studies have assessed the relevance for virulence of different receptors and the interactions with cellular proteins governing the innate immune responses. Research has also analyzed the mechanisms of attenuation. Based on these findings, ongoing clinical trials are exploring new live attenuated vaccines and the use of viruses re-engineered as cancer therapeutics.

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.