Search Results (38)

Hendra virus (HeV) is a highly pathogenic member of the Henipavirus genus (family Paramyxoviridae, order Mononegavirales), for which all basic replication processes are located in the cytoplasm. The HeV matrix (M) protein plays essential roles in viral assembly and budding at the plasma membrane, but also undergoes dynamic nuclear and nucleolar trafficking, accumulating in nucleoli early in infection, before relocalising to the plasma membrane. We previously showed that M targets sub-nucleolar compartments—the fibrillar centre (FC) and dense fibrillar component (DFC)—to modulate rRNA biogenesis by mimicking a process occurring during a nucleolar DNA-damage response (DDR). Here, we show that M protein sub-nucleolar localisation is regulated by ubiquitination, which controls its redistribution between the FC-DFC and granular component (GC). The mutagenesis of a conserved lysine (K258) reported to undergo ubiquitination, combined with the pharmacological modulation of ubiquitination, indicated that a positive charge at K258 is required for M localisation to the FC-DFC, while ubiquitination regulates subsequent egress from the FC-DFC to the GC. M proteins from multiple Henipaviruses exhibited similar ubiquitin-dependent sub-nucleolar trafficking, indicating a conserved mechanism. These findings reveal a novel mechanism regulating viral protein transport between phase-separated sub-nucleolar compartments and highlight ubiquitination as a key modulator of intra-nucleolar trafficking. Full article

In this report, we document and analyze a case in which the Irkut virus (IRKV) (Mononegavirales: Rhabdoviridae) caused a fatal human case following a bat bite in June 2021. Unfortunately, the available data did not permit a detailed taxonomic classification of the carrier bat (Chiroptera). The event occurred in the southwestern part of the Sikhote-Alin mountain region (Russian Far East) covered by the Ussuri taiga forest. The symptoms of the illness began with the following: fever; pronounced psychomotor and motor agitation; tremor of the lower jaw and tongue; aphasia; dyslexia; and dysphagia. These rapidly developed, leading to a severe and fatal encephalitis. The patient was not vaccinated for rabies and did not receive rabies immunoglobulin. Using brain sections prepared from the deceased, molecular diagnostics were performed: immunofluorescence (polyclonal anti-rabies immunoglobulin) indicating the presence of the lyssavirus antigen; and RT-PCR indicating traces of viral RNA. Sectional material (brain) was used for whole-genome sequencing, resulting in a near-complete sequence of the lyssavirus genome. The obtained genomic sequence was identified as the Irkut virus. A comparative analysis of the new sequence and other currently available IRKV sequences (NCBI) revealed differences. Specifically, amino acid differences between antigenic sites in the isolate and those of the rabies vaccine strain used regionally were noted. The patient history and subsequent analysis confirm human IRKV infection following bat contact. Like other fatal cases of IRKV infection described earlier, this case occurred in the southern part of the Russian Far East. Two have occurred in the southwestern part of the Sikhote-Alin mountain region. This indicates the possible existence of an active, natural viral focus. Full article

The dynamic interplay between a multimeric phosphoprotein (P) and polymeric nucleoprotein (N) in complex with the viral RNA is at the heart of the functioning of the RNA-synthesizing machine of negative-sense RNA viruses of the order Mononegavirales. P multimerization and N phosphorylation are often cited as key factors in regulating these interactions, but a detailed understanding of the molecular mechanisms is not yet available. Working with recombinant rabies virus (RABV) N and P proteins and using mainly surface plasmon resonance, we measured the binding interactions of full-length P dimers and of two monomeric fragments of either circular or linear N-RNA complexes, and we analyzed the equilibrium binding isotherms using different models. We found that RABV P binds with nanomolar affinity to both circular and linear N-RNA complexes and that the dimerization of P protein enhances the binding affinity by 15–30-fold as compared to the monomeric fragments, but less than expected for a bivalent ligand, in which the binding domains are connected by a flexible linker. We also showed that the phosphorylation of N at Ser389 creates high-affinity sites on the polymeric N-RNA complex that enhance the binding affinity of P by a factor of about 360. Full article

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. Full article

The documentation of endogenous viral elements (EVEs; virus-derived genetic material integrated into the genome of a nonviral host) has offered insights into how arthropods respond to viral infection via RNA interference pathways. Small non-coding RNAs derived from EVE loci serve to direct RNAi pathways in limiting replication and infection from cognate viruses, thus benefiting the host’s fitness and, potentially, vectorial capacity. Here we use informatic approaches to analyze nine available genome sequences of hard ticks (Acari: Ixodidae; Rhipicephalus sanguineus, R. microplus, R. annulatus, Ixodes ricinus, I. persulcatus, I. scapularis, Hyalomma asiaticum, Haemaphysalis longicornis, and Dermacentor silvarum) to identify endogenous viral elements and to illustrate the shared ancestry of all elements identified. Our results highlight a broad diversity of viral taxa as having given rise to 1234 identified EVEs in ticks, with Mononegavirales (specifically Rhabdoviridae) well-represented in this subset of hard ticks. Further investigation revealed extensive adintovirus integrations in several Ixodes species, the prevalence of Bunyavirales EVEs (notably not observed in mosquitoes), and the presence of several elements similar to known emerging human and veterinary pathogens. These results will inform subsequent work on current and past associations with tick species with regard to the viruses from which their “viral fossils” are derived and may serve as a reference for quality control of various tick-omics data that may suffer from misidentification of EVEs as viral genetic material. Full article

A wide variety of viruses replicate in liquid-like viral factories. Non-segmented negative stranded RNA viruses share a nucleoprotein (N) and a phosphoprotein (P) that together emerge as the main drivers of liquid–liquid phase separation. The respiratory syncytial virus includes the transcription antiterminator M_2-1, which binds RNA and maximizes RNA transcriptase processivity. We recapitulate the assembly mechanism of condensates of the three proteins and the role played by RNA. M_2-1 displays a strong propensity for condensation by itself and with RNA through the formation of electrostatically driven protein–RNA coacervates based on the amphiphilic behavior of M_2-1 and finely tuned by stoichiometry. M_2-1 incorporates into tripartite condensates with N and P, modulating their size through an interplay with P, where M_2-1 is both client and modulator. RNA is incorporated into the tripartite condensates adopting a heterogeneous distribution, reminiscent of the M_2-1-RNA IBAG granules within the viral factories. Ionic strength dependence indicates that M_2-1 behaves differently in the protein phase as opposed to the protein–RNA phase, in line with the subcompartmentalization observed in viral factories. This work dissects the biochemical grounds for the formation and fate of the RSV condensates in vitro and provides clues to interrogate the mechanism under the highly complex infection context. Full article

In poultry, several respiratory viral infections lead to a drop in egg production associated with high economic losses. While the virus–host interactions at the respiratory epithelium are well studied, less is known about these interactions in the oviduct. To investigate possible differences between virus infections at these epithelial structures, we compared the interactions of two important poultry viruses on turkey organ cultures. Two members of the order Mononegavirales, the Avian Metapneumovirus (AMPV) and the Newcastle disease virus (NDV), were selected to conduct the in vitro experiments since these viruses can infect both the trachea and oviduct. In addition, we used different strains of these viruses, a subtype A and a subtype B strain for AMPV and the NDV strains Komarow and Herts’33, to detect possible differences not only between the tissues but also between different viral strains. Turkey tracheal and oviduct organ cultures (TOC and OOC) were prepared to investigate viral replication, antigen localisation, lesion development, and the expression pattern of interferon-λ and importin-α isoforms. All viruses replicated more efficiently in the oviduct than in the tracheal epithelium (p < 0.05). In addition, we observed higher expression levels of both, IFN-λ and importin-α in OOCs compared to TOCs. Our results indicated strain-dependent differences, with the AMPV-B- and Herts’33 strains being more virulent in organ cultures than the AMPV-A- and Komarow strains, based on the higher viral genome loads, more severe histological lesions, and higher upregulation of IFN-λ. Overall, our findings reveal tissue- and virus strain-dependent differences, which may have consequences for disease development in the host tissue and, subsequently, possible treatment strategies. Full article

Citrus leprosis (CL) is the main viral disease affecting the Brazilian citriculture. Sweet orange (Citrus sinensis L. Osbeck) trees affected by CL were identified in small orchards in Southern Brazil. Rod-like particles of 40 × 100 nm and electron lucent viroplasm were observed in the nucleus of infected cells in symptomatic tissues. RNA extracts from three plants, which proved negative by RT-PCR for known CL-causing viruses, were analyzed by high throughput sequencing and Sanger sequencing after RT-PCR. The genomes of bi-segmented ss(−)RNA viruses, with ORFs in a typical organization of members of the genus Dichorhavirus, were recovered. These genomes shared 98–99% nt sequence identity among them but <73% with those of known dichorhavirids, a value below the threshold for new species demarcation within that genus. Phylogenetically, the three haplotypes of the new virus called citrus bright spot virus (CiBSV) are clustered with citrus leprosis virus N, which is a dichorhavirus transmitted by Brevipalpus phoenicis sensu stricto. In CiBSV-infected citrus plants, B. papayensis and B. azores were found, but the virus could only be transmitted to Arabidopsis plants by B. azores. The study provides the first evidence of the role of B. azores as a viral vector and supports the assignment of CiBSV to the tentative new species Dichorhavirus australis. Full article

The order Mononegavirales contains a variety of highly pathogenic viruses that may infect humans, including the families Filoviridae, Bornaviridae, Paramyxoviridae, and Rhabodoviridae. Animal models have historically been important to study virus pathogenicity and to develop medical countermeasures. As these have inherent shortcomings, the rise of microphysiological systems and organoids able to recapitulate hallmarks of the diseases caused by these viruses may have enormous potential to add to or partially replace animal modeling in the future. Indeed, microphysiological systems and organoids are already used in the pharmaceutical R&D pipeline because they are prefigured to overcome the translational gap between model systems and clinical studies. Moreover, they may serve to alleviate ethical concerns related to animal research. In this review, we discuss the value of animal model alternatives in human pathogenic filovirus and bornavirus research. The current animal models and their limitations are presented followed by an overview of existing alternatives, such as organoids and microphysiological systems, which might help answering open research questions. Full article

As for all non-segmented negative RNA viruses, rabies virus has its genome packaged in a linear assembly of nucleoprotein (N), named nucleocapsid. The formation of new nucleocapsids during virus replication in cells requires the production of soluble N protein in complex with its phosphoprotein (P) chaperone. In this study, we reconstituted a soluble heterodimeric complex between an armless N protein of rabies virus (RABV), lacking its N-terminal subdomain (N_NT-ARM), and a peptide encompassing the N⁰ chaperon module of the P protein. We showed that the chaperone module undergoes a disordered−order transition when it assembles with N⁰ and measured an affinity in the low nanomolar range using a competition assay. We solved the crystal structure of the complex at a resolution of 2.3 Å, unveiling the details of the conserved interfaces. MD simulations showed that both the chaperon module of P and RNA-mediated polymerization reduced the ability of the RNA binding cavity to open and close. Finally, by reconstituting a complex with full-length P protein, we demonstrated that each P dimer could independently chaperon two N⁰ molecules. Full article

Morbilliviruses are negative-sense single-stranded monosegmented RNA viruses in the family Paramyxoviridae (order Mononegavirales). Morbilliviruses infect diverse mammals including humans, dogs, cats, small ruminants, seals, and cetaceans, which serve as natural hosts. Here, I report the identification and characterization of novel viruses detected in public RNAseq datasets of South American long-haired and olive field mice. The divergent viruses dubbed Ratón oliváceo morbillivirus (RoMV) detected in renal samples from mice collected from Chile and Argentina are characterized by an unusually large genome including long intergenic regions and the presence of an accessory protein between the F and H genes redounding in a genome architecture consisting in 3′-N-P/V/C-M-F-hp-H-L-5′. Structural and functional annotation, genetic distance, and evolutionary insights suggest that RoMV is a member of a novel species within genus Morbillivirus tentatively named as South American mouse morbillivirus. Phylogenetic analysis suggests that this mouse morbillivirus is closely related to and clusters into a monophyletic group of novel rodent-borne morbilliviruses. This subclade of divergent viruses expands the host range, redefines the genomic organization and provides insights on the evolutionary history of genus Morbillivirus. Full article

Determining the structural organisation of viral replication complexes and unravelling the impact of infection on cellular homeostasis represent important challenges in virology. This may prove particularly useful when confronted with viruses that pose a significant threat to human health, that appear unique within their family, or for which knowledge is scarce. Among Mononegavirales, bornaviruses (family Bornaviridae) stand out due to their compact genomes and their nuclear localisation for replication. The recent recognition of the zoonotic potential of several orthobornaviruses has sparked a surge of interest in improving our knowledge on this viral family. In this work, we provide a complete analysis of the structural organisation of Borna disease virus 1 (BoDV-1) phosphoprotein (P), an important cofactor for polymerase activity. Using X-ray diffusion and diffraction experiments, we revealed that BoDV-1 P adopts a long coiled-coil α-helical structure split into two parts by an original β-strand twist motif, which is highly conserved across the members of whole Orthobornavirus genus and may regulate viral replication. In parallel, we used BioID to determine the proximal interactome of P in living cells. We confirmed previously known interactors and identified novel proteins linked to several biological processes such as DNA repair or mRNA metabolism. Altogether, our study provides important structure/function cues, which may improve our understanding of BoDV-1 pathogenesis. Full article

Borna disease virus 1 (BoDV-1) is a neurotropic RNA virus belonging to the family Bornaviridae within the order Mononegavirales. Whereas BoDV-1 causes neurological and behavioral disorders, called Borna disease (BD), in a wide range of mammals, its virulence in humans has been debated for several decades. However, a series of case reports in recent years have established the nature of BoDV-1 as a zoonotic pathogen that causes fatal encephalitis in humans. Although many virological properties of BoDV-1 have been revealed to date, the mechanism by which it causes fatal encephalitis in humans remains unclear. In addition, there are no effective vaccines or antiviral drugs that can be used in clinical practice. A reverse genetics approach to generating replication-competent recombinant viruses from full-length cDNA clones is a powerful tool that can be used to not only understand viral properties but also to develop vaccines and antiviral drugs. The rescue of recombinant BoDV-1 (rBoDV-1) was first reported in 2005. However, due to the slow nature of the replication of this virus, the rescue of high-titer rBoDV-1 required several months, limiting the use of this system. This review summarizes the history of the reverse genetics and artificial replication systems for orthobornaviruses and explores the recent progress in efforts to rescue rBoDV-1. Full article

Nipah virus (NiV) is a zoonotic paramyxovirus with a fatality rate of up to 92% in humans. While several pathogenic mechanisms used by NiV to counteract host immune defense responses have been described, all of the processes that take place in cells during infection are not fully characterized. Here, we describe the formation of ordered intracellular structures during NiV infection. We observed that these structures are formed specifically during NiV infection, but not with other viruses from the same Mononegavirales order (namely Ebola virus) or from other orders such as Bunyavirales (Junín virus). We also determined the kinetics of the appearance of these structures and their cellular localization at the cellular periphery. Finally, we confirmed the presence of these NiV-specific ordered structures using structured illumination microscopy (SIM), as well as their localization by transmission electron microscopy (TEM), scanning electron microscopy (SEM), and correlative light and electron microscopy (CLEM). Herein, we describe a cytopathogenic mechanism that provides a new insight into NiV biology. These newly described ordered structures could provide a target for novel antiviral approaches. Full article

Mononegavirales is an order of viruses with a genome in the form of a non-segmented negative-strand RNA that encodes several proteins. The functional polymerase complex of these viruses is composed of two proteins: a large protein (L) and a phosphoprotein (P). The replication of viruses from this order depends on Hsp90 chaperone activity. Previous studies have demonstrated that Hsp90 inhibition results in the degradation of mononegaviruses L protein, with exception of the rabies virus, for which the degradation of P protein was observed. Here, we demonstrated that Hsp90 inhibition does not affect the expression of rabies L and P proteins, but it inhibits binding of the P protein and L protein into functional viral polymerase. Rabies and the vesicular stomatitis virus, but not the measles virus, L proteins can be expressed independently of the presence of a P protein and in the presence of an Hsp90 inhibitor. Our results suggest that the interaction of L proteins with P proteins and Hsp90 in the process of polymerase maturation may be a process specific to particular viruses. Full article