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Single-domain antibodies, referred to as VHH (variable heavy chains of heavy chain-only antibodies) or in their commercial name as nanobodies, are potent tools for the detection of target proteins in biological samples. They have the advantage of being highly stable, specific, and sensitive, with affinities reaching the nanomolar range. We utilized this tool to develop a rapid detection method that discriminates cells infected with Rift Valley fever virus (RVFV), based on the intracellular detection of the viral nonstructural NSm protein localized on the outer membrane of mitochondria. Here we describe how NSm-specific VHHs have been produced, cloned, and characterized, highlighting their value in RVFV research and diagnosis. This work may also raise interest in other potential applications such as antiviral therapy., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Rift Valley fever virus (RVFV) is a pathogenic arthropod-borne virus that can cause serious illness in both ruminants and humans. The virus can be transmitted by an arthropod bite or contact with contaminated fluids or tissues. Two live-attenuated veterinary vaccines-the Smithburn (SB) and Clone 13 (Cl.13)-are currently used during epizootic events in Africa. However, their residual pathogenicity (i.e., SB) or potential of reversion (i.e., Cl.13) causes important adverse effects, strongly limiting their use in the field. In this study, we infected immunocompetent mice with SB or Cl.13 by a subcutaneous or an intranasal inoculation. Interestingly, we found that, unlike the subcutaneous infection, the intranasal inoculation led to a high mortality rate. In addition, we detected high titers and viral N antigen levels in the brain of both the SB- and Cl.13-infected mice. Overall, we unveil a clear correlation between the pathogenicity and the route of administration of both SB and Cl.13, with the intranasal inoculation leading to a stronger neurovirulence and higher mortality rate than the subcutaneous infection.

The dengue virus nonstructural protein 1 (NS1) is a secreted virulence factor that modulates complement, activates immune cells and alters endothelial barriers. The molecular basis of these events remains incompletely understood. Here we describe a functional high affinity complex formed between NS1 and human high-density lipoproteins (HDL). Collapse of the soluble NS1 hexamer upon binding to the lipoprotein particle leads to the anchoring of amphipathic NS1 dimeric subunits into the HDL outer layer. The stable complex can be visualized by electron microscopy as a spherical HDL with rod-shaped NS1 dimers protruding from the surface. We further show that the assembly of NS1-HDL complexes triggers the production of pro-inflammatory cytokines in human primary macrophages while NS1 or HDL alone do not. Finally, we detect NS1 in complex with HDL and low-density lipoprotein (LDL) particles in the plasma of hospitalized dengue patients and observe NS1-apolipoprotein E-positive complexes accumulating overtime. The functional reprogramming of endogenous lipoprotein particles by NS1 as a means to exacerbate systemic inflammation during viral infection provides a new paradigm in dengue pathogenesis., (© 2022 The Authors.)

Background: Human encephalitis represents a medical challenge from a diagnostic and therapeutic point of view. We investigated the cause of 2 fatal cases of encephalitis of unknown origin in immunocompromised patients., Methods: Untargeted metatranscriptomics was applied on the brain tissue of 2 patients to search for pathogens (viruses, bacteria, fungi, or protozoans) without a prior hypothesis., Results: Umbre arbovirus, an orthobunyavirus never previously identified in humans, was found in 2 patients. In situ hybridization and reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) showed that Umbre virus infected neurons and replicated at high titers. The virus was not detected in cerebrospinal fluid by RT-qPCR. Viral sequences related to Koongol virus, another orthobunyavirus close to Umbre virus, were found in Culex pipiens mosquitoes captured in the south of France where the patients had spent some time before the onset of symptoms, demonstrating the presence of the same clade of arboviruses in Europe and their potential public health impact. A serological survey conducted in the same area did not identify individuals positive for Umbre virus. The absence of seropositivity in the population may not reflect the actual risk of disease transmission in immunocompromised individuals., Conclusions: Umbre arbovirus can cause encephalitis in immunocompromised humans and is present in Europe., (© The Author(s) 2020. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Rift Valley fever virus (RVFV) is a highly pathogenic zoonotic arbovirus endemic in many African countries and the Arabian Peninsula. Animal infections cause high rates of mortality and abortion among sheep, goats, and cattle. In humans, an estimated 1 to 2% of RVFV infections result in severe disease (encephalitis, hepatitis, or retinitis) with a high rate of lethality when associated with hemorrhagic fever. The RVFV NSs protein, which is the main virulence factor, counteracts the host innate antiviral response to favor viral replication and spread. However, the mechanisms underlying RVFV-induced cytopathic effects and the role of NSs in these alterations remain for the most part unknown. In this work, we have analyzed the effects of NSs expression on the actin cytoskeleton while conducting infections with the NSs-expressing virulent (ZH548) and attenuated (MP12) strains of RVFV and the non-NSs-expressing avirulent (ZH548ΔNSs) strain, as well as after the ectopic expression of NSs. In macrophages, fibroblasts, and hepatocytes, NSs expression prevented the upregulation of Abl2 (a major regulator of the actin cytoskeleton) expression otherwise induced by avirulent infections and identified here as part of the antiviral response. The presence of NSs was also linked to an increased mobility of ZH548-infected cells compared to ZH548ΔNSs-infected fibroblasts and to strong changes in cell morphology in nonmigrating hepatocytes, with reduction of lamellipodia, cell spreading, and dissolution of adherens junctions reminiscent of the ZH548-induced cytopathic effects observed in vivo Finally, we show evidence of the presence of NSs within long actin-rich structures associated with NSs dissemination from NSs-expressing toward non-NSs-expressing cells. IMPORTANCE Rift Valley fever virus (RVFV) is a dangerous human and animal pathogen that was ranked by the World Health Organization in 2018 as among the eight pathogens of most concern for being likely to cause wide epidemics in the near future and for which there are no, or insufficient, countermeasures. The focus of this work is to address the question of the mechanisms underlying RVFV-induced cytopathic effects that participate in RVFV pathogenicity. We demonstrate here that RVFV targets cell adhesion and the actin cytoskeleton at the transcriptional and cellular level, affecting cell mobility and inducing cell shape collapse, along with distortion of cell-cell adhesion. All these effects may participate in RVFV-induced pathogenicity, facilitate virulent RVFV dissemination, and thus constitute interesting potential targets for future development of antiviral therapeutic strategies that, in the case of RVFV, as with several other emerging arboviruses, are presently lacking., (Copyright © 2020 American Society for Microbiology.)

Amyloid fibrils result from the aggregation of host cell-encoded proteins, many giving rise to specific human illnesses such as Alzheimer's disease. Here we show that the major virulence factor of Rift Valley fever virus, the protein NSs, forms filamentous structures in the brain of mice and affects mortality. NSs assembles into nuclear and cytosolic disulfide bond-dependent fibrillary aggregates in infected cells. NSs structural arrangements exhibit characteristics typical for amyloids, such as an ultrastructure of 12 nm-width fibrils, a strong detergent resistance, and interactions with the amyloid-binding dye Thioflavin-S. The assembly dynamics of viral amyloid-like fibrils can be visualized in real-time. They form spontaneously and grow in an amyloid fashion within 5 hours. Together, our results demonstrate that viruses can encode amyloid-like fibril-forming proteins and have strong implications for future research on amyloid aggregation and toxicity in general.

RNA viruses present an extraordinary threat to human health, given their sudden and unpredictable appearance, the potential for rapid spread among the human population, and their ability to evolve resistance to antiviral therapies. The recent emergence of chikungunya virus, Zika virus, and Ebola virus highlights the struggles to contain outbreaks. A significant hurdle is the availability of antivirals to treat the infected or protect at-risk populations. While several compounds show promise in vitro and in vivo, these outbreaks underscore the need to accelerate drug discovery. The replication of several viruses has been described to rely on host polyamines, small and abundant positively charged molecules found in the cell. Here, we describe the antiviral effects of two molecules that alter polyamine levels: difluoromethylornithine (DFMO; also called eflornithine), which is a suicide inhibitor of ornithine decarboxylase 1 (ODC1), and diethylnorspermine (DENSpm), an activator of spermidine/spermine N 1 -acetyltransferase (SAT1). We show that reducing polyamine levels has a negative effect on diverse RNA viruses, including several viruses involved in recent outbreaks, in vitro and in vivo These findings highlight the importance of the polyamine biosynthetic pathway to viral replication, as well as its potential as a target in the development of further antivirals or currently available molecules, such as DFMO., Importance: RNA viruses present a significant hazard to human health, and combatting these viruses requires the exploration of new avenues for targeting viral replication. Polyamines, small positively charged molecules within the cell, have been demonstrated to facilitate infection for a few different viruses. Our study demonstrates that diverse RNA viruses rely on the polyamine pathway for replication and highlights polyamine biosynthesis as a promising drug target., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Rift Valley fever virus (RVFV) is an enzootic virus circulating in Africa that is transmitted to its vertebrate host by a mosquito vector and causes severe clinical manifestations in humans and ruminants. RVFV has a tripartite genome of negative or ambisense polarity. The M segment contains five in-frame AUG codons that are alternatively used for the synthesis of two major structural glycoproteins, GN and GC, and at least two accessory proteins, NSm, a 14-kDa cytosolic protein, and P78/NSm-GN, a 78-kDa glycoprotein. To determine the relative contribution of P78 and NSm to RVFV infectivity, AUG codons were knocked out to generate mutant viruses expressing various sets of the M-encoded proteins. We found that, in the absence of the second AUG codon used to express NSm, a 13-kDa protein corresponding to an N-terminally truncated form of NSm, named NSm', was synthesized from AUG 3. None of the individual accessory proteins had any significant impact on RVFV virulence in mice. However, a mutant virus lacking both NSm and NSm' was strongly attenuated in mice and grew to reduced titers in murine macrophages, a major target cell type of RVFV. In contrast, P78 was not associated with reduced viral virulence in mice, yet it appeared as a major determinant of virus dissemination in mosquitoes. This study demonstrates how related accessory proteins differentially contribute to RVFV propagation in mammalian and arthropod hosts.