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Biotechnology and Bioengineering Nov 2021Pathogen surface antigens are at the forefront of the viral strategy when invading host organisms. These antigens, including membrane proteins (MPs), are broadly...
Pathogen surface antigens are at the forefront of the viral strategy when invading host organisms. These antigens, including membrane proteins (MPs), are broadly targeted by the host immune response. Obtaining these MPs in a soluble and stable form constitutes a real challenge, regardless of the application purposes (e.g. quantification/characterization assays, diagnosis, and preventive and curative strategies). A rapid process to obtain a native-like antigen by solubilization of a full-length MP directly from a pathogen is reported herein. Rabies virus (RABV) was used as a model for this demonstration and its full-length G glycoprotein (RABV-G) was stabilized with amphipathic polymers, named amphipols (APols). The stability of RABV-G trapped in APol A8-35 (RABV-G/A8-35) was evaluated under different stress conditions (temperature, agitation, and light exposure). RABV-G/A8-35 in liquid form exhibited higher unfolding temperature (+6°C) than in detergent and was demonstrated to be antigenically stable over 1 month at 5°C and 25°C. Kinetic modeling of antigenicity data predicted antigenic stability of RABV-G/A8-35 in a solution of up to 1 year at 5°C. The RABV-G/A8-35 complex formulated in an optimized buffer composition and subsequently freeze-dried displayed long-term stability for 2-years at 5, 25, and 37°C. This study reports for the first time that a natural full-length MP extracted from a virus, complexed to APols and subsequently freeze-dried, displayed long-term antigenic stability, without requiring storage under refrigerated conditions.
Topics: Antigens, Viral; Detergents; Freeze Drying; Protein Stability; Rabies virus; Viral Envelope Proteins
PubMed: 34297405
DOI: 10.1002/bit.27900 -
Virulence Dec 2022Rabies is an important zoonotic disease caused by the rabies virus (RABV). Currently, no effective treatment is available for this condition. The prevention and control...
Rabies is an important zoonotic disease caused by the rabies virus (RABV). Currently, no effective treatment is available for this condition. The prevention and control of rabies mainly depend on effective vaccination. Therefore, it is crucial to enhance the immune responses induced by the rabies vaccine. Virus neutralizing antibodies (VNA) induced by rabies vaccines are important for the clearance of RABV. Interleukin-25 (IL-25) has been demonstrated to activate T helper type 2 cells that contribute to humoral immune responses. The IL-25 gene was inserted into the genome of RABV, and the immunogenicity of recombinant RABV with IL-25 gene was investigated to develop more efficient rabies vaccines. Here, we found that the expression of IL-25 did not affect RABV production and pathogenicity . However, recombinant RABV expression of IL-25 induced a better VNA level than the parental virus in mice. In addition, expression of IL-25 enhanced the IgG1 level induced by RABV. Furthermore, mice immunized with recombinant RABV showed a higher survival rate and milder clinical signs than those immunized with the parent strain after challenge with CVS-11. Thus, these results showed that IL-25 could enhance the humoral immune responses induced by RABV, suggesting that IL-25 can be used as a viral vaccine adjuvant.
Topics: Animals; Antibodies, Viral; Immunity, Humoral; Interleukin-17; Mice; Rabies; Rabies Vaccines; Rabies virus
PubMed: 35999776
DOI: 10.1080/21505594.2022.2116146 -
Antiviral Research Jan 2019Rabies virus transmits from animals to humans and causes encephalitis. Every year more than 15 million people receive a post exposure prophylaxis (PEP) treatment that is...
Rabies virus transmits from animals to humans and causes encephalitis. Every year more than 15 million people receive a post exposure prophylaxis (PEP) treatment that is highly effective in the prevention of rabies disease. However, when clinical symptoms appear, for example in people who did not receive PEP, rabies is almost invariably fatal. Due to the limited access to PEP in some target populations, mostly in Asia and in Africa, rabies causes at least 59,000 deaths a year. PEP is not effective after the onset of symptoms and attempts to develop a treatment for clinical rabies have been unsuccessful. After screening a library of 385 FDA-approved drugs, we found that pyrimethamine inhibits rabies infection in vitro through the inhibition of adenosine synthesis. In addition, this compound shows a synergistic interaction with ribavirin. Unfortunately, in rabies infected-mice, pyrimethamine showed no efficacy. One possible explanation may be that the antiviral effect is negated by the observed interference of pyrimethamine with the innate immune response.
Topics: Adenosine; Animals; Antiviral Agents; Drug Synergism; Mice; Mice, Inbred BALB C; Pyrimethamine; Rabies virus; Ribavirin; Small Molecule Libraries; Virus Replication
PubMed: 30367894
DOI: 10.1016/j.antiviral.2018.10.016 -
Journal of Virology Jan 2022Rabies is an old zoonotic disease caused by rabies virus (RABV), but the pathogenic mechanism of RABV is still not completely understood. Lipid droplets (LDs) have been...
Rabies is an old zoonotic disease caused by rabies virus (RABV), but the pathogenic mechanism of RABV is still not completely understood. Lipid droplets (LDs) have been reported to play a role in pathogenesis of several viruses. However, their role in RABV infection remains unclear. Here, we initially found that RABV infection upregulated LD production in multiple cells and mouse brains. After treatment with atorvastatin, a specific inhibitor of LDs, RABV replication in N2a cells decreased. Then we found that RABV infection could upregulate N-myc downstream regulated gene-1 (NDRG1), which in turn enhanced the expression of diacylglycerol acyltransferase 1/2 (DGAT1/2). DGAT1/2 could elevate cellular triglyceride synthesis and ultimately promote intracellular LD formation. Furthermore, we found that RABV-M and RABV-G, which were mainly involved in the viral budding process, could colocalize with LDs, indicating that RABV might utilize LDs as a carrier to facilitate viral budding and eventually increase virus production. Taken together, our study reveals that lipid droplets are beneficial for RABV replication, and their biogenesis is regulated via the NDRG1-DGAT1/2 pathway, which provides novel potential targets for developing anti-RABV drugs. Lipid droplets have been proven to play an important role in viral infections, but their role in RABV infection has not yet been elaborated. Here, we find that RABV infection upregulates the generation of LDs by enhancing the expression of N-myc downstream regulated gene-1 (NDRG1). Then NDRG1 elevated cellular triglycerides synthesis by increasing the activity of diacylglycerol acyltransferase 1/2 (DGAT1/2), which promotes the biogenesis of LDs. RABV-M and RABV-G, which are the major proteins involved in viral budding, could utilize LDs as a carrier for transport to cell membrane, resulting in enhanced virus budding. Our findings will extend the knowledge of lipid metabolism in RABV infection and help to explore potential therapeutic targets for RABV.
Topics: Animals; Anticholesteremic Agents; Atorvastatin; Cell Cycle Proteins; Diacylglycerol O-Acyltransferase; Intracellular Signaling Peptides and Proteins; Lipid Droplets; Mice; Neurons; Rabies; Rabies virus; Triglycerides; Viral Structural Proteins; Virus Release; Virus Replication
PubMed: 34757839
DOI: 10.1128/JVI.01473-21 -
Journal of Clinical Microbiology Mar 2023Molecular analysis of rabies virus can provide accurate diagnosis and information on its genetic diversity. The transportation of rabies brain samples from remote areas...
Molecular analysis of rabies virus can provide accurate diagnosis and information on its genetic diversity. The transportation of rabies brain samples from remote areas to a central laboratory is challenging owing to biohazard risks and decomposability. We investigated the utility of used lateral flow devices (LFDs) for subsequent molecular analysis and assessed the necessary storage temperatures. Using RNA extracted from used LFD strips, we performed conventional reverse transcription-PCR (RT-PCR) using an LN34 primer set to amplify short fragments (165 bp) for rabies virus detection and the P1-304 primer set to amplify long fragments of the entire N gene amplicon (1,506 bp) for phylogenetic analysis. Among 71 used LFDs stored in a refrigerator and 64 used LFDs stored at room temperature, the LN34 assay showed high sensitivities (96.2% and 100%, respectively) for the diagnosis of rabies, regardless of the storage temperature. A significant reduction in the sensitivity of rabies diagnosis was observed when using the P1-304 primer set for used LFDs stored at room temperature compared to those stored at refrigeration temperature (20.9% versus 100%; < 0.05). Subsequent sequencing and phylogenetic analysis were successfully performed using the amplicons generated by the P1-304 RT-PCR assays. Used LFDs are thus promising resources for rabies virus RNA detection and sequence analysis. Virus detection via RT-PCR, amplifying a short fragment, was possible regardless of the storage temperature of the used LFDs. However, refrigerated storage is recommended for RT-PCR amplification of long fragments for phylogenetic analysis.
Topics: Humans; Rabies virus; Rabies; Phylogeny; RNA, Viral; Polymerase Chain Reaction; Sensitivity and Specificity; Reverse Transcriptase Polymerase Chain Reaction
PubMed: 36840574
DOI: 10.1128/jcm.01543-22 -
Viruses Dec 2022As 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...
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), and a peptide encompassing the N chaperon module of the P protein. We showed that the chaperone module undergoes a disordered-order transition when it assembles with N 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 N molecules.
Topics: Rabies virus; Nucleoproteins; Protein Binding; Nucleocapsid Proteins; Molecular Chaperones; Phosphoproteins; RNA; RNA, Viral
PubMed: 36560817
DOI: 10.3390/v14122813 -
Neuroscience Bulletin Mar 2020
Topics: Animals; Dependovirus; Nerve Net; Neuroanatomical Tract-Tracing Techniques; Rabies virus
PubMed: 32065368
DOI: 10.1007/s12264-020-00472-z -
Clinical and Translational Medicine Jan 2022Neurotropic virus infection can cause serious damage to the central nervous system (CNS) in both humans and animals. The complexity of the CNS poses unique challenges to...
BACKGROUND
Neurotropic virus infection can cause serious damage to the central nervous system (CNS) in both humans and animals. The complexity of the CNS poses unique challenges to investigate the infection of these viruses in the brain using traditional techniques.
METHODS
In this study, we explore the use of fluorescence micro-optical sectioning tomography (fMOST) and single-cell RNA sequencing (scRNA-seq) to map the spatial and cellular distribution of a representative neurotropic virus, rabies virus (RABV), in the whole brain. Mice were inoculated with a lethal dose of a recombinant RABV encoding enhanced green fluorescent protein (EGFP) under different infection routes, and a three-dimensional (3D) view of RABV distribution in the whole mouse brain was obtained using fMOST. Meanwhile, we pinpointed the cellular distribution of RABV by utilizing scRNA-seq.
RESULTS
Our fMOST data provided the 3D view of a neurotropic virus in the whole mouse brain, which indicated that the spatial distribution of RABV in the brain was influenced by the infection route. Interestingly, we provided evidence that RABV could infect multiple nuclei related to fear independent of different infection routes. More surprisingly, our scRNA-seq data revealed that besides neurons RABV could infect macrophages and the infiltrating macrophages played at least three different antiviral roles during RABV infection.
CONCLUSION
This study draws a comprehensively spatial and cellular map of typical neurotropic virus infection in the mouse brain, providing a novel and insightful strategy to investigate the pathogenesis of RABV and other neurotropic viruses.
Topics: Animals; Brain; Disease Models, Animal; Mice; Rabies; Rabies virus; Single-Cell Analysis; Tomography, Optical
PubMed: 35051311
DOI: 10.1002/ctm2.700 -
PloS One 2017Near complete rabies virus N gene sequences (1,110 nt) were determined for 82 isolates obtained from different regions of Russia between 2008 and 2016. These sequences...
Near complete rabies virus N gene sequences (1,110 nt) were determined for 82 isolates obtained from different regions of Russia between 2008 and 2016. These sequences were analyzed together with 108 representative GenBank sequences from 1977-2016 using the Bayesian coalescent approach. The timing of the major evolutionary events was estimated. Most of the isolates represented the steppe rabies virus group C, which was found over a vast geographic region from Central Russia to Mongolia and split into three groups (C0-C2) with discrete geographic prevalence. A single strain of the steppe rabies virus lineage was isolated in the far eastern part of Russia (Primorsky Krai), likely as a result of a recent anthropogenic introduction. For the first time the polar rabies virus group A2, previously reported in Alaska, was described in the northern part of European Russia and at the Franz Josef Land. Phylogenetic analysis suggested that all currently circulating rabies virus groups in the Russian Federation were introduced within the few last centuries, with most of the groups spreading in the 20th century. The dating of evolutionary events was highly concordant with the historical epidemiological data.
Topics: Genome, Viral; Phylogeny; Rabies; Rabies virus; Russia
PubMed: 28225771
DOI: 10.1371/journal.pone.0171855 -
PloS One 2023Monosynaptically restricted rabies viruses have been used for more than a decade for synaptic connectivity tracing. However, the verisimilitude of quantitative...
Monosynaptically restricted rabies viruses have been used for more than a decade for synaptic connectivity tracing. However, the verisimilitude of quantitative conclusions drawn from these experiments is largely unknown. The primary reason is the simple metrics commonly used, which generally disregard the effect of starter cell numbers. Here we present an experimental dataset with a broad range of starter cell numbers and explore their relationship with the number of input cells across the brain using descriptive statistics and modelling. We show that starter cell numbers strongly affect input fraction and convergence index measures, making quantitative comparisons unreliable. Furthermore, we suggest a principled way to analyse rabies derived connectivity data by taking advantage of the starter vs input cell relationship that we describe and validate across independent datasets.
Topics: Humans; Rabies virus; Rabies; Brain
PubMed: 36996085
DOI: 10.1371/journal.pone.0278053