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Journal of Virology Feb 2023Identification of bona fide functional receptors and elucidation of the mechanism of receptor-mediated virus entry are important to reveal targets for developing...
Identification of bona fide functional receptors and elucidation of the mechanism of receptor-mediated virus entry are important to reveal targets for developing therapeutics against rabies virus (RABV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Our previous studies suggest that metabotropic glutamate receptor subtype 2 (mGluR2) functions as an entry receptor for RABV , and is an important internalization factor for SARS-CoV-2 and . Here, we demonstrate that mGluR2 facilitates RABV internalization and infection . We found that transferrin receptor 1 (TfR1) interacts with mGluR2 and internalizes with mGluR2 and RABV in the same clathrin-coated pit. Knockdown of TfR1 blocks agonist-triggered internalization of mGluR2. Importantly, TfR1 also interacts with the SARS-CoV-2 spike protein and is important for SARS-CoV-2 internalization. Our findings identify a novel axis (mGluR2-TfR1 axis) used by RABV and SARS-CoV-2 for entry, and reveal TfR1 as a potential target for therapeutics against RABV and SARS-CoV-2. We previously found that metabotropic glutamate receptor subtype 2 (mGluR2) is an entry receptor for RABV , and an important internalization factor for SARS-CoV-2 and . However, whether mGluR2 is required for RABV infection was unknown. In addition, how mGluR2 mediates the internalization of RABV and SARS-CoV-2 needed to be resolved. Here, we found that mGluR2 gene knockout mice survived a lethal challenge with RABV. To our knowledge, mGluR2 is the first host factor to be definitively shown to play an important role in RABV street virus infection . We further found that transferrin receptor protein 1 (TfR1) directly interacts and cooperates with mGluR2 to regulate the endocytosis of RABV and SARS-CoV-2. Our study identifies a novel axis (mGluR2-TfR1 axis) used by RABV and SARS-CoV-2 for entry and opens a new door for the development of therapeutics against RABV and SARS-CoV-2.
Topics: Animals; Humans; Mice; COVID-19; Rabies; Rabies virus; Receptors, Metabotropic Glutamate; Receptors, Transferrin; SARS-CoV-2; Spike Glycoprotein, Coronavirus; Virus Internalization
PubMed: 36779763
DOI: 10.1128/jvi.01611-22 -
Emerging Microbes & Infections Dec 2022Rabies virus has existed for thousands of years and is circulating in many species. In the present study, a total of 2896 rabies viruses isolated worldwide were...
Rabies virus has existed for thousands of years and is circulating in many species. In the present study, a total of 2896 rabies viruses isolated worldwide were phylogenetically classified into ten clusters based on the G gene sequence, and these clusters showed a close relationship with the hosts and regions that they were isolated from. Eighty-three representative G sequences were selected from ten clusters and were used to construct pseudoviruses using the VSV vector. The phylogenetic relationships, infectivity and antigenicity of the representative 83 pseudotyped rabies viruses were comprehensively analyzed. Eighty three pseudoviruses were divided into four antigentic clusters (GAgV), of which GAgV4 showed poor neutralization to all immunized sera. Further analysis showed that almost all strains in the GAgV4 were isolated from wild animals in the America, especially bats and skunks. No significant relationship in terms of phylogeny, infectivity and antigenicity was proved. Amino acid mutations at residues 231and 436 can affect the infectivity, while mutations at residues 113, 164 and 254 may affect the sensitivity to immunized animal sera, especially residue 254. We recommend close monitoring of infectivity and antigenicity, which should be more precise than simple genetic analysis.
Topics: Animals; Animals, Wild; Chiroptera; Phylogeny; Rabies virus
PubMed: 35570580
DOI: 10.1080/22221751.2022.2078742 -
Biologicals : Journal of the... Jul 2022We have obtained an attenuated rabies virus CTN181-3. In this paper, we make a comprehensive studies on CTN181-3. CTN181-3 showed no pathogenicity by i. c. or o. i....
We have obtained an attenuated rabies virus CTN181-3. In this paper, we make a comprehensive studies on CTN181-3. CTN181-3 showed no pathogenicity by i. c. or o. i. inoculation in 3-week-old mice, lower pathogenic in 2-week-old mice, and no virulence by o. i. inoculation in 8-week-old golden hamsters. CTN181-3 showed high immunogenicity, which produced high level neutralizing antibodies, 100% sero-conversation and >5.0 IU/ml GMT by one dose i. m. or o. i. vaccination in mice and golden hamsters. Cellular immune response by one dose i. m. or o. i. inoculation was detected. Especially in PEP, reduced dose of vaccination resulted in 50% (one dose) and 100% (2 doses) protections in golden hamsters. Molecular basis of the attenuation indicated that eight substitutions compared to its parental virus strain CTN-1, among them the two substitutions at the G276 (Leu→Val) and L1496 (Met→Trp) were the critical attenuated site. The phenotypic and genotypic characteristics of CTN181-3 were highly stable, no reversion was occurred when the virus was multiple passaged in suckling mice brains, guinea pig submandibular glands or BSR/Vero cell cultures. The gene homology compared to the Chinese rabies isolates showed much higher than rabies vaccine strains used in China, suggesting CTN181-3 is a promising and suitable oral rabies vaccine candidate strain.
Topics: Animals; Antibodies, Viral; Chlorocebus aethiops; Cricetinae; Guinea Pigs; Mesocricetus; Mice; Rabies; Rabies Vaccines; Rabies virus; Vero Cells
PubMed: 35786353
DOI: 10.1016/j.biologicals.2022.06.004 -
Acta Neuropathologica Communications Nov 2020The highly neurotropic rabies virus (RABV) enters peripheral neurons at axon termini and requires long distance axonal transport and trans-synaptic spread between...
The highly neurotropic rabies virus (RABV) enters peripheral neurons at axon termini and requires long distance axonal transport and trans-synaptic spread between neurons for the infection of the central nervous system (CNS). Recent 3D imaging of field RABV-infected brains revealed a remarkably high proportion of infected astroglia, indicating that highly virulent field viruses are able to suppress astrocyte-mediated innate immune responses and virus elimination pathways. While fundamental for CNS invasion, in vivo field RABV spread and tropism in peripheral tissues is understudied. Here, we used three-dimensional light sheet and confocal laser scanning microscopy to investigate the in vivo distribution patterns of a field RABV clone in cleared high-volume tissue samples after infection via a natural (intramuscular; hind leg) and an artificial (intracranial) inoculation route. Immunostaining of virus and host markers provided a comprehensive overview of RABV infection in the CNS and peripheral nerves after centripetal and centrifugal virus spread. Importantly, we identified non-neuronal, axon-ensheathing neuroglia (Schwann cells, SCs) in peripheral nerves of the hind leg and facial regions as a target cell population of field RABV. This suggests that virus release from axons and infected SCs is part of the RABV in vivo cycle and may affect RABV-related demyelination of peripheral neurons and local innate immune responses. Detection of RABV in axon-surrounding myelinating SCs after i.c. infection further provided evidence for anterograde spread of RABV, highlighting that RABV axonal transport and spread of infectious virus in peripheral nerves is not exclusively retrograde. Our data support a new model in which, comparable to CNS neuroglia, SC infection in peripheral nerves suppresses glia-mediated innate immunity and delays antiviral host responses required for successful transport from the peripheral infection sites to the brain.
Topics: Animals; Axonal Transport; Axons; Brain; Imaging, Three-Dimensional; Immunity, Innate; Mice; Microscopy, Confocal; Neuroglia; Neurons; Peripheral Nerves; RNA, Viral; Rabies; Rabies virus; Schwann Cells; Viral Tropism
PubMed: 33228789
DOI: 10.1186/s40478-020-01074-6 -
International Journal of Molecular... Apr 2023Rabies is a highly fatal disease, and it is vital to find effective ways to manage and control infection. There is a need for new effective antiviral drugs that are...
Rabies is a highly fatal disease, and it is vital to find effective ways to manage and control infection. There is a need for new effective antiviral drugs that are particularly effective treatments for rabies. Deoxynivalenol (DON) is known mainly for its toxicity, but at the molecular level, it can inhibit RNA and DNA replication, and there is increasing evidence that different doses of DON have a positive effect on inhibiting virus replication. Based on this, we evaluated the effect of DON on inhibiting the rabies virus in vitro. The inhibitory effect of DON on rabies virus activity was dose- and time-dependent, and 0.25 μg/mL of DON could inhibit 99% of rabies virus activity within 24 h. Furthermore, DON could inhibit the adsorption, entry, replication, and release of rabies virus but could not inactivate the virus. The inhibitory effect of DON on rabies virus may be achieved by promoting apoptosis. Our study provides a new perspective for the study of anti-rabies virus and expands the direction of action of mycotoxins.
Topics: Mycotoxins; Rabies virus; Trichothecenes; Virus Replication
PubMed: 37175500
DOI: 10.3390/ijms24097793 -
Viruses Feb 2020Currently, no rabies virus-specific antiviral drugs are available. Ranpirnase has strong antitumor and antiviral properties associated with its ribonuclease activity....
Currently, no rabies virus-specific antiviral drugs are available. Ranpirnase has strong antitumor and antiviral properties associated with its ribonuclease activity. TMR-001, a proprietary bulk drug substance solution of ranpirnase, was evaluated against rabies virus in three cell types: mouse neuroblastoma, BSR (baby hamster kidney cells), and bat primary fibroblast cells. When TMR-001 was added to cell monolayers 24 h preinfection, rabies virus release was inhibited for all cell types at three time points postinfection. TMR-001 treatment simultaneous with infection and 24 h postinfection effectively inhibited rabies virus release in the supernatant and cell-to-cell spread with 50% inhibitory concentrations of 0.2-2 nM and 20-600 nM, respectively. TMR-001 was administered at 0.1 mg/kg via intraperitoneal, intramuscular, or intravenous routes to Syrian hamsters beginning 24 h before a lethal rabies virus challenge and continuing once per day for up to 10 days. TMR-001 at this dose, formulation, and route of delivery did not prevent rabies virus transit from the periphery to the central nervous system in this model ( = 32). Further aspects of local controlled delivery of other active formulations or dose concentrations of TMR-001 or ribonuclease analogues should be investigated for this class of drugs as a rabies antiviral therapeutic.
Topics: Animals; Antiviral Agents; Cell Line; Cells, Cultured; Chiroptera; Cricetinae; Female; Fibroblasts; Mesocricetus; Mice; Rabies; Rabies virus; Ribonucleases; Virus Release; Virus Replication
PubMed: 32033253
DOI: 10.3390/v12020177 -
Frontiers in Veterinary Science 2022Rabies is a fatal encephalitis caused by the rabies virus. The diagnosis of the disease depends in large part on the exposure history of the victim and clinical...
Rabies is a fatal encephalitis caused by the rabies virus. The diagnosis of the disease depends in large part on the exposure history of the victim and clinical manifestations of the disease. Rapid rabies diagnosis is an important step in its prevention and control. Therefore, for accurate and timely diagnosis and prevention of rabies, we developed nanomaterials for a novel photoelectrochemical biosensing approach (PBA) for the rapid and reliable diagnosis of rabies virus. This approach uses high-efficiency exciton energy transfer between cadmium telluride quantum dots and Au nanoparticles and is low cost, and easy to miniaturize. By constructing PBA, rabies virus can be detected quickly and with a high degree of sensitivity and specificity; the minimum detection concentration limit for rabies virus is approximately 2.16 ffu/mL of rabies virus particles, or 2.53 × 10 fg/mL of rabies virus RNA. PBA could also detect rabies virus in the brain and lung tissue from rabid dogs and mice with better sensitivity than RT-PCR.
PubMed: 36619964
DOI: 10.3389/fvets.2022.1079916 -
Bioinformation 2023Rabies virus is a zoonotic pathogen that causes lethal encephalitis with a case fatality rate of almost 100% in unvaccinated individuals. The currently available...
Rabies virus is a zoonotic pathogen that causes lethal encephalitis with a case fatality rate of almost 100% in unvaccinated individuals. The currently available vaccines against rabies are composed of inactivated viral particles that only confer a short-term immune response. It is well-known that the entry of rabies virus into host cells is mediated by a trimeric glycoprotein presents on the surface of viral envelope. As the sole viral surface protein, this trimeric glycoprotein represents a promising molecular target to design new vaccines and neutralizing antibodies against rabies virus. Epitope mapping studies had identified several antigenic sites on the surface of trimeric pre-fusion glycoprotein of rabies virus. Therefore, it is of interest to screen the rabies virus glycoprotein by different web-based immuno-informatics tools to identify potential B-cells and T-cells linear epitopes. Here, we present a construct of peptide vaccine that consists of these predicted linear epitopes of rabies virus glycoprotein together with appropriate linkers and adjuvant. Various online prediction tools, molecular docking and dynamics simulation assume that the vaccine construct may be stable, safe and effective. However, validation of these in-silico results is necessary both in vitro and in vivo setting.
PubMed: 37814687
DOI: 10.6026/97320630019167 -
Journal of Virology Jun 2020Rabies virus (RABV) causes a severe and fatal neurological disease, but morbidity is vaccine preventable and treatable prior to the onset of clinical symptoms. However,...
Rabies virus (RABV) causes a severe and fatal neurological disease, but morbidity is vaccine preventable and treatable prior to the onset of clinical symptoms. However, immunoglobulin (IgG)-based rabies postexposure prophylaxis (PEP) is expensive, restricting access to life-saving treatment, especially for patients in low-income countries where the clinical need is greatest, and does not confer cross-protection against newly emerging phylogroup II lyssaviruses. Toward identifying a cost-effective replacement for the IgG component of rabies PEP, we developed and implemented a high-throughput screening protocol utilizing a single-cycle RABV reporter strain. A large-scale screen and subsequent direct and orthogonal counterscreens identified a first-in-class direct-acting RABV inhibitor, GRP-60367, with a specificity index (SI) of >100,000. Mechanistic characterization through time-of-addition studies, transient cell-to-cell fusion assays, and chimeric vesicular stomatitis virus (VSV) recombinants expressing the RABV glycoprotein (G) demonstrated that GRP-60367 inhibits entry of a subset of RABV strains. Resistance profiling of the chemotype revealed hot spots in conserved hydrophobic positions of the RABV G protein fusion loop that were confirmed in transient cell-to-cell fusion assays. Transfer of RABV G genes with signature resistance mutations into a recombinant VSV backbone resulted in the recovery of replication-competent virions with low susceptibility to the inhibitor. This work outlines a tangible strategy for mechanistic characterization and resistance profiling of RABV drug candidates and identified a novel, well-behaved molecular probe chemotype that specifically targets the RABV G protein and prevents G-mediated viral entry. Rabies PEP depends on anti-RABV IgG, which is expensive and in limited supply in geographical areas with the highest disease burden. Replacing the IgG component with a cost-effective and shelf-stable small-molecule antiviral could address this unmet clinical need by expanding access to life-saving medication. This study has established a robust protocol for high-throughput anti-RABV drug screens and identified a chemically well-behaved, first-in-class hit with nanomolar anti-RABV potency that blocks RABV G protein-mediated viral entry. Resistance mapping revealed a druggable site formed by the G protein fusion loops that has not previously emerged as a target for neutralizing antibodies. Discovery of this RABV entry inhibitor establishes a new molecular probe to advance further mechanistic and structural characterization of RABV G that may aid in the design of a next-generation clinical candidate against RABV.
Topics: Animals; Antibodies, Neutralizing; Antibodies, Viral; Antiviral Agents; Cell Line; Cross Protection; Drug Evaluation, Preclinical; Humans; Peptide Library; Rabies; Rabies Vaccines; Rabies virus; Vesicular stomatitis Indiana virus; Vesiculovirus; Viral Fusion Proteins
PubMed: 32321812
DOI: 10.1128/JVI.00321-20 -
Pathogens (Basel, Switzerland) Feb 2022In 2019, the World Health Organization (WHO) and the Pan-American Health Organization (PAHO) recognized Mexico as a country free of human rabies transmitted by dogs....
In 2019, the World Health Organization (WHO) and the Pan-American Health Organization (PAHO) recognized Mexico as a country free of human rabies transmitted by dogs. Nevertheless, the sylvatic cycle remains as a public health concern in the country. Although cougars () are not reservoirs of any rabies virus variant (RVV), these felines could act as vectors at the top of the food chain, and their relationships with other organisms must be considered important for the regulatory effect on their prey's populations. In this study, genetic and antigenic characterization was performed on all cougar rabies cases diagnosed at the Rabies Laboratory Network of the Ministry of Health (RLNMH) in Mexico from 2000 to 2021. Samples from other species, a skunk, a horse () (attacked by a cougar), and a gray fox (), were included as reference. Rabies cases in cougars were restricted to two Northern states of Mexico (Sonora and Chihuahua). Five out of six samples of cougars were RVV7 (Arizona gray fox RVV) and one from Sonora was RVV1. Interestingly, there is no evidence of RVV1 in dogs in the Northern states since the 1990s but skunk species now harbor this RVV1 in this region of the country.
PubMed: 35215207
DOI: 10.3390/pathogens11020265