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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 in vitro, and is an important internalization factor for SARS-CoV-2 in vitro and in vivo. Here, we demonstrate that mGluR2 facilitates RABV internalization in vitro and infection in vivo. 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. IMPORTANCE We previously found that metabotropic glutamate receptor subtype 2 (mGluR2) is an entry receptor for RABV in vitro, and an important internalization factor for SARS-CoV-2 in vitro and in vivo. However, whether mGluR2 is required for RABV infection in vivo 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 in vivo. 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.
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COVID-19 , Virus de la Rabia , Receptores de Glutamato Metabotrópico , Receptores de Transferrina , SARS-CoV-2 , Internalización del Virus , Animales , Humanos , Ratones , Rabia/metabolismo , Virus de la Rabia/fisiología , Receptores de Glutamato Metabotrópico/metabolismo , Receptores de Transferrina/metabolismo , SARS-CoV-2/fisiología , Glicoproteína de la Espiga del Coronavirus/metabolismoRESUMEN
Rabies virus (RABV) is a prototypical neurotropic virus that causes rabies in human and animals with an almost 100% mortality rate. Once RABV enters the central nervous system, no treatment is proven to prevent death. RABV glycoprotein (G) interacts with cell surface receptors and then enters cells via clathrin-mediated endocytosis (CME); however, the key host factors involved remain largely unknown. Here, we identified transferrin receptor 1 (TfR1), a classic receptor that undergoes CME, as an entry factor for RABV. TfR1 interacts with RABV G and is involved in the endocytosis of RABV. An antibody against TfR1 or the TfR1 ectodomain soluble protein significantly blocked RABV infection in HEK293 cells, N2a cells, and mouse primary neuronal cells. We further found that the endocytosis of TfR1 is coupled with the endocytosis of RABV and that TfR1 and RABV are transported to early and late endosomes. Our results suggest that RABV hijacks the transport pathway of TfR1 for entry, thereby deepening our understanding of the entry mechanism of RABV. IMPORTANCE For most viruses, cell entry involves engagement with many distinct plasma membrane components, each of which is essential. After binding to its specific receptor(s), rabies virus (RABV) enters host cells through the process of clathrin-mediated endocytosis. However, whether the receptor-dependent clathrin-mediated endocytosis of RABV requires other plasma membrane components remain largely unknown. Here, we demonstrate that transferrin receptor 1 (TfR1) is a functional entry factor for RABV infection. The endocytosis of RABV is coupled with the endocytosis of TfR1. Our results indicate that RABV hijacks the transport pathway of TfR1 for entry, which deepens our understanding of the entry mechanism of RABV.
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Virus de la Rabia , Rabia , Receptores de Transferrina , Internalización del Virus , Animales , Humanos , Ratones , Clatrina/metabolismo , Células HEK293 , Rabia/metabolismo , Virus de la Rabia/metabolismo , Receptores de Transferrina/metabolismo , Línea Celular , EndocitosisRESUMEN
The continuous emergence of severe acute respiratory coronavirus 2 (SARS-CoV-2) variants and the increasing number of breakthrough infection cases among vaccinated people support the urgent need for research and development of antiviral drugs. Viral entry is an intriguing target for antiviral drug development. We found that diltiazem, a blocker of the L-type calcium channel Cav1.2 pore-forming subunit (Cav1.2 α1c) and an FDA-approved drug, inhibits the binding and internalization of SARS-CoV-2, and decreases SARS-CoV-2 infection in cells and mouse lung. Cav1.2 α1c interacts with SARS-CoV-2 spike protein and ACE2, and affects the attachment and internalization of SARS-CoV-2. Our finding suggests that diltiazem has potential as a drug against SARS-CoV-2 infection and that Cav1.2 α1c is a promising target for antiviral drug development for COVID-19.
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Tratamiento Farmacológico de COVID-19 , COVID-19 , Diltiazem/farmacología , Pulmón/efectos de los fármacos , SARS-CoV-2/efectos de los fármacos , Células A549 , Animales , COVID-19/patología , COVID-19/virología , Células Cultivadas , Chlorocebus aethiops , Diltiazem/uso terapéutico , Modelos Animales de Enfermedad , Femenino , Células HEK293 , Células HeLa , Humanos , Pulmón/patología , Pulmón/virología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , SARS-CoV-2/fisiología , Células Vero , Acoplamiento Viral/efectos de los fármacos , Internalización del Virus/efectos de los fármacosRESUMEN
Rabies virus (RABV) matrix protein (M) plays crucial roles in viral transcription, replication, assembly, and budding; however, its function during the early stage of virus replication remains unknown. Here, we mapped the protein interactome between RABV M and human host factors using a proteomic approach, finding a link to the V-type proton ATPase catalytic subunit A (ATP6V1A), which is located in the endosomes where RABV first enters. By downregulating or upregulating ATP6V1A expression in HEK293T cells, we found that ATP6V1A facilitated RABV replication. We further found that ATP6V1A was involved in the dissociation of incoming viral M proteins during viral uncoating. Coimmunoprecipitation demonstrated that M interacted with the full length or middle domain of ATP6V1A, which was dependent on the lysine residue at position 256 and the glutamic acid residue at position 279. RABV growth and uncoating in ATP6V1A-depleted cells was restored by trans-complementation with the full length or interaction domain of ATP6V1A. Moreover, stably overexpressed ATP6V1A enhanced RABV growth in Vero cells, which are used for the production of rabies vaccine. Our findings identify a new partner for RABV M proteins and establish a new role of ATP6V1A by promoting virion uncoating during RABV replication.
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ATPasas de Translocación de Protón Vacuolares/metabolismo , Animales , Supervivencia Celular/genética , Supervivencia Celular/fisiología , Chlorocebus aethiops , Células HEK293 , Humanos , Inmunoprecipitación , Espectrometría de Masas , Plásmidos/genética , Proteómica , Interferencia de ARN , Rabia/inmunología , Rabia/prevención & control , Vacunas Antirrábicas/inmunología , Vacunas Antirrábicas/uso terapéutico , Virus de la Rabia/inmunología , Virus de la Rabia/patogenicidad , ATPasas de Translocación de Protón Vacuolares/genética , Células Vero , Replicación Viral/genética , Replicación Viral/fisiologíaRESUMEN
Online forumpost evaluationis an effective way for instructors to assess students' knowledge understanding and writing mechanics. Manually evaluating massive posts costs a lot of time. Automatically grading online posts could significantly alleviate instructors' burden. Similar text assessment tasks like Automated Text Scoring evaluate the writing quality of independent texts or relevance between text and prompt. And Automatic Short Answer Grading measures the semantic matching of short answers according to given problems and correct answers. Different from existing tasks, we propose a novel task, Automated Post Scoring (APS), which grades all online discussion posts in each thread of each student with given topics and quoted posts. APS evaluates not only the writing quality of posts automatically but also the relevance to topics. To measure the relevance, we model the semantic consistency between posts and topics. Supporting arguments are also extracted from quoted posts to enhance posts evaluation. Specifically, we propose a mixture model including a hierarchical text model to measure the writing quality, a semantic matching model to model topic relevance, and a semantic representation model to integrate quoted posts. We also construct a new dataset called Online Discussion Dataset containing 2,542 online posts from 694 students of a social science course. The proposed models are evaluated on the dataset with correlation and residual based evaluation metrics. Compared with measuring posts alone, experimental results demonstrate that incorporating topics and quoted posts could improve the performance of APS by a large margin, more than 9 percent on QWK.
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Rabies virus (RABV) is a widespread pathogen that causes fatal disease in humans and animals. It has been suggested that multiple host factors are involved in RABV host entry. Here, we showed that RABV uses integrin ß1 (ITGB1) for cellular entry. RABV infection was drastically decreased after ITGB1 short interfering RNA knockdown and moderately increased after ITGB1 overexpression in cells. ITGB1 directly interacts with RABV glycoprotein. Upon infection, ITGB1 is internalized into cells and transported to late endosomes together with RABV. The infectivity of cell-adapted RABV in cells and street RABV in mice was neutralized by ITGB1 ectodomain soluble protein. The role of ITGB1 in RABV infection depends on interaction with fibronectin in cells and mice. We found that Arg-Gly-Asp (RGD) peptide and antibody to ITGB1 significantly blocked RABV infection in cells in vitro and street RABV infection in mice via intramuscular inoculation but not the intracerebral route. ITGB1 also interacts with nicotinic acetylcholine receptor, which is the proposed receptor for peripheral RABV infection. Our findings suggest that ITGB1 is a key cellular factor for RABV peripheral entry and is a potential therapeutic target for postexposure treatment against rabies.IMPORTANCE Rabies is a severe zoonotic disease caused by rabies virus (RABV). However, the nature of RABV entry remains unclear, which has hindered the development of therapy for rabies. It is suggested that modulations of RABV glycoprotein and multiple host factors are responsible for RABV invasion. Here, we showed that integrin ß1 (ITGB1) directly interacts with RABV glycoprotein, and both proteins are internalized together into host cells. Differential expression of ITGB1 in mature muscle and cerebral cortex of mice led to A-4 (ITGB1-specific antibody), and RGD peptide (competitive inhibitor for interaction between ITGB1 and fibronectin) blocked street RABV infection via intramuscular but not intracerebral inoculation in mice, suggesting that ITGB1 plays a role in RABV peripheral entry. Our study revealed this distinct cellular factor in RABV infection, which may be an attractive target for therapeutic intervention.
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Integrina beta1/metabolismo , Virus de la Rabia/metabolismo , Rabia/metabolismo , Proteínas Virales de Fusión/metabolismo , Internalización del Virus , Animales , Endosomas/genética , Endosomas/metabolismo , Endosomas/virología , Fibronectinas/genética , Fibronectinas/metabolismo , Células HEK293 , Humanos , Integrina beta1/genética , Ratones , Oligopéptidos/farmacología , Rabia/tratamiento farmacológico , Rabia/genética , Rabia/patología , Virus de la Rabia/genética , Proteínas Virales de Fusión/genéticaRESUMEN
Rabies virus (RABV) invades the central nervous system and nearly always causes fatal disease in humans. How RABV interacts with host neuron membrane receptors to become internalized and cause rabid symptoms is not yet fully understood. Here, we identified a novel receptor of RABV, which RABV uses to infect neurons. We found that metabotropic glutamate receptor subtype 2 (mGluR2), a member of the G protein-coupled receptor family that is abundant in the central nervous system, directly interacts with RABV glycoprotein to mediate virus entry. RABV infection was drastically decreased after mGluR2 siRNA knock-down in cells. Antibodies to mGluR2 blocked RABV infection in cells in vitro. Moreover, mGluR2 ectodomain soluble protein neutralized the infectivity of RABV cell-adapted strains and a street strain in cells (in vitro) and in mice (in vivo). We further found that RABV and mGluR2 are internalized into cells and transported to early and late endosomes together. These results suggest that mGluR2 is a functional cellular entry receptor for RABV. Our findings may open a door to explore and understand the neuropathogenesis of rabies.
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Virus de la Rabia/patogenicidad , Rabia/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Internalización del Virus , Animales , Línea Celular , Humanos , Ratones , Rabia/fisiopatologíaRESUMEN
Bovine ephemeral fever (BEF) is caused by the arthropod-borne bovine ephemeral fever virus (BEFV), which is a member of the family Rhabdoviridae and the genus Ephemerovirus. BEFV causes an acute febrile infection in cattle and water buffalo. In this study, a recombinant Newcastle disease virus (NDV) expressing the glycoprotein (G) of BEFV (rL-BEFV-G) was constructed, and its biological characteristics in vitro and in vivo, pathogenicity, and immune response in mice and cattle were evaluated. BEFV G enabled NDV to spread from cell to cell. rL-BEFV-G remained nonvirulent in poultry and mice compared with vector LaSota virus. rL-BEFV-G triggered a high titer of neutralizing antibodies against BEFV in mice and cattle. These results suggest that rL-BEFV-G might be a suitable candidate vaccine against BEF.
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Anticuerpos Neutralizantes/biosíntesis , Anticuerpos Antivirales/biosíntesis , Virus de la Fiebre Efímera Bovina/genética , Fiebre Efímera/prevención & control , Virus de la Enfermedad de Newcastle/genética , Vacunas Virales/genética , Animales , Bovinos , Embrión de Pollo , Cricetinae , Perros , Fiebre Efímera/inmunología , Fiebre Efímera/virología , Virus de la Fiebre Efímera Bovina/efectos de los fármacos , Virus de la Fiebre Efímera Bovina/inmunología , Células Epiteliales/virología , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/inmunología , Glicoproteínas/administración & dosificación , Glicoproteínas/genética , Glicoproteínas/inmunología , Inmunización , Células de Riñón Canino Madin Darby , Ratones , Ratones Endogámicos BALB C , Virus de la Enfermedad de Newcastle/inmunología , Virus Reordenados/genética , Virus Reordenados/inmunología , Vacunas Sintéticas , Proteínas Virales/administración & dosificación , Proteínas Virales/genética , Proteínas Virales/inmunología , Vacunas Virales/administración & dosificación , Vacunas Virales/inmunologíaRESUMEN
BACKGROUND: Vesicular stomatitis virus (VSV) causes severe losses to the animal husbandry industry. In this study, a recombinant Newcastle disease virus (NDV) expressing the glycoprotein (G) of VSV (rL-VSV-G) was constructed and its pathogenicity and immune protective efficacy in mouse were evaluated. RESULTS: In pathogenicity evaluation test, the analysis of the viral distribution in mouse organs and body weight change showed that rL-VSV-G was safe in mice. In immune protection assay, the recombinant rL-VSV-G triggered a high titer of neutralizing antibodies against VSV. After challenge, the wild-type (wt) VSV viral load in mouse organs was lower in rL-VSV-G group than that in rLaSota groups. wt VSV was not detected in the blood, liver, or kidneys of mice, whereas it was found in these tissues in control groups. The mice body weight had no significant change after challenge in the rL-VSV-G group. Additionally, suckling mice produced from female mice immunized with rL-VSV-G were partially protected from wt VSV challenge. CONCLUSIONS: These results demonstrated that rL-VSV-G may be a suitable candidate vaccine against vesicular stomatitis (VS).
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Expresión Génica , Vectores Genéticos/genética , Glicoproteínas de Membrana/genética , Virus de la Enfermedad de Newcastle/genética , Estomatitis Vesicular/inmunología , Virus de la Estomatitis Vesicular Indiana/genética , Virus de la Estomatitis Vesicular Indiana/inmunología , Proteínas del Envoltorio Viral/genética , Animales , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Embrión de Pollo , Modelos Animales de Enfermedad , Orden Génico , Glicoproteínas de Membrana/inmunología , Ratones , Enfermedades de las Aves de Corral , Estomatitis Vesicular/mortalidad , Estomatitis Vesicular/prevención & control , Proteínas del Envoltorio Viral/inmunologíaRESUMEN
BACKGROUND: West Nile virus (WNV) is an emerging zoonotic pathogen which is harmful to human and animal health. Effective vaccination in susceptible hosts should protect against WNV infection and significantly reduce viral transmission between animals and from animals to humans. A versatile vaccine suitable for different species that can be delivered via flexible routes remains an essential unmet medical need. In this study, we developed a recombinant avirulent Newcastle disease virus (NDV) LaSota strain expressing WNV premembrane/envelope (PrM/E) proteins (designated rLa-WNV-PrM/E) and evaluated its immunogenicity in mice, horses, chickens, ducks and geese. RESULTS: Mouse immunization experiments disclosed that rLa-WNV-PrM/E induces significant levels of WNV-neutralizing antibodies and E protein-specific CD4+ and CD8+ T-cell responses. Moreover, recombinant rLa-WNV-PrM/E elicited significant levels of WNV-specific IgG in horses upon delivery via intramuscular immunization, and in chickens, ducks and geese via intramuscular, oral or intranasal immunization. CONCLUSIONS: Our results collectively support the utility of rLa-WNV-PrM/E as a promising WNV veterinary vaccine candidate for mammals and poultry.
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Mamíferos/inmunología , Virus de la Enfermedad de Newcastle/genética , Enfermedades de las Aves de Corral/inmunología , Vacunas Virales/inmunología , Fiebre del Nilo Occidental/prevención & control , Virus del Nilo Occidental/inmunología , Animales , Anticuerpos Antivirales/inmunología , Pollos , Femenino , Vectores Genéticos/genética , Vectores Genéticos/inmunología , Humanos , Mamíferos/virología , Ratones , Virus de la Enfermedad de Newcastle/metabolismo , Enfermedades de las Aves de Corral/prevención & control , Enfermedades de las Aves de Corral/virología , Vacunación , Proteínas del Envoltorio Viral/administración & dosificación , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/inmunología , Vacunas Virales/administración & dosificación , Vacunas Virales/genética , Fiebre del Nilo Occidental/inmunología , Fiebre del Nilo Occidental/virología , Virus del Nilo Occidental/genéticaRESUMEN
Peste des petits ruminants (PPR) and foot-and-mouth disease (FMD) are both highly contagious diseases of small domestic and wild ruminants caused by the PPR virus (PPRV) and the FMD virus (FMDV). In this study, a recombinant PPRV expressing the FMDV VP1 gene (rPPRV/VP1) was generated and FMDV VP1 expression did not impair replication of the recombinant virus in vitro and immunogenicity in inducing neutralizing antibody against PPR in goats. Vaccination with one dose of rPPRV/VP1 induced FMDV neutralizing antibody in goats and protected them from challenge with virulent FMDV. Our results suggest that the recombinant PPRV expressing the FMDV VP1 protein is a potential dual live vectored vaccine against PPRV and FMDV.
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Virus de la Fiebre Aftosa/inmunología , Fiebre Aftosa/prevención & control , Enfermedades de las Cabras/prevención & control , Peste de los Pequeños Rumiantes/prevención & control , Virus de la Peste de los Pequeños Rumiantes/inmunología , Proteínas Virales/genética , Vacunas Virales/inmunología , Animales , Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , Fiebre Aftosa/virología , Virus de la Fiebre Aftosa/genética , Enfermedades de las Cabras/virología , Cabras , Inmunidad Activa/efectos de los fármacos , Pruebas de Neutralización/veterinaria , Peste de los Pequeños Rumiantes/virología , Virus de la Peste de los Pequeños Rumiantes/genética , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunología , Proteínas Virales/metabolismo , Vacunas Virales/genéticaRESUMEN
Background: Lung cancer (LC) is one of the most common malignant tumors in the world and the leading cause of cancer-related deaths, which seriously threatens human life and health as well as brings a heavy burden to the society. In recent years, the tumor microenvironment (TME) has become an emerging research field and hotspot affecting tumor pathogenesis and therapeutic approaches. However, to date, there has been no bibliometric analysis of lung cancer and the tumor microenvironment from 2014 to 2023.This study aims to comprehensively summarize the current situation and development trends in the field from a bibliometric perspective. Methods: The publications about lung cancer and the tumor microenvironment from 2014 to 2023 were extracted from the Web of Science Core Collection (WoSCC). The Microsoft Excel, Origin, R-bibliometrix, CiteSpace, and VOSviewer software are comprehensively used to scientifically analyze the data. Results: Totally, 763 publications were identified in this study. A rapid increase in the number of publications was observed after 2018. More than 400 organizations published these publications in 36 countries or regions. China and the United States have significant influence in this field. Zhou, CC and Frontiers in Immunology are the most productive authors and journals respectively. Besides, the most frequently cited references were those on lung cancer pathogenesis, clinical trials, and treatment modalities. It suggests that novel lung cancer treatment models mainly based on the TME components, such as cancer-associated fibroblasts (CAFs) may lead to future research trends. Conclusions: The field of lung cancer and the tumor microenvironment research is still in the beginning stages. Gene expression, molecular pathways, therapeutic modalities, and novel detection technologies in this field have been widely studied by researchers. This is the first bibliometric study to comprehensively summarize the research trend and development regarding lung cancer and tumor microenvironment over the last decade. The result of our research provides the updated perspective for scholars to understand the key information and cutting-edge hotspots in this field, as well as to identify future research directions.
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Jumping, a fundamental survival behavior observed in organisms, serves as a vital mechanism for adapting to the surrounding environment and overcoming significant obstacles within a given terrain. Here, we present a light-controlled soft jumping actuator inspired by asphondylia, which employs a closed-loop structure and utilizes a liquid crystal elastomer (LCE). Photo-mechanical coupling highlights the significant influence of the light source on the actuator's jumping behavior. Manipulating the light intensity, the relative position of stimulus and light lock, and the concentration of disperse red 1 (DR1) allows precise control over both the maximum take-off velocity and jump height. Furthermore, tailoring the size of the LCE actuator offers a means of regulating jumping behavior. Upon exposure to 460 nm LED irradiation, our actuator achieves remarkable performance, with a maximum jumping height of 10 body length (BL) and take-off velocity of 62 BL/s. These actuators accumulate and rapidly release energy, enabling the effective transportation of microcargos across substantial distances. Our research yields valuable insights into the realm of soft robotics, underscoring the pivotal importance of photo-mechanical coupling in the field of soft robotics, thereby serving as a catalyst for inspiring continued exploration of agile and capable systems by prestoring elastic energy.
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OBJECTIVE: To assess the diagnostic efficacy of the CEUS LI-RADS combined with a model constructed on the basis of age, sex, AFP, and PIVKA-II (ASAP) for the diagnosis of HCC in high-risk patients. METHODS: This retrospective study included 366 liver lesions from 366 patients who underwent liver CEUS. All liver lesions were characterized and categorized according to CEUS LI-RADS v2017. Two modified methods were applied: LR-3/4/M nodules accompanied by AFP > 200 ng/mL (Criterion 2) or ASAP model score > 0.5256 and CA 19-9 in the normal range (Criterion 3) were recategorized as LR-5. The reference criteria included histopathological or comprehensive imaging and the clinical follow-up results. The diagnostic performance was evaluated and compared by the sensitivity, specificity, PPV, and NPV. RESULTS: The incidence of HCC in LR-3, LR-4, LR-5, and LR-M was 33.3% (4/12), 86.4% (38/44), 98.5% (191/194) and 82.7% (81/98), respectively. After using Criterion 2 compared to CEUS LI-RADS v2017, the sensitivity of the modified LR-5 for diagnosing HCC increased from 60.8% to 70.7% (p < 0.01) with little effect on its specificity (94.2% vs. 92.3%, p = 1.00) or PPV (98.5% vs. 98.2%, p = 0.86). After using Criterion 3, the sensitivity of the modified LR-5 for the diagnosis of HCC was further improved to 86.9% (p < 0.01), and its specificity and PPV were not significantly changed (92.3% and 98.6%, both p > 0.05). CONCLUSION: CEUS LI-RADS combined with the serum biomarker-based ASAP model improved the sensitivity of LR-5 in diagnosing HCC with little effect on its specificity and PPV.
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SARS-CoV-2 and filovirus enter cells via the cell surface angiotensin-converting enzyme 2 (ACE2) or the late-endosome Niemann-Pick C1 (NPC1) as a receptor. Here, we screened 974 natural compounds and identified Tubeimosides I, II, and III as pan-coronavirus and filovirus entry inhibitors that target NPC1. Using in-silico, biochemical, and genomic approaches, we provide evidence that NPC1 also binds SARS-CoV-2 spike (S) protein on the receptor-binding domain (RBD), which is blocked by Tubeimosides. Importantly, NPC1 strongly promotes productive SARS-CoV-2 entry, which we propose is due to its influence on fusion in late endosomes. The Tubeimosides' antiviral activity and NPC1 function are further confirmed by infection with SARS-CoV-2 variants of concern (VOC), SARS-CoV, and MERS-CoV. Thus, NPC1 is a critical entry co-factor for highly pathogenic human coronaviruses (HCoVs) in the late endosomes, and Tubeimosides hold promise as a new countermeasure for these HCoVs and filoviruses.
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Ebolavirus , Receptores Virales , Humanos , Unión Proteica , Receptores Virales/metabolismo , Proteína Niemann-Pick C1/metabolismo , Ebolavirus/fisiología , Internalización del Virus , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Glicoproteína de la Espiga del Coronavirus/metabolismoRESUMEN
BACKGROUND: Using reverse genetics, we generated a recombinant low-pathogenic LaSota strain Newcastle disease virus (NDV) expressing the glycoprotein (GP) of Ebola virus (EBOV), designated rLa-EBOVGP, and evaluated its biological characteristic in vivo and in vitro. RESULTS: The introduction and expression of the EBOV GP gene did not increase the virulence of the NDV vector in poultry or mice. EBOV GP was incorporated into the particle of the vector virus and the recombinant virus rLa-EBOVGP infected cells and spread within them independently of exogenous trypsin. rLa-EBOVGP is more resistant to NDV antiserum than the vector NDV and is moderately sensitive to EBOV GP antiserum. More importantly, infection with rLa-EBOVGP was markedly inhibited by IPA3, indicating that rLa-EBOVGP uses macropinocytosis as the major internalization pathway for cell entry. CONCLUSIONS: The results demonstrate that EBOV GP in recombinant NDV particles functions independently to mediate the viral infection of the host cells and alters the cell-entry pathway.
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Endocitosis , Virus de la Enfermedad de Newcastle/fisiología , Proteínas del Envoltorio Viral/metabolismo , Internalización del Virus , Animales , Línea Celular , Cricetinae , Ratones , Virus de la Enfermedad de Newcastle/genética , Virus de la Enfermedad de Newcastle/patogenicidad , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Genética Inversa , Análisis de Supervivencia , Proteínas del Envoltorio Viral/genética , VirulenciaRESUMEN
The 3C-like protease (3CLpro) is an essential enzyme for the replication of SARS-CoV-2 and other coronaviruses and thus is a target for coronavirus drug discovery. Nearly all inhibitors of coronavirus 3CLpro reported so far are covalent inhibitors. Here, we report the development of specific, noncovalent inhibitors of 3CLpro. The most potent one, WU-04, effectively blocks SARS-CoV-2 replications in human cells with EC50 values in the 10-nM range. WU-04 also inhibits the 3CLpro of SARS-CoV and MERS-CoV with high potency, indicating that it is a pan-inhibitor of coronavirus 3CLpro. WU-04 showed anti-SARS-CoV-2 activity similar to that of PF-07321332 (Nirmatrelvir) in K18-hACE2 mice when the same dose was administered orally. Thus, WU-04 is a promising drug candidate for coronavirus treatment.
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Effective, safe, and affordable rabies vaccines are still being sought. Newcastle disease virus (NDV), an avian paramyxovirus, has shown promise as a vaccine vector for mammals. Here, we generated a recombinant avirulent NDV La Sota strain expressing the rabies virus glycoprotein (RVG) and evaluated its potential to serve as a vaccine against rabies. The recombinant virus, rL-RVG, retained its high-growth property in chicken eggs, with titers of up to 109·8 50% egg infective doses (EID50)/ml of allantoic fluid. RVG expression enabled rL-RVG to spread from cell to cell in a rabies virus-like manner, and RVG was incorporated on the surface of the rL-RVG viral particle. RVG incorporation did not alter the trypsin-dependent infectivity of the NDV vector in mammalian cells. rL-RVG and La Sota NDV showed similar levels of sensitivity to a neutralization antibody against NDV and similar levels of resistance to a neutralization antibody against rabies virus. Animal studies demonstrated that rL-RVG is safe in several species, including cats and dogs, when administered as multiple high doses of recombinant vaccine. Intramuscular vaccination with rL-RVG induced a substantial rabies virus neutralization antibody response and provided complete protection from challenge with circulating rabies virus strains. Most importantly, rL-RVG induced strong and long-lasting protective neutralization antibody responses to rabies virus in dogs and cats. A low vaccine dose of 108·³ EID50 completely protected dogs from challenge with a circulating strain of rabies virus for more than a year. This is the first study to demonstrate that immunization with an NDV-vectored vaccine can induce long-lasting, systemic protective immunity against rabies.
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Anticuerpos Neutralizantes/biosíntesis , Virus de la Enfermedad de Newcastle/genética , Virus de la Enfermedad de Newcastle/inmunología , Vacunas Antirrábicas , Virus de la Rabia/inmunología , Rabia/prevención & control , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/inmunología , Animales , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/biosíntesis , Anticuerpos Antivirales/inmunología , Western Blotting , Gatos , Línea Celular Tumoral , Chlorocebus aethiops , Cricetinae , Perros , Técnica del Anticuerpo Fluorescente , Regulación Viral de la Expresión Génica , Glicoproteínas/biosíntesis , Glicoproteínas/genética , Glicoproteínas/inmunología , Humanos , Ratones , Virus de la Enfermedad de Newcastle/fisiología , Rabia/inmunología , Vacunas Antirrábicas/administración & dosificación , Vacunas Antirrábicas/genética , Vacunas Antirrábicas/inmunología , Virus de la Rabia/genética , Vacunación , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/inmunología , Células Vero , Proteínas del Envoltorio Viral/biosíntesisRESUMEN
Influenza prophylaxis would benefit from a simple method to administer influenza vaccine into skin without the need for hypodermic needles. In this study, solid metal microneedle arrays (MNs) were investigated as a system for cutaneous vaccine delivery using influenza virus antigen. The MNs with 5 monument-shaped microneedles per array were produced and coated with inactivated influenza virus A/PR/8/34 (IIV). As much as 10 microg of viral proteins could be coated onto an array of 5 microneedles, and the coated IIV was delivered into skin at high efficiency within minutes. The coated MNs were used to immunize mice in comparison with conventional intramuscular injection at the same dose. Analysis of immune responses showed that a single immunization with IIV-coated MNs induced strong antibody responses against influenza virus, with significant levels of hemagglutination inhibition activities (>1:40), which were comparable to those induced by conventional intramuscular immunization. Moreover, mice immunized by a single dose of IIV coated on MNs were effectively protected against lethal challenge by a high dose of mouse-adapted influenza virus A/PR/8/34. These results show that MNs are highly effective as a simple method of vaccine delivery to elicit protective immune responses against virus infection.
Asunto(s)
Inmunización/métodos , Vacunas contra la Influenza/administración & dosificación , Orthomyxoviridae/inmunología , Administración Cutánea , Animales , Anticuerpos/química , Citocinas/metabolismo , Ensayo de Inmunoadsorción Enzimática , Femenino , Hemaglutinación , Glicoproteínas Hemaglutininas del Virus de la Influenza/química , Virus de la Influenza A/inmunología , Ratones , Ratones Endogámicos BALB C , Vacunas/químicaRESUMEN
Severe acute respiratory syndrome (SARS) is a highly contagious zoonotic disease caused by SARS coronavirus (SARS-CoV). Since its outbreak in Guangdong Province of China in 2002, SARS has caused 8096 infections and 774 deaths by December 31st, 2003. Although there have been no more SARS cases reported in human populations since 2004, the recent emergence of a novel coronavirus disease (COVID-19) indicates the potential of the recurrence of SARS and other coronavirus disease among humans. Thus, developing a rapid response SARS vaccine to provide protection for human populations is still needed. Spike (S) protein of SARS-CoV can induce neutralizing antibodies, which is a pivotal immunogenic antigen for vaccine development. Here we constructed a recombinant chimeric vesicular stomatitis virus (VSV) VSVΔG-SARS, in which the glycoprotein (G) gene is replaced with the SARS-CoV S gene. VSVΔG-SARS maintains the bullet-like shape of the native VSV, with the heterogeneous S protein incorporated into its surface instead of G protein. The results of safety trials revealed that VSVΔG-SARS is safe and effective in mice at a dose of 1 â× â106 TCID50. More importantly, only a single-dose immunization of 2 â× â107 TCID50 can provide high-level neutralizing antibodies and robust T cell responses to non-human primate animal models. Thus, our data indicate that VSVΔG-SARS can be used as a rapid response vaccine candidate. Our study on the recombinant VSV-vectored SARS-CoV vaccines can accumulate experience and provide a foundation for the new coronavirus disease in the future.