RESUMO
Ebolavirus (EBOV) belongs to a family of highly pathogenic viruses that cause severe hemorrhagic fever in humans. EBOV replication requires the activity of the viral polymerase complex, which includes the cofactor and Interferon antagonist VP35. We previously showed that the covalent ubiquitination of VP35 promotes virus replication by regulating interactions with the polymerase complex. In addition, VP35 can also interact non-covalently with ubiquitin (Ub); however, the function of this interaction is unknown. Here, we report that VP35 interacts with free (unanchored) K63-linked polyUb chains. Ectopic expression of Isopeptidase T (USP5), which is known to degrade unanchored polyUb chains, reduced VP35 association with Ub and correlated with diminished polymerase activity in a minigenome assay. Using computational methods, we modeled the VP35-Ub non-covalent interacting complex, identified the VP35-Ub interacting surface, and tested mutations to validate the interface. Docking simulations identified chemical compounds that can block VP35-Ub interactions leading to reduced viral polymerase activity. Treatment with the compounds reduced replication of infectious EBOV in cells and in vivo in a mouse model. In conclusion, we identified a novel role of unanchored polyUb in regulating Ebola virus polymerase function and discovered compounds that have promising anti-Ebola virus activity.
Assuntos
Ebolavirus , Doença pelo Vírus Ebola , Proteínas do Nucleocapsídeo , Ubiquitina , Replicação Viral , Animais , Humanos , Camundongos , Ebolavirus/genética , Ubiquitina/metabolismo , Proteínas Virais Reguladoras e Acessórias , Replicação Viral/genéticaRESUMO
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-a new coronavirus that has led to a worldwide pandemic1-has a furin cleavage site (PRRAR) in its spike protein that is absent in other group-2B coronaviruses2. To explore whether the furin cleavage site contributes to infection and pathogenesis in this virus, we generated a mutant SARS-CoV-2 that lacks the furin cleavage site (ΔPRRA). Here we report that replicates of ΔPRRA SARS-CoV-2 had faster kinetics, improved fitness in Vero E6 cells and reduced spike protein processing, as compared to parental SARS-CoV-2. However, the ΔPRRA mutant had reduced replication in a human respiratory cell line and was attenuated in both hamster and K18-hACE2 transgenic mouse models of SARS-CoV-2 pathogenesis. Despite reduced disease, the ΔPRRA mutant conferred protection against rechallenge with the parental SARS-CoV-2. Importantly, the neutralization values of sera from patients with coronavirus disease 2019 (COVID-19) and monoclonal antibodies against the receptor-binding domain of SARS-CoV-2 were lower against the ΔPRRA mutant than against parental SARS-CoV-2, probably owing to an increased ratio of particles to plaque-forming units in infections with the former. Together, our results demonstrate a critical role for the furin cleavage site in infection with SARS-CoV-2 and highlight the importance of this site for evaluating the neutralization activities of antibodies.
Assuntos
COVID-19/virologia , Furina/metabolismo , Mutação , SARS-CoV-2/genética , SARS-CoV-2/patogenicidade , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Sequência de Aminoácidos , Animais , Anticorpos Neutralizantes/imunologia , COVID-19/patologia , COVID-19/fisiopatologia , Linhagem Celular , Chlorocebus aethiops , Cricetinae , Feminino , Humanos , Pneumopatias/patologia , Pneumopatias/fisiopatologia , Pneumopatias/virologia , Masculino , Camundongos , Camundongos Transgênicos , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Proteólise , SARS-CoV-2/química , SARS-CoV-2/metabolismo , Serina Endopeptidases/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Células Vero , Replicação Viral/genéticaRESUMO
The common marmoset (Callithrix jacchus) is increasingly recognized as an ideal nonhuman primate (NHP) at high biocontainment due to its smaller size and relative ease of handling. Here, we evaluated the susceptibility and pathogenesis of Nipah virus Bangladesh strain (NiVB) infection in marmosets at biosafety level 4. Infection via the intranasal and intratracheal route resulted in fatal disease in all 4 infected marmosets. Three developed pulmonary edema and hemorrhage as well as multifocal hemorrhagic lymphadenopathy, while 1 recapitulated neurologic clinical manifestations and cardiomyopathy on gross pathology. Organ-specific innate and inflammatory responses were characterized by RNA sequencing in 6 different tissues from infected and control marmosets. Notably, a unique transcriptome was revealed in the brainstem of the marmoset exhibiting neurological signs. Our results provide a more comprehensive understanding of NiV pathogenesis in an accessible and novel NHP model, closely reflecting clinical disease as observed in NiV patients.
Assuntos
Infecções por Henipavirus , Vírus Nipah , Edema Pulmonar , Animais , Callithrix , BangladeshRESUMO
Interferons (IFN) are essential antiviral cytokines that establish the cellular antiviral state through upregulation of hundreds of interferon-stimulated genes (ISGs), most of which have uncharacterized functions and mechanisms. We identified cholesterol-25-hydroxylase (CH25H) as a broadly antiviral ISG. CH25H converts cholesterol to a soluble antiviral factor, 25-hydroxycholesterol (25HC). 25HC treatment in cultured cells broadly inhibited growth of enveloped viruses including VSV, HSV, HIV, and MHV68 and acutely pathogenic EBOV, RVFV, RSSEV, and Nipah viruses under BSL4 conditions. It suppressed viral growth by blocking membrane fusion between virus and cell. In animal models, Ch25h-deficient mice were more susceptible to MHV68 lytic infection. Moreover, administration of 25HC in humanized mice suppressed HIV replication and reversed T cell depletion. Thus, our studies demonstrate a unique mechanism by which IFN achieves its antiviral state through the production of a natural oxysterol to inhibit viral entry and implicate membrane-modifying oxysterols as potential antiviral therapeutics.
Assuntos
Antivirais/farmacologia , Hidroxicolesteróis/metabolismo , Interferons/farmacologia , Esteroide Hidroxilases/metabolismo , Internalização do Vírus/efeitos dos fármacos , Animais , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Membrana Celular/virologia , Vírus de DNA/efeitos dos fármacos , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Hidroxicolesteróis/farmacologia , Fusão de Membrana/efeitos dos fármacos , Camundongos , Camundongos Knockout , Vírus de RNA/efeitos dos fármacos , Esteroide Hidroxilases/genética , Proteínas Virais/metabolismoRESUMO
BACKGROUND: More children presenting to Emergency Departments (EDs) with acute infections are now directly referred for outpatient parenteral antibiotic therapy (OPAT). Sparse data exist on what clinical features in these children are associated with OPAT failure. We hypothesised that children who were younger or presented with systemic features of infection would be more likely to need admission. METHODS: We conducted a service evaluation over a 5-year period (12 September 2015-12 September 2020) at a single UK tertiary centre paediatric ED formally known as the Royal Hospital for Sick Children Edinburgh. All children referred from the ED for OPAT with ceftriaxone were included. OPAT failure was defined as a decision by a senior clinician of need for admission. Univariate statistical testing and multivariate logistic regression modelling were performed. RESULTS: 754 children received OPAT in the ED over a 5-year period. 95 children (13%) required admission for inpatient management. Need for admission was independently associated with having a positive blood culture (adjusted OR (aOR) 8.9; 95% CI 1.49 to 47; p=0.01) or an ultrasound performed (aOR 6.8; 95% CI 3.74 to 12.7; p<0.001). We observed no significant association between age and systemic features (fever, white cell count or C reactive protein) with need for admission in our multivariate analysis. CONCLUSION: In children presenting with acute infections to our paediatric ED who were deemed suitable by senior clinicians to be managed using OPAT with ceftriaxone, younger age (above 3 months) and the presence of systemic features were not independently associated with need for admission. Overall, our service was safe and no child came to harm from early ambulation during this evaluation.
RESUMO
Marburg virus (MARV; family Filoviridae) causes sporadic outbreaks of Marburg hemorrhagic fever in sub-Saharan Africa with case fatality rates reaching 90%. Wild-type filoviruses, including MARV and the closely related Ebola virus, are unable to suppress the type I interferon response in rodents, and therefore require adaptation of the viruses to cause disease in immunocompetent animals. In the current study, we demonstrate that STAT2 knockout Syrian hamsters are susceptible to infection with different wild-type MARV variants. MARV Musoke causes a robust and systemic infection resulting in lethal disease. Histopathological findings share features similar to those observed in human patients and other animal models of filovirus infection. Reverse-transcription polymerase chain reaction analysis of host transcripts shows a dysregulation of the innate immune response. Our results demonstrate that the STAT2 knockout hamster represents a novel small animal model of severe MARV infection and disease without the requirement for virus adaptation.
Assuntos
Doença do Vírus de Marburg/etiologia , Fator de Transcrição STAT2/fisiologia , Animais , Cricetinae , Citocinas/biossíntese , Modelos Animais de Doenças , Suscetibilidade a Doenças , Feminino , Masculino , Doença do Vírus de Marburg/imunologia , Doença do Vírus de Marburg/patologiaRESUMO
Background: Nipah virus (NiV) is a paramyxovirus (genus Henipavirus) that can cause severe respiratory illness and encephalitis in humans. Transmission occurs through consumption of NiV-contaminated foods, and contact with NiV-infected animals or human body fluids. However, it is unclear whether aerosols derived from aforesaid sources or others also contribute to transmission, and current knowledge on NiV-induced pathogenicity after small-particle aerosol exposure is still limited. Methods: Infectivity, pathogenicity, and real-time dissemination of aerosolized NiV in Syrian hamsters was evaluated using NiV-Malaysia (NiV-M) and/or its recombinant expressing firefly luciferase (rNiV-FlucNP). Results: Both viruses had an equivalent pathogenicity in hamsters, which developed respiratory and neurological symptoms of disease, similar to using intranasal route, with no direct correlations to the dose. We showed that virus replication was predominantly initiated in the lower respiratory tract and, although delayed, also intensely in the oronasal cavity and possibly the brain, with gradual increase of signal in these regions until at least day 5-6 postinfection. Conclusion: Hamsters infected with small-particle aerosolized NiV undergo similar clinical manifestations of the disease as previously described using liquid inoculum, and exhibit histopathological lesions consistent with NiV patient reports. NiV droplets could therefore play a role in transmission by close contact.
Assuntos
Aerossóis/administração & dosagem , Infecções por Henipavirus , Vírus Nipah/patogenicidade , Administração por Inalação , Animais , Cricetinae , Modelos Animais de Doenças , Infecções por Henipavirus/diagnóstico por imagem , Infecções por Henipavirus/patologia , Infecções por Henipavirus/transmissão , Infecções por Henipavirus/virologia , Luciferases de Vaga-Lume/genética , Luciferases de Vaga-Lume/metabolismo , Pulmão/diagnóstico por imagem , Pulmão/patologia , Pulmão/virologia , Mesocricetus , Imagem Óptica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Nipah virus (NiV) is a zoonotic emerging paramyxovirus that can cause fatal respiratory illness or encephalitis in humans. Despite many efforts, the molecular mechanisms of NiV-induced acute lung injury (ALI) remain unclear. We previously showed that NiV replicates to high titers in human lung grafts in NOD-SCID/γ mice, resulting in a robust inflammatory response. Interestingly, these mice can undergo human immune system reconstitution by the bone marrow, liver, and thymus (BLT) reconstitution method, in addition to lung tissue engraftment, giving altogether a realistic model to study human respiratory viral infections. Here, we characterized NiV Bangladesh strain (NiV-B) infection of human lung grafts from human immune system-reconstituted mice in order to identify the overall effect of immune cells on NiV pathogenesis of the lung. We show that NiV-B replicated to high titers in human lung grafts and caused similar cytopathic effects irrespective of the presence of human leukocytes in mice. However, the human immune system interfered with virus spread across lung grafts, responded to infection by leukocyte migration to small airways and alveoli of the lung grafts, and accelerated oxidative stress in lung grafts. In addition, the presence of human leukocytes increased the expression of cytokines and chemokines that regulate inflammatory influx to sites of infection and tissue damage. These results advance our understanding of how the immune system limits NiV dissemination and contributes to ALI and inform efforts to identify therapeutic targets.IMPORTANCE Nipah virus (NiV) is an emerging paramyxovirus that can cause a lethal respiratory and neurological disease in humans. Only limited data are available on NiV pathogenesis in the human lung, and the relative contribution of the innate immune response and NiV to acute lung injury (ALI) is still unknown. Using human lung grafts in a human immune system-reconstituted mouse model, we showed that the NiV Bangladesh strain induced cytopathic lesions in lung grafts similar to those described in patients irrespective of the donor origin or the presence of leukocytes. However, the human immune system interfered with virus spread, responded to infection by leukocyte infiltration in the small airways and alveolar area, induced oxidative stress, and triggered the production of cytokines and chemokines that regulate inflammatory influx by leukocytes in response to infection. Understanding how leukocytes interact with NiV and cause ALI in human lung xenografts is crucial for identifying therapeutic targets.
Assuntos
Lesão Pulmonar Aguda/patologia , Infecções por Henipavirus/patologia , Leucócitos/imunologia , Pulmão/patologia , Vírus Nipah/crescimento & desenvolvimento , Estresse Oxidativo , Animais , Citocinas/análise , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos SCIDRESUMO
Incorporation of reporter genes within virus genomes is an indispensable tool for interrogation of virus biology and pathogenesis. In previous work, we incorporated a fluorophore into a viral ORF by attaching it to the viral gene via a P2A ribosomal skipping sequence. This recombinant Nipah virus, however, was attenuated in vitro relative to WT virus. In this work, we determined that inefficient ribosomal skipping was a major contributing factor to this attenuation. Inserting a GSG linker before the P2A sequence resulted in essentially complete skipping, significantly improved growth in vitro, and WT lethality in vivo. To the best of our knowledge, this represents the first time a recombinant virus of Mononegavirales with integration of a reporter into a viral ORF has been compared with the WT virus in vivo. Incorporating the GSG linker for improved skipping efficiency whenever functionally important is a critical consideration for recombinant virus design.
Assuntos
Genes Reporter , Engenharia Genética/métodos , Infecções por Henipavirus/genética , Vírus Nipah/genética , Proteínas Ribossômicas/genética , Sequência de Aminoácidos , Animais , Feminino , Regulação da Expressão Gênica , Infecções por Henipavirus/mortalidade , Infecções por Henipavirus/patologia , Infecções por Henipavirus/virologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Mesocricetus , Dados de Sequência Molecular , Mutagênese Insercional , Vírus Nipah/patogenicidade , Faloidina/genética , Faloidina/metabolismo , Proteínas Ribossômicas/metabolismo , Ribossomos/genética , Ribossomos/metabolismo , Análise de Sobrevida , Transcrição Gênica , Replicação Viral/genética , Proteína Vermelha FluorescenteRESUMO
UNLABELLED: Rift Valley fever (RVF) is a mosquito-borne zoonotic disease endemic to Africa and characterized by a high rate of abortion in ruminants and hemorrhagic fever, encephalitis, or blindness in humans. RVF is caused by Rift Valley fever virus (RVFV; family Bunyaviridae, genus Phlebovirus), which has a tripartite negative-stranded RNA genome (consisting of the S, M, and L segments). Further spread of RVF into countries where the disease is not endemic may affect the economy and public health, and vaccination is an effective approach to prevent the spread of RVFV. A live-attenuated MP-12 vaccine is one of the best-characterized RVF vaccines for safety and efficacy and is currently conditionally licensed for use for veterinary purposes in the United States. Meanwhile, as of 2015, no other RVF vaccine has been conditionally or fully licensed for use in the United States. The MP-12 strain is derived from wild-type pathogenic strain ZH548, and its genome encodes 23 mutations in the three genome segments. However, the mechanism of MP-12 attenuation remains unknown. We characterized the attenuation of wild-type pathogenic strain ZH501 carrying a mutation(s) of the MP-12 S, M, or L segment in a mouse model. Our results indicated that MP-12 is attenuated by the mutations in the S, M, and L segments, while the mutations in the M and L segments confer stronger attenuation than those in the S segment. We identified a combination of 3 amino acid changes, Y259H (Gn), R1182G (Gc), and R1029K (L), that was sufficient to attenuate ZH501. However, strain MP-12 with reversion mutations at those 3 sites was still highly attenuated. Our results indicate that MP-12 attenuation is supported by a combination of multiple partial attenuation mutations and a single reversion mutation is less likely to cause a reversion to virulence of the MP-12 vaccine. IMPORTANCE: Rift Valley fever (RVF) is a mosquito-transmitted viral disease that is endemic to Africa and that has the potential to spread into other countries. Vaccination is considered an effective way to prevent the disease, and the only available veterinary RVF vaccine in the United States is a live-attenuated MP-12 vaccine, which is conditionally licensed. Strain MP-12 is different from its parental pathogenic RVFV strain, strain ZH548, because of the presence of 23 mutations. This study determined the role of individual mutations in the attenuation of the MP-12 strain. We found that full attenuation of MP-12 occurs by a combination of multiple mutations. Our findings indicate that a single reversion mutation will less likely cause a major reversion to virulence of the MP-12 vaccine.
Assuntos
Febre do Vale de Rift/patologia , Febre do Vale de Rift/virologia , Vírus da Febre do Vale do Rift/genética , Vírus da Febre do Vale do Rift/fisiologia , Vacinas Virais/genética , Vacinas Virais/imunologia , Animais , Análise Mutacional de DNA , Modelos Animais de Doenças , Feminino , Camundongos , Mutação de Sentido Incorreto , Vírus da Febre do Vale do Rift/imunologia , Vírus da Febre do Vale do Rift/patogenicidade , Vacinas Atenuadas/genética , Vacinas Atenuadas/imunologia , VirulênciaRESUMO
UNLABELLED: The New World arenavirus Junin virus (JUNV) is the causative agent of Argentine hemorrhagic fever (AHF), a potentially deadly disease endemic to central regions of Argentina. The live-attenuated Candid #1 (Can) strain of JUNV is currently used to vaccinate the human population at risk. However, the mechanism of attenuation of this strain is still largely unknown. Therefore, the identification and functional characterization of viral genetic determinants dictating JUNV virulence or attenuation would significantly improve the understanding of the mechanisms underlying AHF and facilitate the development of novel, more effective, and safer vaccines. Here, we utilized a reverse genetics approach to generate recombinant JUNV (rJUNV) strains encoding different gene combinations of the pathogenic Romero (Rom) and attenuated Can strains of JUNV. All strains of rJUNV exhibited in vitro growth kinetics similar to those of their parental counterparts. Analysis of virulence of the rJUNV in a guinea pig model of lethal infection that closely reproduces the features of AHF identified the envelope glycoproteins (GPs) as the major determinants of pathogenesis and attenuation of JUNV. Accordingly, rJUNV strains expressing the full-length GPs of Rom and Can exhibited virulent and attenuated phenotypes, respectively, in guinea pigs. Mutation F427I in the transmembrane region of JUNV envelope glycoprotein GP2 has been shown to attenuate the neurovirulence of JUNV in suckling mice. We document that in the guinea pig model of AHF, mutation F427I in GP2 is also highly attenuating but insufficient to prevent virus dissemination and development of mild clinical and pathological symptoms, indicating that complete attenuation of JUNV requires additional mutations present in Can glycoprotein precursor (GPC). IMPORTANCE: Development of antiviral strategies against viral hemorrhagic fevers, including AHF, is one of the top priorities within the Implementation Plan of the U.S. Department of Health and Human Services Public Health Emergency Medical Countermeasures Enterprise. Live-attenuated Candid #1 strain, derived from the 44th mouse brain passage of the prototype XJ strain of JUNV, has been demonstrated to be safe, immunogenic, and highly protective and is currently licensed for human use in Argentina. However, the bases for the attenuated phenotype of Candid #1 have not been established. Therefore, the identification and functional characterization of viral genetic factors implicated in JUNV pathogenesis and attenuation would significantly improve the understanding of the molecular mechanisms underlying AHF and facilitate the development of novel antiviral strategies.
Assuntos
Glicoproteínas/metabolismo , Febre Hemorrágica Americana/virologia , Vírus Junin/fisiologia , Proteínas do Envelope Viral/metabolismo , Animais , Modelos Animais de Doenças , Glicoproteínas/genética , Cobaias , Febre Hemorrágica Americana/patologia , Vírus Junin/genética , Genética Reversa , Proteínas do Envelope Viral/genética , Virulência , Fatores de VirulênciaRESUMO
UNLABELLED: Nipah virus (NiV) and Hendra virus (HeV) are closely related henipaviruses of the Paramyxovirinae. Spillover from their fruit bat reservoirs can cause severe disease in humans and livestock. Despite their high sequence similarity, NiV and HeV exhibit apparent differences in receptor and tissue tropism, envelope-mediated fusogenicity, replicative fitness, and other pathophysiologic manifestations. To investigate the molecular basis for these differences, we first established a highly efficient reverse genetics system that increased rescue titers by ≥3 log units, which offset the difficulty of generating multiple recombinants under constraining biosafety level 4 (BSL-4) conditions. We then replaced, singly and in combination, the matrix (M), fusion (F), and attachment glycoprotein (G) genes in mCherry-expressing recombinant NiV (rNiV) with their HeV counterparts. These chimeric but isogenic rNiVs replicated well in primary human endothelial and neuronal cells, indicating efficient heterotypic complementation. The determinants of budding efficiency, fusogenicity, and replicative fitness were dissociable: HeV-M budded more efficiently than NiV-M, accounting for the higher replicative titers of HeV-M-bearing chimeras at early times, while the enhanced fusogenicity of NiV-G-bearing chimeras did not correlate with increased replicative fitness. Furthermore, to facilitate spatiotemporal studies on henipavirus pathogenesis, we generated a firefly luciferase-expressing NiV and monitored virus replication and spread in infected interferon alpha/beta receptor knockout mice via bioluminescence imaging. While intraperitoneal inoculation resulted in neuroinvasion following systemic spread and replication in the respiratory tract, intranasal inoculation resulted in confined spread to regions corresponding to olfactory bulbs and salivary glands before subsequent neuroinvasion. This optimized henipavirus reverse genetics system will facilitate future investigations into the growing numbers of novel henipavirus-like viruses. IMPORTANCE: Nipah virus (NiV) and Hendra virus (HeV) are recently emergent zoonotic and highly lethal pathogens with pandemic potential. Although differences have been observed between NiV and HeV replication and pathogenesis, the molecular basis for these differences has not been examined. In this study, we established a highly efficient system to reverse engineer changes into replication-competent NiV and HeV, which facilitated the generation of reporter-expressing viruses and recombinant NiV-HeV chimeras with substitutions in the genes responsible for viral exit (the M gene, critical for assembly and budding) and viral entry (the G [attachment] and F [fusion] genes). These chimeras revealed differences in the budding and fusogenic properties of the M and G proteins, respectively, which help explain previously observed differences between NiV and HeV. Finally, to facilitate future in vivo studies, we monitored the replication and spread of a bioluminescent reporter-expressing NiV in susceptible mice; this is the first time such in vivo imaging has been performed under BSL-4 conditions.
Assuntos
Modelos Animais de Doenças , Vírus Hendra/fisiologia , Infecções por Henipavirus/virologia , Vírus Nipah/fisiologia , Internalização do Vírus , Liberação de Vírus , Animais , Teste de Complementação Genética , Humanos , Camundongos Knockout , Recombinação Genética , Genética Reversa , Tropismo ViralRESUMO
LJ001 is a lipophilic thiazolidine derivative that inhibits the entry of numerous enveloped viruses at non-cytotoxic concentrations (IC50 ≤ 0.5 µM), and was posited to exploit the physiological difference between static viral membranes and biogenic cellular membranes. We now report on the molecular mechanism that results in LJ001's specific inhibition of virus-cell fusion. The antiviral activity of LJ001 was light-dependent, required the presence of molecular oxygen, and was reversed by singlet oxygen ((1)O2) quenchers, qualifying LJ001 as a type II photosensitizer. Unsaturated phospholipids were the main target modified by LJ001-generated (1)O2. Hydroxylated fatty acid species were detected in model and viral membranes treated with LJ001, but not its inactive molecular analog, LJ025. (1)O2-mediated allylic hydroxylation of unsaturated phospholipids leads to a trans-isomerization of the double bond and concurrent formation of a hydroxyl group in the middle of the hydrophobic lipid bilayer. LJ001-induced (1)O2-mediated lipid oxidation negatively impacts on the biophysical properties of viral membranes (membrane curvature and fluidity) critical for productive virus-cell membrane fusion. LJ001 did not mediate any apparent damage on biogenic cellular membranes, likely due to multiple endogenous cytoprotection mechanisms against phospholipid hydroperoxides. Based on our understanding of LJ001's mechanism of action, we designed a new class of membrane-intercalating photosensitizers to overcome LJ001's limitations for use as an in vivo antiviral agent. Structure activity relationship (SAR) studies led to a novel class of compounds (oxazolidine-2,4-dithiones) with (1) 100-fold improved in vitro potency (IC50<10 nM), (2) red-shifted absorption spectra (for better tissue penetration), (3) increased quantum yield (efficiency of (1)O2 generation), and (4) 10-100-fold improved bioavailability. Candidate compounds in our new series moderately but significantly (p≤0.01) delayed the time to death in a murine lethal challenge model of Rift Valley Fever Virus (RVFV). The viral membrane may be a viable target for broad-spectrum antivirals that target virus-cell fusion.
Assuntos
Antivirais/farmacologia , Oxazóis/farmacologia , Rodanina/análogos & derivados , Vírus da Febre do Vale do Rift/efeitos dos fármacos , Tionas/farmacologia , Ligação Viral/efeitos dos fármacos , Internalização do Vírus/efeitos dos fármacos , Animais , Antivirais/química , Disponibilidade Biológica , Membrana Celular/efeitos dos fármacos , Membrana Celular/virologia , Camundongos , Camundongos Endogâmicos BALB C , Fosfolipídeos/química , Fosfolipídeos/metabolismo , Fármacos Fotossensibilizantes/farmacologia , Rodanina/farmacologia , Febre do Vale de Rift/tratamento farmacológico , Oxigênio Singlete/química , Relação Estrutura-AtividadeRESUMO
Lassa fever (LF) is a potentially lethal human disease that is caused by the arenavirus Lassa virus (LASV). Annually, around 300,000 infections with up to 10,000 deaths occur in regions of Lassa fever endemicity in West Africa. Here we demonstrate that mice lacking a functional STAT1 pathway are highly susceptible to infection with LASV and develop lethal disease with pathology similar to that reported in humans.
Assuntos
Febre Lassa/virologia , Vírus Lassa/patogenicidade , Fator de Transcrição STAT1/fisiologia , África Ocidental , Animais , Células Cultivadas , Chlorocebus aethiops , Humanos , Rim/metabolismo , Rim/virologia , Febre Lassa/genética , Febre Lassa/mortalidade , Camundongos , Camundongos Knockout , Receptor de Interferon alfa e beta/fisiologia , Taxa de Sobrevida , Células VeroRESUMO
Favipiravir is a ribonucleoside analogue that has been explored as a therapeutic for the treatment of Ebola Virus Disease (EVD). Promising data from rodent models has informed nonhuman primate trials, as well as evaluation in patients during the 2013-2016 West African EVD outbreak of favipiravir treatment. However, mixed results from these studies hindered regulatory approval of favipiravir for the indication of EVD. This study examined the influence of route of administration, duration of treatment, and treatment schedule of favipiravir in immune competent mouse and guinea pig models using rodent-adapted Zaire ebolavirus (EBOV). A dose of 300 mg/kg/day of favipiravir with an 8-day treatment was found to be fully effective at preventing lethal EVD-like disease in BALB/c mice regardless of route of administration (oral, intraperitoneal, or subcutaneous) or whether it was provided as a once-daily dose or a twice-daily split dose. Preclinical data generated in guinea pigs demonstrates that an 8-day treatment of 300 mg/kg/day of favipiravir reduces mortality following EBOV challenge regardless of route of treatment or duration of treatments for 8, 11, or 15 days. This work supports the future translational development of favipiravir as an EVD therapeutic.
Assuntos
Amidas , Antivirais , Modelos Animais de Doenças , Ebolavirus , Doença pelo Vírus Ebola , Camundongos Endogâmicos BALB C , Pirazinas , Animais , Amidas/uso terapêutico , Amidas/administração & dosagem , Amidas/farmacologia , Cobaias , Pirazinas/administração & dosagem , Pirazinas/uso terapêutico , Doença pelo Vírus Ebola/tratamento farmacológico , Camundongos , Ebolavirus/efeitos dos fármacos , Antivirais/administração & dosagem , Antivirais/uso terapêutico , Feminino , Vias de Administração de Medicamentos , Esquema de MedicaçãoRESUMO
Rift Valley fever is a mosquito-borne zoonotic disease endemic to sub-Saharan Africa. Rift Valley fever virus (RVFV; genus Phlebovirus, family Bunyaviridae) causes high rates of abortion and fetal malformation in pregnant ruminants, and haemorrhagic fever, neurological disorders or blindness in humans. The MP-12 strain is a highly efficacious and safe live-attenuated vaccine candidate for both humans and ruminants. However, MP-12 lacks a marker to differentiate infected from vaccinated animals. In this study, we originally aimed to characterize the efficacy of a recombinant RVFV MP-12 strain encoding Toscana virus (TOSV) NSs gene in place of MP-12 NSs (rMP12-TOSNSs). TOSV NSs promotes the degradation of dsRNA-dependent protein kinase (PKR) and inhibits interferon-ß gene up-regulation without suppressing host general transcription. Unexpectedly, rMP12-TOSNSs increased death in vaccinated outbred mice and inbred BALB/c or C57BL/6 mice. Immunohistochemistry showed diffusely positive viral antigens in the thalamus, hypothalamus and brainstem, including the medulla. No viral antigens were detected in spleen or liver, which is similar to the antigen distribution of moribund mice infected with MP-12. These results suggest that rMP12-TOSNSs retains neuroinvasiveness in mice. Our findings demonstrate that rMP12-TOSNSs causes neuroinvasion without any hepatic disease and will be useful for studying the neuroinvasion mechanism of RVFV and TOSV.
Assuntos
Encéfalo/virologia , Doenças do Sistema Nervoso/virologia , Febre do Vale de Rift/prevenção & controle , Vírus da Febre do Flebótomo Napolitano/genética , Vírus da Febre do Flebótomo Napolitano/patogenicidade , Vacinas Atenuadas/efeitos adversos , Proteínas não Estruturais Virais/metabolismo , Vacinas Virais/efeitos adversos , Animais , Linhagem Celular , Chlorocebus aethiops , Feminino , Humanos , Imunoglobulina G/sangue , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Febre do Vale de Rift/imunologia , Vírus da Febre do Vale do Rift/imunologia , Vírus da Febre do Flebótomo Napolitano/imunologia , Vacinação , Vacinas Atenuadas/administração & dosagem , Vacinas Atenuadas/imunologia , Células Vero , Proteínas não Estruturais Virais/imunologia , Vacinas Virais/administração & dosagem , Vacinas Virais/imunologiaRESUMO
Lassa virus (LASV) is the causative agent of Lassa hemorrhagic fever (LF) in humans, a deadly disease endemic to West Africa that results in 5,000 to 10,000 deaths annually. Here we present results demonstrating that functional type I and type II interferon (IFN) signaling is required for efficient control of LASV dissemination and clearance.
Assuntos
Interferons/imunologia , Febre Lassa/imunologia , Vírus Lassa/imunologia , Animais , Feminino , Humanos , Febre Lassa/virologia , Vírus Lassa/fisiologia , Masculino , Camundongos , Camundongos Knockout , Receptores de Interferon/genética , Receptores de Interferon/imunologiaRESUMO
Ebola virus (EBOV) causes Ebola virus disease (EVD), which is characterized by hemorrhagic fever with high mortality rates in humans. EBOV sexual transmission has been a concern since the 2014-2016 outbreak in Africa, as persistent infection in the testis and transmission to women was demonstrated. The only study related to establishing an intravaginal small animal infection model was recently documented in IFNAR-/- mice using wild-type and mouse-adapted EBOV (maEBOV), and resulted in 80% mortality, supporting epidemiological data. However, this route of transmission is still poorly understood in women, and the resulting EVD from it is understudied. Here, we contribute to this field of research by providing data from immunocompetent BALB/c mice. We demonstrate that progesterone priming increased the likelihood of maEBOV vaginal infection and of exhibiting the symptoms of disease and seroconversion. However, our data suggest subclinical infection, regardless of the infective dose. We conclude that maEBOV can infect BALB/c mice through vaginal inoculation, but that this route of infection causes significantly less disease compared to intraperitoneal injection at a similar dose, which is consistent with previous studies using other peripheral routes of inoculation in that animal model. Our data are inconsistent with the disease severity described in female patients, therefore suggesting that BALB/c mice are unsuitable for modeling typical EVD following vaginal challenge with maEBOV. Further studies are required to determine the mechanisms by which EVD is attenuated in BALB/c mice, using maEBOV via the vaginal route, as in our experimental set-up.
Assuntos
Ebolavirus , Doença pelo Vírus Ebola , Humanos , Masculino , Animais , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Vagina , Modelos AnimaisRESUMO
The genetic diversities of mammalian tick-borne flaviviruses are poorly understood. We used next-generation sequencing (NGS) to deep sequence different viruses and strains belonging to this group of flaviviruses, including Central European tick-borne encephalitis virus (TBEV-Eur), Far Eastern TBEV (TBEV-FE), Langat (LGTV), Powassan (POWV), Deer Tick (DTV), Kyasanur Forest Disease (KFDV), Alkhurma hemorrhagic fever (AHFV), and Omsk hemorrhagic fever (OHFV) viruses. DTV, AHFV, and KFDV had the lowest genetic diversity, while POWV strains LEIV-5530 and LB, OHFV, TBEV-Eur, and TBEV-FE had higher genetic diversities. These findings are compatible with the phylogenetic relationships between the viruses. For DTV and POWV, the amount of genetic diversity could be explained by the number of tick vector species and amplification hosts each virus can occupy, with low diversity DTV having a more limited vector and host pool, while POWV with higher genetic diversities has been isolated from different tick species and mammals. It is speculated that high genetic diversity may contribute to the survival of the virus as it encounters these different environments.
Assuntos
Vírus da Encefalite Transmitidos por Carrapatos , Encefalite Transmitida por Carrapatos , Animais , Filogenia , Vírus da Encefalite Transmitidos por Carrapatos/genética , Mamíferos , Variação GenéticaRESUMO
Rift Valley fever (RVF) is a mosquito-borne zoonosis endemic to Africa and the Arabian Peninsula, which causes large outbreaks among humans and ruminants. Single dose vaccinations using live-attenuated RVF virus (RVFV) support effective prevention of viral spread in endemic countries. Due to the segmented nature of RVFV genomic RNA, segments of vaccine strain-derived genomic RNA could be incorporated into wild-type RVFV within co-infected mosquitoes or animals. Rationally designed vaccine candidate RVax-1 displays protective epitopes fully identical to the previously characterized MP-12 vaccine. Additionally, all genome segments of RVax-1 contribute to the attenuation phenotype, which prevents the formation of pathogenic reassortant strains. This study demonstrated that RVax-1 cannot replicate efficiently in orally fed Aedes aegypti mosquitoes, while retaining strong immunogenicity and protective efficacy in an inbred mouse model, which were indistinguishable from the MP-12 vaccine. These findings support further development of RVax-1 as the next generation MP-12-based vaccine for prevention of Rift Valley fever in humans and animals.