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Enterovirus D68 (EV-D68), a respiratory RNA virus in the family Picornaviridae, is implicated as a potential etiological agent for acute flaccid myelitis in preteen adolescents. The absence of a specific therapeutic intervention necessitates the development of an effective animal model for EV-D68. The AG129 mouse strain, characterized by the double knockout of IFN-α/ß and IFN-γ receptors on the 129 genetic background, has been proposed as a suitable model for EV-D68. The goals of this study were to assess the effect of a nonmouse-adapted EV-D68 strain (US/MO/14-18947, NR-49129) in AG129 (IFN-α/ß and IFN-γ receptors null), A129 (IFN-α/ß receptor null), G129 (IFN-γ receptor null), and the 129 background strain (129S2/SvPasCrl) when infected intraperitoneally at 10 d of age. Both AG129 and A129 strains demonstrated similar clinical signs (paralysis, paresis, lethargy, dyspnea [characterized by prominent abdominal respiration], and morbidity requiring euthanasia) induced by EV-D68. While G129 and 129S2 strains also exhibited susceptibility to EV-D68, the severity of clinical signs was less than in AG129 and A129 strains, and many survived to the experimental endpoint. Histopathological and immunohistochemical data confirmed EV-D68 tropism for the skeletal muscle and spinal cord and suggest that the dyspnea observed in infected mice could be attributed, in part, to lesions in the diaphragmatic skeletal muscles. These findings contribute valuable insights into the pathogenesis of EV-D68 infection in this mouse model and provide investigators with key information on virus dose and mouse strain selection when using this mouse model to evaluate candidate EV-D68 therapeutics.
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Immunodeficient NSG mice are reported to be less responsive to buprenorphine analgesia. Here, we used NSG mice to compare the efficacy of the commonly used dose of carprofen (5 mg/kg) with 5 and 10 times that dose (25 and 50 mg/kg) for attenuating postoperative mechanical and thermal hypersensitivity following an incisional pain model. Male and female NSG mice (n = 45) were randomly assigned to one of 4 groups and received daily subcutaneous injections for 3 d: saline (5 mL/kg), 5 mg/kg carprofen (Carp5), 25 mg/kg carprofen (Carp25), and 50 mg/kg carprofen (Carp50). Mechanical and thermal hypersensitivity were assessed 24 h before and at 4, 24, and 48 h after surgery. Plasma carprofen concentrations were measured in a separate group of mice (n = 56) on days 0 (at 2, 4, 12, and 23 h), 1, and 2 after the first, second, and third doses, respectively. Toxicity was assessed through daily fecal occult blood testing (n = 27) as well as gross and histopathologic evaluation (n = 15). Our results indicated that the saline group showed both mechanical and thermal hypersensitivity throughout the study. Carp5 did not attenuate mechanical or thermal hypersensitivity at any time point. Carp25 attenuated mechanical and thermal (except for the 4-h time point) hypersensitivity. Carp50 attenuated only thermal hypersensitivity at 24 h. Fecal occult blood was detected in 1 of 8 Carp25-treated mice at 48 and 72 h. Histopathologic abnormalities (gastric ulceration, ulcerative enteritis, and renal lesions) were observed in some Carp50-treated mice. Plasma carprofen concentrations were dose and time dependent. Our results indicate that Carp25 attenuated postoperative mechanical and thermal hypersensitivity more effectively than Carp5 or Carp50 in NSG mice with incisional pain. Therefore, we recommend providing carprofen at 25 mg/kg SID for incisional pain procedures using immunodeficient NSG mouse.
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Carbazoles , Dolor Postoperatorio , Animales , Ratones , Femenino , Dolor Postoperatorio/tratamiento farmacológico , Dolor Postoperatorio/prevención & control , Masculino , Carbazoles/administración & dosificación , Hiperalgesia/tratamiento farmacológico , Antiinflamatorios no Esteroideos/administración & dosificación , Relación Dosis-Respuesta a DrogaRESUMEN
While the immunodeficient status of NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) and NSG-related mice provides utility for numerous research models, it also results in increased susceptibility to opportunistic pathogens. Over a 9-week period, a high rate of mortality was reported in a housing room of NSG and NSG-related mice. Diagnostics were performed to determine the underlying etiopathogenesis. Mice submitted for evaluation included those found deceased (n = 2), cage mates of deceased mice with or without diarrhea (n = 17), and moribund mice (n = 8). Grossly, mice exhibited small intestinal and cecal dilation with abundant gas and/or digesta (n = 18), serosal hemorrhage and congestion (n = 6), or were grossly normal (n = 3). Histologically, there was erosive to ulcerative enterocolitis (n = 7) of the distal small and large intestine or widespread individual epithelial cell death with luminal sloughing (n = 13) and varying degrees of submucosal edema and mucosal hyperplasia. Cecal dysbiosis, a reduction in typical filamentous bacteria coupled with overgrowth of bacterial rods, was identified in 18 of 24 (75%) mice. Clostridium spp. and Paeniclostridium sordellii were identified in 13 of 23 (57%) and 7 of 23 (30%) mice, respectively. Clostridium perfringens (7 of 23, 30%) was isolated most frequently. Toxinotyping of C. perfringens positive mice (n = 2) identified C. perfringens type A. Luminal immunoreactivity to several clostridial species was identified within lesioned small intestine by immunohistochemistry. Clinicopathologic findings were thus associated with overgrowth of various clostridial species, though direct causality could not be ascribed. A diet shift preceding the mortality event may have contributed to loss of intestinal homeostasis.
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Infecciones por Clostridium , Enterocolitis , Animales , Ratones , Enterocolitis/veterinaria , Enterocolitis/microbiología , Enterocolitis/patología , Infecciones por Clostridium/veterinaria , Infecciones por Clostridium/patología , Infecciones por Clostridium/microbiología , Modelos Animales de Enfermedad , Ratones Endogámicos NOD , Femenino , Clostridium/aislamiento & purificación , Disbiosis/veterinaria , Disbiosis/patología , Masculino , Ciego/patología , Ciego/microbiologíaRESUMEN
The development of transgenic mouse models that express genes of interest in specific cell types has transformed our understanding of basic biology and disease. However, generating these models is time- and resource-intensive. Here we describe a model system, SELective Expression and Controlled Transduction In Vivo (SELECTIV), that enables efficient and specific expression of transgenes by coupling adeno-associated virus (AAV) vectors with Cre-inducible overexpression of the multi-serotype AAV receptor, AAVR. We demonstrate that transgenic AAVR overexpression greatly increases the efficiency of transduction of many diverse cell types, including muscle stem cells, which are normally refractory to AAV transduction. Superior specificity is achieved by combining Cre-mediated AAVR overexpression with whole-body knockout of endogenous Aavr, which is demonstrated in heart cardiomyocytes, liver hepatocytes and cholinergic neurons. The enhanced efficacy and exquisite specificity of SELECTIV has broad utility in development of new mouse model systems and expands the use of AAV for gene delivery in vivo.
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Técnicas de Transferencia de Gen , Vectores Genéticos , Ratones , Animales , Vectores Genéticos/genética , Ratones Transgénicos , Terapia Genética , Transgenes , Dependovirus/genética , Transducción GenéticaRESUMEN
Enterovirus A71 (EV-A71) causes hand, foot, and mouth disease outbreaks with neurological complications and deaths. We previously isolated an EV-A71 variant in the stool, cerebrospinal fluid, and blood of an immunocompromised patient who had a leucine-to-arginine substitution on the VP1 capsid protein, resulting in increased heparin sulfate binding. We show here that this mutation increases the virus's pathogenicity in orally infected mice with depleted B cells, which mimics the patient's immune status, and increases susceptibility to neutralizing antibodies. However, a double mutant with even greater heparin sulfate affinity is not pathogenic, suggesting that increased heparin sulfate affinity may trap virions in peripheral tissues and reduce neurovirulence. This research sheds light on the increased pathogenicity of variant with heparin sulfate (HS)-binding ability in individuals with decreased B cell immunity.
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Enterovirus Humano A , Infecciones por Enterovirus , Enterovirus , Humanos , Animales , Ratones , Enterovirus/genética , Enterovirus Humano A/genética , Antígenos Virales/metabolismo , Heparitina Sulfato/metabolismo , Heparina/metabolismoRESUMEN
Buprenorphine is perhaps the most prescribed analgesic for management of postoperative pain in mice. Although various buprenorphine formulations are effective in commonly used immunocompetent mouse strains, a knowledge gap exists regarding its efficacy in immunodeficient mice. Here we used a plantar incision to evaluate the efficacy of 3 buprenorphine formulations for attenuating postoperative mechanical and thermal hypersensitivity in the immunodeficient NSG mouse strain. We also characterized the pharmacokinetics of these formulations over a 72-h period. We hypothesized that all 3 buprenorphine formulations evaluated-the standard preparation and 2 extended-release products (Bup-HCl, Bup-ER, and Bup-XR, respectively)-would attenuate postoperative mechanical and thermal hypersensitivity resulting from a plantar incision in NSG mice. Male and female NSG mice (n = 48) were allocated to 4 treatment groups: saline (0.9% NaCl, 5 mL/kg SC once); Bup-HCl (0.1 mg/kg SC, BID for 2 d); Bup-ER (1.0 mg/kg SC once); and Bup-XR (3.25 mg/kg SC once). Mechani- cal and thermal hypersensitivity assessments were conducted 24 h before surgery and at 4, 8, 24, 48, and 72 h afterward. All groups of mice showed mechanical and thermal hypersensitivity within the first 24 h after surgery. Behavioral pain indicators (guarding, toe-touching [intermittent partial weight bearing], licking the incision, vocalizations) were observed in some mice from each group at every postoperative time point. Plasma buprenorphine was measured in a separate group of mice and concentrations surpassed the suggested therapeutic level (1.0 ng/mL) for less than 4 h for Bup-HCl, for at least 24 h for Bup-ER, and for 72 h for Bup-XR. Our results indicate that at the dosages studied, these buprenorphine formulations do not adequately attenuate postoperative mechanical and thermal hypersensitivity in the plantar incisional model in NSG mice. These findings support the need for strain-specific analgesic protocols for mice used in research.
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Buprenorfina , Analgésicos/uso terapéutico , Analgésicos Opioides/uso terapéutico , Animales , Preparaciones de Acción Retardada , Femenino , Masculino , Ratones , Dimensión del Dolor/métodos , Dolor Postoperatorio/tratamiento farmacológico , Dolor Postoperatorio/veterinaria , Solución Salina/uso terapéuticoRESUMEN
Lysosomes are key degradative compartments of the cell. Transport to lysosomes relies on GlcNAc-1-phosphotransferase-mediated tagging of soluble enzymes with mannose 6-phosphate (M6P). GlcNAc-1-phosphotransferase deficiency leads to the severe lysosomal storage disorder mucolipidosis II (MLII). Several viruses require lysosomal cathepsins to cleave structural proteins and thus depend on functional GlcNAc-1-phosphotransferase. We used genome-scale CRISPR screens to identify lysosomal enzyme trafficking factor (LYSET, also named TMEM251) as essential for infection by cathepsin-dependent viruses including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). LYSET deficiency resulted in global loss of M6P tagging and mislocalization of GlcNAc-1-phosphotransferase from the Golgi complex to lysosomes. Lyset knockout mice exhibited MLII-like phenotypes, and human pathogenic LYSET alleles failed to restore lysosomal sorting defects. Thus, LYSET is required for correct functioning of the M6P trafficking machinery and mutations in LYSET can explain the phenotype of the associated disorder.
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COVID-19 , Lisosomas , Mucolipidosis , Proteínas , Animales , COVID-19/genética , Catepsinas/metabolismo , Humanos , Lisosomas/metabolismo , Manosa/metabolismo , Ratones , Ratones Noqueados , Mucolipidosis/genética , Mucolipidosis/metabolismo , Proteínas/genética , Transferasas (Grupos de Otros Fosfatos Sustitutos)RESUMEN
Affordable and effective antiviral therapies are needed worldwide, especially against agents such as dengue virus that are endemic in underserved regions. Many antiviral compounds have been studied in cultured cells but are unsuitable for clinical applications due to pharmacokinetic profiles, side effects, or inconsistent efficacy across dengue serotypes. Such tool compounds can, however, aid in identifying clinically useful treatments. Here, computational screening (Rapid Overlay of Chemical Structures) was used to identify entries in an in silico database of safe-in-human compounds (SWEETLEAD) that display high chemical similarities to known inhibitors of dengue virus. Inhibitors of the dengue proteinase NS2B/3, the dengue capsid, and the host autophagy pathway were used as query compounds. Three FDA-approved compounds that resemble the tool molecules structurally, cause little toxicity, and display strong antiviral activity in cultured cells were selected for further analysis. Pyrimethamine (50% inhibitory concentration [IC50] = 1.2 µM), like the dengue proteinase inhibitor ARDP0006 to which it shows structural similarity, inhibited intramolecular NS2B/3 cleavage. Lack of toxicity early in infection allowed testing in mice, in which pyrimethamine also reduced viral loads. Niclosamide (IC50 = 0.28 µM), like dengue core inhibitor ST-148, affected structural components of the virion and inhibited early processes during infection. Vandetanib (IC50 = 1.6 µM), like cellular autophagy inhibitor spautin-1, blocked viral exit from cells and could be shown to extend survival in vivo Thus, three FDA-approved compounds with promising utility for repurposing to treat dengue virus infections and their potential mechanisms were identified using computational tools and minimal phenotypic screening.IMPORTANCE No antiviral therapeutics are currently available for dengue virus infections. By computationally overlaying the three-dimensional (3D) chemical structures of compounds known to inhibit dengue virus over those of compounds known to be safe in humans, we identified three FDA-approved compounds that are attractive candidates for repurposing as antivirals. We identified targets for two previously identified antiviral compounds and revealed a previously unknown potential anti-dengue compound, vandetanib. This computational approach to analyze a highly curated library of structures has the benefits of speed and cost efficiency. It also leverages mechanistic work with query compounds used in biomedical research to provide strong hypotheses for the antiviral mechanisms of the safer hit compounds. This workflow to identify compounds with known safety profiles can be expanded to any biological activity for which a small-molecule query compound has been identified, potentially expediting the translation of basic research to clinical interventions.
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Antivirales/farmacología , Virus del Dengue/efectos de los fármacos , Dengue/virología , Animales , Bases de Datos Farmacéuticas , Dengue/tratamiento farmacológico , Virus del Dengue/genética , Virus del Dengue/fisiología , Evaluación Preclínica de Medicamentos , Reposicionamiento de Medicamentos , Humanos , Ratones , Ratones Endogámicos C57BL , Carga Viral/efectos de los fármacos , Replicación Viral/efectos de los fármacosRESUMEN
Enteroviruses (EVs) comprise a large genus of positive-sense, single-stranded RNA viruses whose members cause a number of important and widespread human diseases, including poliomyelitis, myocarditis, acute flaccid myelitis and the common cold. How EVs co-opt cellular functions to promote replication and spread is incompletely understood. Here, using genome-scale CRISPR screens, we identify the actin histidine methyltransferase SET domain containing 3 (SETD3) as critically important for viral infection by a broad panel of EVs, including rhinoviruses and non-polio EVs increasingly linked to severe neurological disease such as acute flaccid myelitis (EV-D68) and viral encephalitis (EV-A71). We show that cytosolic SETD3, independent of its methylation activity, is required for the RNA replication step in the viral life cycle. Using quantitative affinity purification-mass spectrometry, we show that SETD3 specifically interacts with the viral 2A protease of multiple enteroviral species, and we map the residues in 2A that mediate this interaction. 2A mutants that retain protease activity but are unable to interact with SETD3 are severely compromised in RNA replication. These data suggest a role of the viral 2A protein in RNA replication beyond facilitating proteolytic cleavage. Finally, we show that SETD3 is essential for in vivo replication and pathogenesis in multiple mouse models for EV infection, including CV-A10, EV-A71 and EV-D68. Our results reveal a crucial role of a host protein in viral pathogenesis, and suggest targeting SETD3 as a potential mechanism for controlling viral infections.
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Enterovirus/metabolismo , Enterovirus/patogenicidad , Histona Metiltransferasas/metabolismo , Metiltransferasas/metabolismo , Animales , Sistemas CRISPR-Cas , Enfermedades Virales del Sistema Nervioso Central/virología , Modelos Animales de Enfermedad , Encefalitis Viral , Enterovirus/genética , Infecciones por Enterovirus/virología , Histona Metiltransferasas/genética , Ratones , Mielitis/virología , Enfermedades Neuromusculares/virología , Proteolisis , Proteínas Virales , Replicación ViralRESUMEN
For more than 50 years, the methylation of mammalian actin at histidine 73 has been known to occur1. Despite the pervasiveness of His73 methylation, which we find is conserved in several model animals and plants, its function remains unclear and the enzyme that generates this modification is unknown. Here we identify SET domain protein 3 (SETD3) as the physiological actin His73 methyltransferase. Structural studies reveal that an extensive network of interactions clamps the actin peptide onto the surface of SETD3 to orient His73 correctly within the catalytic pocket and to facilitate methyl transfer. His73 methylation reduces the nucleotide-exchange rate on actin monomers and modestly accelerates the assembly of actin filaments. Mice that lack SETD3 show complete loss of actin His73 methylation in several tissues, and quantitative proteomics analysis shows that actin His73 methylation is the only detectable physiological substrate of SETD3. SETD3-deficient female mice have severely decreased litter sizes owing to primary maternal dystocia that is refractory to ecbolic induction agents. Furthermore, depletion of SETD3 impairs signal-induced contraction in primary human uterine smooth muscle cells. Together, our results identify a mammalian histidine methyltransferase and uncover a pivotal role for SETD3 and actin His73 methylation in the regulation of smooth muscle contractility. Our data also support the broader hypothesis that protein histidine methylation acts as a common regulatory mechanism.
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Actinas/química , Actinas/metabolismo , Distocia/enzimología , Distocia/prevención & control , Histidina/química , Histidina/metabolismo , Metiltransferasas/metabolismo , Animales , Línea Celular , Femenino , Histona Metiltransferasas , Histonas , Tamaño de la Camada/genética , Masculino , Metilación , Metiltransferasas/deficiencia , Metiltransferasas/genética , Ratones , Modelos Moleculares , Músculo Liso/citología , Músculo Liso/fisiología , Embarazo , Proteómica , Contracción Uterina , Útero/citología , Útero/fisiologíaRESUMEN
Many positive-strand RNA viruses translate their genomes as single polyproteins that are processed by host and viral proteinases to generate all viral protein products. Among these is dengue virus, which encodes the serine proteinase NS2B/3 responsible for seven different cleavages in the polyprotein. NS2B/3 has been the subject of many directed screens to find chemical inhibitors, of which the compound ARDP0006 is among the most effective at inhibiting viral growth. We show that at least three cleavages in the dengue polyprotein are exclusively intramolecular. By definition, such a cis-acting defect cannot be rescued in trans This creates the possibility that a drug-susceptible or inhibited proteinase can be genetically dominant, inhibiting the outgrowth of drug-resistant virus via precursor accumulation. Indeed, an NS3-G459L variant that is incapable of cleavage at the internal NS3 junction dominantly inhibited negative-strand RNA synthesis of wild-type virus present in the same cell. This internal NS3 cleavage site is the junction most inhibited by ARDP0006, making it likely that the accumulation of toxic precursors, not inhibition of proteolytic activity per se, explains the antiviral efficacy of this compound in restraining viral growth. We argue that intramolecularly cleaving proteinases are promising drug targets for viruses that encode polyproteins. The most effective inhibitors will specifically target cleavage sites required for processing precursors that exert trans-dominant inhibition.
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Virus del Dengue/metabolismo , Proteolisis , ARN Viral/biosíntesis , Serina Endopeptidasas/metabolismo , Proteínas no Estructurales Virales/metabolismo , Animales , Línea Celular , Cricetinae , Virus del Dengue/genética , ARN Viral/genética , Serina Endopeptidasas/genética , Proteínas no Estructurales Virales/genéticaRESUMEN
There is an urgent need for strategies to combat dengue virus (DENV) infection; a major global threat. We reported that the cellular kinases AAK1 and GAK regulate intracellular trafficking of multiple viruses and that sunitinib and erlotinib, approved anticancer drugs with potent activity against these kinases, protect DENV-infected mice from mortality. Nevertheless, further characterization of the therapeutic potential and underlying mechanism of this approach is required prior to clinical evaluation. Here, we demonstrate that sunitinib/erlotinib combination achieves sustained suppression of systemic infection at approved dose in DENV-infected IFN-α/ß and IFN-γ receptor-deficient mice. Nevertheless, treatment with these blood-brain barrier impermeable drugs delays, yet does not prevent, late-onset paralysis; a common manifestation in this immunodeficient mouse model but not in humans. Sunitinib and erlotinib treatment also demonstrates efficacy in human primary monocyte-derived dendritic cells. Additionally, DENV infection induces expression of AAK1 transcripts, but not GAK, via single-cell transcriptomics, and these kinases are important molecular targets underlying the anti-DENV effect of sunitinib and erlotinib. Lastly, sunitinib/erlotinib combination alters inflammatory cytokine responses in DENV-infected mice. These findings support feasibility of repurposing sunitinib/erlotinib combination as a host-targeted antiviral approach and contribute to understanding its mechanism of antiviral action.
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Antivirales/uso terapéutico , Virus del Dengue/efectos de los fármacos , Dengue/tratamiento farmacológico , Reposicionamiento de Medicamentos , Clorhidrato de Erlotinib/uso terapéutico , Sunitinib/uso terapéutico , Animales , Células Cultivadas , Células Dendríticas/efectos de los fármacos , Células Dendríticas/virología , Virus del Dengue/fisiología , Estudios de Factibilidad , Femenino , Perfilación de la Expresión Génica , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones SCID , Proteínas Serina-Treonina Quinasas/genética , Análisis de la Célula Individual , Replicación Viral/efectos de los fármacosRESUMEN
Determinants and mechanisms of cell attachment and entry steer adeno-associated virus (AAV) in its utility as a gene therapy vector. Thus far, a systematic assessment of how diverse AAV serotypes engage their proteinaceous receptor AAVR (KIAA0319L) to establish transduction has been lacking, despite potential implications for cell and tissue tropism. Here, a large set of human and simian AAVs as well as in silico-reconstructed ancestral AAV capsids were interrogated for AAVR usage. We identified a distinct AAV capsid lineage comprised of AAV4 and AAVrh32.33 that can bind and transduce cells in the absence of AAVR, independent of the multiplicity of infection. Virus overlay assays and rescue experiments in nonpermissive cells demonstrate that these AAVs are unable to bind to or use the AAVR protein for entry. Further evidence for a distinct entry pathway was observed in vivo, as AAVR knockout mice were equally as permissive to transduction by AAVrh32.33 as wild-type mice upon systemic injection. We interestingly observe that some AAV capsids undergo a low level of transduction in the absence of AAVR, both in vitro and in vivo, suggesting that some capsids may have a multimodal entry pathway. In aggregate, our results demonstrate that AAVR usage is conserved among all primate AAVs except for those of the AAV4 lineage, and a non-AAVR pathway may be available to other serotypes. This work furthers our understanding of the entry of AAV, a vector system of broad utility in gene therapy.IMPORTANCE Adeno-associated virus (AAV) is a nonpathogenic virus that is used as a vehicle for gene delivery. Here, we have identified several situations in which transduction is retained in both cell lines and a mouse model in the absence of a previously defined entry receptor, AAVR. Defining the molecular determinants of the infectious pathway of this highly relevant viral vector system can help refine future applications and therapies with this vector.
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Cápside/metabolismo , Dependovirus , Vectores Genéticos , Transducción Genética , Internalización del Virus , Animales , Línea Celular , Dependovirus/genética , Dependovirus/metabolismo , Ratones , Ratones Noqueados , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismoRESUMEN
Zika virus has garnered great attention over the last several years, as outbreaks of the disease have emerged throughout the Western Hemisphere. Until quite recently Zika virus was considered a fairly benign virus, with limited clinical severity in both people and animals. The size and scope of the outbreak in the Western Hemisphere has allowed for the identification of severe clinical disease that is associated with Zika virus infection, most notably microcephaly among newborns, and an association with Guillian-Barré syndrome in adults. This recent association with severe clinical disease, of which further analysis strongly suggested causation by Zika virus, has resulted in a massive increase in the amount of both basic and applied research of this virus. Both small and large animal models are being used to uncover the pathogenesis of this emerging disease and to develop vaccine and therapeutic strategies. Here we review the animal-model-based Zika virus research that has been performed to date.
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Modelos Animales de Enfermedad , Infección por el Virus Zika , Animales , Síndrome de Guillain-Barré/virología , Macaca , Ratones , Microcefalia/virología , Proyectos de InvestigaciónRESUMEN
The role of host type I IFN signaling and its interaction with other immune pathways during bacterial infections is incompletely understood. Type II IFN signaling plays a key role during numerous bacterial infections including granulocytic anaplasmosis (GA) caused by Anaplasma phagocytophilum infection. The function of combined type I and type II IFN signaling and their potential synergism during GA and similar tick-borne diseases is a topic of current research investigation. The goal of this study was to evaluate 2 mouse models of absent type I/type II IFN signaling in experimental A. phagocytophilum infection to determine the effects of background strain. Mice lacking both type I and type II IFN receptor signaling (IFNAR-/-/IFNGR-/-) on either the 129/SvEv or C57BL/6J genetic background were evaluated at days 0, 6, 8, and 12 of infection. Pathogen burden in multiple organs was largely similar between strains of infected mice, with few significant differences. Background strain influenced the immune response to infection. Mice of the 129/SvEv strain developed more severe hematologic abnormalities, particularly more severe leukocytosis with marked neutrophilia and lymphocytosis, throughout acute infection. Histopathologic changes occurred in infected mice of both strains and varied in severity by organ. 129/SvEv mice developed more severe pathologic changes in spleen and bone marrow, whereas C57BL/6J mice developed more severe renal pathology. This work highlights the importance of mouse background strain in dictating pathophysiologic response to infection and informs future work regarding the loss of type I and type II IFN signaling on the immune response during GA.
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Anaplasmosis/genética , Interferón Tipo I/genética , Interferón gamma/genética , Anaplasmosis/inmunología , Anaplasmosis/patología , Animales , Modelos Animales de Enfermedad , Predisposición Genética a la Enfermedad , Interferón Tipo I/metabolismo , Interferón gamma/metabolismo , Ratones Endogámicos C57BL , Ratones Endogámicos , Carga de Parásitos , Transducción de Señal/genéticaRESUMEN
PURPOSE: It is well known that cancers exploit immune checkpoints (programmed death 1 receptor (PD-1) and its ligand (PD-L1)) to evade anti-tumor immune responses. Although immune checkpoint (IC) blockade is a promising approach, not all patients respond. Hence, imaging of tumor-infiltrating lymphocytes (TILs) is of high specific interest, as they are known to express PD-1 during activation and subsequent exhaustion in the tumor microenvironment and are thought to be potentially predictive of therapeutic responses to IC blockade. PROCEDURES: We developed immune-tracers for positron emission tomography (PET) to image hPD-1 status of human peripheral blood mononuclear cells (hPBMCs) adoptively transferred to NOD-scid IL-2Rγnull (NSG) mice (hNSG) bearing A375 human skin melanoma tumors. The anti-PD-1 human antibody (IgG; keytruda) was labeled with either Zr-89 or Cu-64 radiometals to image PD-1-expressing human TILs in vivo. RESULTS: [89Zr] Keytruda (groups = 2; NSG-ctl (control) and hNSG-nblk (non-blocking), n = 3-5, 3.2 ± 0.4 MBq/15-16 µg/200 µl) and [64Cu] Keytruda (groups = 3; NSG-ctl, NSG-blk (blocking), and hNSG-nblk; n = 4, 7.4 ± 0.4 MBq /20-25 µg/200 µl) were administered in mice. PET-CT scans were performed over 1-144 h ([89Zr] Keytruda) and 1-48 h ([64Cu] Keytruda) on mice. hNSG mice exhibited a high tracer uptake in the spleen, lymphoid organs and tumors. At 24 h, human TILs homing into melanoma of hNSG-nblk mice exhibited high signal (mean %ID/g ± SD) of 3.8 ± 0.4 ([89Zr] Keytruda), and 6.4 ± 0.7 ([64Cu] Keytruda), which was 1.5- and 3-fold higher uptake compared to NSG-ctl mice (p = 0.01), respectively. Biodistribution measurements of hNSG-nblk mice performed at 144 h ([89Zr] Keytruda) and 48 h ([64Cu] Keytruda) p.i. revealed tumor to muscle ratios as high as 45- and 12-fold, respectively. CONCLUSIONS: Our immunoPET study clearly demonstrates specific imaging of human PD-1-expressing TILs within the tumor and lymphoid tissues. This suggests these anti-human-PD-1 tracers could be clinically translatable to monitor cancer treatment response to IC blockade therapy.
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Anticuerpos Monoclonales Humanizados/química , Antígeno B7-H1/metabolismo , Linfocitos Infiltrantes de Tumor/metabolismo , Linfocitos Infiltrantes de Tumor/patología , Tomografía de Emisión de Positrones , Animales , Línea Celular Tumoral , Cromatografía Líquida de Alta Presión , Modelos Animales de Enfermedad , Humanos , Ratones , Tomografía Computarizada por Tomografía de Emisión de Positrones , Radiofármacos , Distribución TisularRESUMEN
Global health is threatened by emerging viral infections, which largely lack effective vaccines or therapies. Targeting host pathways that are exploited by multiple viruses could offer broad-spectrum solutions. We previously reported that AAK1 and GAK, kinase regulators of the host adaptor proteins AP1 and AP2, are essential for hepatitis C virus (HCV) infection, but the underlying mechanism and relevance to other viruses or in vivo infections remained unknown. Here, we have discovered that AP1 and AP2 cotraffic with HCV particles in live cells. Moreover, we found that multiple viruses, including dengue and Ebola, exploit AAK1 and GAK during entry and infectious virus production. In cultured cells, treatment with sunitinib and erlotinib, approved anticancer drugs that inhibit AAK1 or GAK activity, or with more selective compounds inhibited intracellular trafficking of HCV and multiple unrelated RNA viruses with a high barrier to resistance. In murine models of dengue and Ebola infection, sunitinib/erlotinib combination protected against morbidity and mortality. We validated sunitinib- and erlotinib-mediated inhibition of AAK1 and GAK activity as an important mechanism of antiviral action. Additionally, we revealed potential roles for additional kinase targets. These findings advance our understanding of virus-host interactions and establish a proof of principle for a repurposed, host-targeted approach to combat emerging viruses.
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Antineoplásicos/farmacología , Antivirales/farmacología , Clorhidrato de Erlotinib/farmacología , Indoles/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Pirroles/farmacología , Complejo 1 de Proteína Adaptadora/metabolismo , Complejo 2 de Proteína Adaptadora/metabolismo , Animales , Línea Celular Tumoral , Dengue/prevención & control , Dengue/virología , Virus del Dengue/efectos de los fármacos , Virus del Dengue/metabolismo , Evaluación Preclínica de Medicamentos , Sinergismo Farmacológico , Ebolavirus/efectos de los fármacos , Ebolavirus/metabolismo , Femenino , Fiebre Hemorrágica Ebola/prevención & control , Fiebre Hemorrágica Ebola/virología , Hepacivirus/efectos de los fármacos , Hepacivirus/metabolismo , Hepatitis C/prevención & control , Hepatitis C/virología , Humanos , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Masculino , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/metabolismo , Transporte de Proteínas , Sunitinib , Internalización del Virus/efectos de los fármacosRESUMEN
Neonatal mice (that is, pups younger than 6 d) must be exposed to CO2 for as long as 50 min to achieve euthanasia. Alternatively, other inhalant anesthetic agents have been used to euthanize laboratory rodent species. We investigated the efficacy of isoflurane at saturated vapor pressure to euthanize neonatal mice. Neonatal mice (n = 76; age, 1 or 2 d) were exposed to isoflurane in a sealed, quart-size (0.95-L) plastic bag at room temperature. Righting and withdrawal reflexes were absent in less than 2 min. After 30 min of exposure to isoflurane, pups were removed and monitored for recovery. All pups were cyanotic and showed no detectable signs of life when they were removed from the bag. However, after 30 to 120 min after removal from the bag, 24% of isoflurane-overexposed pups began gasping and then resumed normal respiration and regained a normal pink coloration. These results demonstrate that isoflurane overexposure at saturated vapor pressure for 30 min is insufficient to euthanize neonatal mice and that isoflurane overexposure must be followed by a secondary means of euthanasia.
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Isoflurano/administración & dosificación , Ratones , Anestésicos por Inhalación/administración & dosificación , Animales , Animales Recién Nacidos , Dióxido de Carbono/administración & dosificación , Eutanasia Animal/métodos , Ratones Endogámicos C57BL , ReflejoRESUMEN
UNLABELLED: Dengue virus is a major human pathogen responsible for 400 million infections yearly. As with other RNA viruses, daunting challenges to antiviral design exist due to the high error rates of RNA-dependent RNA synthesis. Indeed, treatment of dengue virus infection with a nucleoside analog resulted in the expected genetic selection of resistant viruses in tissue culture and in mice. However, when the function of the oligomeric core protein was inhibited, no detectable selection of drug resistance in tissue culture or in mice was detected, despite the presence of drug-resistant variants in the population. Suppressed selection of drug-resistant virus correlated with cooligomerization of the targeted drug-susceptible and drug-resistant core proteins. The concept of "dominant drug targets," in which inhibition of oligomeric viral assemblages leads to the formation of drug-susceptible chimeras, can therefore be used to prevent the outgrowth of drug resistance during dengue virus infection. IMPORTANCE: Drug resistance is a major hurdle in the development of effective antivirals, especially those directed at RNA viruses. We have found that one can use the concept of the genetic dominance of defective subunits to "turn cousins into enemies," i.e., to thwart the outgrowth of drug-resistant viral genomes as soon as they are generated. This requires deliberate targeting of larger assemblages, which would otherwise rarely be considered by antiviral researchers.
Asunto(s)
Virus del Dengue/efectos de los fármacos , Virus del Dengue/crecimiento & desarrollo , Farmacorresistencia Viral , Supresión Genética , Animales , Antivirales/administración & dosificación , Antivirales/farmacología , Ratones , Selección GenéticaRESUMEN
We report the isolation of a novel helicobacter isolated from the caecum of the Siberian hamster (Phodopus sungorus). Sequence analysis showed 97% sequence similarity to Helicobacter ganmani. In addition, we report the co-infection of these Siberian hamsters with a Campylobacter sp. and a second Helicobacter sp. with 99% sequence similarity to Helicobacter sp. flexispira taxon 8 (Helicobacter bilis), a species isolated previously from patients with bacteraemia. Gross necropsy and histopathology did not reveal any overt pathological lesions of the liver and gastrointestinal tract that could be attributed to the Helicobacter or Campylobacter spp. infections. This is the first helicobacter to be identified in the Siberian hamster and the first report of co-infection of Helicobacter spp. and Campylobacter sp. in asymptomatic Siberian hamsters.