RESUMEN
OBJECTIVES: In many jurisdictions, ethical concerns require surrogate humane endpoints to replace death in small animal models of acute lung injury. Heterogenous selection and reporting of surrogate endpoints render interpretation and generalizability of findings between studies difficult. We aimed to establish expert-guided consensus among preclinical scientists and laboratory animal veterinarians on selection and reporting of surrogate endpoints, monitoring of these models, and the use of analgesia. DESIGN: A three-round consensus process, using modified Delphi methodology, with researchers who use small animal models of acute lung injury and laboratory animal veterinarians who provide care for these animals. Statements on the selection and reporting of surrogate endpoints, monitoring, and analgesia were generated through a systematic search of MEDLINE and Embase. Participants were asked to suggest any additional potential statements for evaluation. SETTING: A web-based survey of participants representing the two stakeholder groups (researchers, laboratory animal veterinarians). Statements were rated on level of evidence and strength of support by participants. A final face-to-face meeting was then held to discuss results. SUBJECTS: None. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Forty-two statements were evaluated, and 29 were rated as important, with varying strength of evidence. The majority of evidence was based on rodent models of acute lung injury. Endpoints with strong support and evidence included temperature changes and body weight loss. Behavioral signs and respiratory distress also received support but were associated with lower levels of evidence. Participants strongly agreed that analgesia affects outcomes in these models and that none may be necessary following nonsurgical induction of acute lung injury. Finally, participants strongly supported transparent reporting of surrogate endpoints. A prototype composite score was also developed based on participant feedback. CONCLUSIONS: We provide a preliminary framework that researchers and animal welfare committees may adapt for their needs. We have identified knowledge gaps that future research should address.
Asunto(s)
Lesión Pulmonar Aguda/fisiopatología , Comités de Atención Animal/organización & administración , Bienestar del Animal/normas , Animales de Laboratorio , Consenso , Animales , Biomarcadores , Humanos , Modelos Animales , Veterinarios/normasRESUMEN
Variants in the leucine-rich repeat kinase-2 (LRRK2) gene are associated with Parkinson's disease, leprosy, and Crohn's disease, three disorders with inflammation as an important component. Because of its high expression in granulocytes and CD68-positive cells, LRRK2 may have a function in innate immunity. We tested this hypothesis in two ways. First, adult mice were intravenously inoculated with Salmonella typhimurium, resulting in sepsis. Second, newborn mouse pups were intranasally infected with reovirus (serotype 3 Dearing), which induced encephalitis. In both mouse models, wild-type Lrrk2 expression was protective and showed a sex effect, with female Lrrk2-deficient animals not controlling infection as well as males. Mice expressing Lrrk2 carrying the Parkinson's disease-linked p.G2019S mutation controlled infection better, with reduced bacterial growth and longer animal survival during sepsis. This gain-of-function effect conferred by the p.G2019S mutation was mediated by myeloid cells and was abolished in animals expressing a kinase-dead Lrrk2 variant, p.D1994S. Mouse pups with reovirus-induced encephalitis that expressed the p.G2019S Lrrk2 mutation showed increased mortality despite lower viral titers. The p.G2019S mutant Lrrk2 augmented immune cell chemotaxis and generated more reactive oxygen species during virulent infection. Reovirus-infected brains from mice expressing the p.G2019S mutant Lrrk2 contained higher concentrations of α-synuclein. Animals expressing one or two p.D1994S Lrrk2 alleles showed lower mortality from reovirus-induced encephalitis. Thus, Lrrk2 alleles may alter the course of microbial infections by modulating inflammation, and this may be dependent on the sex and genotype of the host as well as the type of pathogen.
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Alelos , Infecciones/enzimología , Infecciones/genética , Inflamación/genética , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/genética , Caracteres Sexuales , Animales , Encéfalo/patología , Encéfalo/virología , Quimiotaxis , Encefalitis/virología , Femenino , Humanos , Infecciones/inmunología , Infecciones/patología , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/deficiencia , Leucocitos/enzimología , Masculino , Ratones Endogámicos C57BL , Mutación/genética , Especies Reactivas de Oxígeno/metabolismo , Reoviridae/fisiología , Salmonella typhimurium/crecimiento & desarrollo , Sepsis/microbiología , Análisis de Supervivencia , alfa-Sinucleína/metabolismoRESUMEN
The influenza A virus RNA polymerase cleaves the 5' ends of host RNAs and uses these RNA fragments as primers for viral mRNA synthesis. We performed deep sequencing of the 5' host-derived ends of the eight viral mRNAs of influenza A/Puerto Rico/8/1934 (H1N1) virus in infected A549 cells, and compared the population to those of A/Hong Kong/1/1968 (H3N2) and A/WSN/1933 (H1N1). In the three strains, the viral RNAs target different populations of host RNAs. Host RNAs are cap-snatched based on their abundance, and we found that RNAs encoding proteins involved in metabolism are overrepresented in the cap-snatched populations. Because this overrepresentation could be a reflection of the host response early after infection, and thus of the increased availability of these transcripts, our results suggest that host RNAs are cap-snatched mainly based on their abundance without preferential targeting.
Asunto(s)
Células Epiteliales/virología , Interacciones Huésped-Patógeno , Subtipo H1N1 del Virus de la Influenza A/genética , Subtipo H3N2 del Virus de la Influenza A/genética , Caperuzas de ARN/genética , Caperuzas de ARN/metabolismo , Replicación Viral , Línea Celular , Variación Genética , Humanos , Análisis de Secuencia de ADNRESUMEN
Influenza A virus (IAV) non-structural protein 1 (NS1) suppresses host innate immune responses by inhibiting type I interferon (IFN) production. We provide evidence that residues F103 and M106 in the CPSF4-binding domain of A/HK/1/68 [H3N2] NS1 contribute to post-transcriptional inhibition of antiviral IFN-stimulated genes (ISGs), thereby suppressing an antiviral type I IFN response. Recombinant (r) IAVs encoding F103L and M106I mutations in NS1 replicate to significantly lower viral titers in human A549 lung epithelial cells and primary type II alveolar cells. In A549 cells, rIAVs encoding these mutant NS1s induce higher levels of IFN-ß production and are more sensitive to the antiviral effects of IFN-ß treatment. qPCR characterization of polysomal mRNA, in the presence or absence of IFN-ß treatment, identified a greater proportion of heavy polysome-associated ISGs including EIF2AK2, OAS1, and MxA in A549 cells infected with rIAVs encoding these CPSF4-binding mutant NS1s, in contrast to rIAV encoding wildtype NS1.
Asunto(s)
Factor de Especificidad de Desdoblamiento y Poliadenilación/metabolismo , Subtipo H3N2 del Virus de la Influenza A/metabolismo , Gripe Humana/metabolismo , Interferones/genética , Iniciación de la Cadena Peptídica Traduccional , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/metabolismo , Secuencias de Aminoácidos , Factor de Especificidad de Desdoblamiento y Poliadenilación/genética , Interacciones Huésped-Patógeno , Humanos , Subtipo H3N2 del Virus de la Influenza A/química , Subtipo H3N2 del Virus de la Influenza A/genética , Gripe Humana/genética , Gripe Humana/virología , Interferones/metabolismo , Unión Proteica , Proteínas no Estructurales Virales/genéticaRESUMEN
Braak and Del Tredici have proposed that typical Parkinson disease (PD) has its origins in the olfactory bulb and gastrointestinal tract. However, the role of the olfactory system has insufficiently been explored in the pathogeneses of PD and Alzheimer disease (AD) in laboratory models. Here, we demonstrate applications of a new method to process mouse heads for microscopy by sectioning, mounting, and staining whole skulls ('holocranohistochemistry'). This technique permits the visualization of the olfactory system from the nasal cavity to mitral cells and dopamine-producing interneurons of glomeruli in the olfactory bulb. We applied this method to two specific goals: first, to visualize PD- and AD-linked gene expression in the olfactory system, where we detected abundant, endogenous α-synuclein and tau expression in the olfactory epithelium. Furthermore, we observed amyloid-ß plaques and proteinase-K-resistant α-synuclein species, respectively, in cranial nerve-I of APP- and human SNCA-over-expressing mice. The second application of the technique was to the modeling of gene-environment interactions in the nasal cavity of mice. We tracked the infection of a neurotropic respiratory-enteric-orphan virus from the nose pad into cranial nerves-I (and -V) and monitored the ensuing brain infection. Given its abundance in the olfactory epithelia, we questioned whether α-synuclein played a role in innate host defenses to modify the outcome of infections. Indeed, Snca-null mice were more likely to succumb to viral encephalitis versus their wild-type littermates. Moreover, using a bacterial sepsis model, Snca-null mice were less able to control infection after intravenous inoculation with Salmonella typhimurium. Together, holocranohistochemistry enabled new discoveries related to α-synuclein expression and its function in mice. Future studies will address: the role of Mapt and mutant SNCA alleles in infection paradigms; the contribution of xenobiotics in the initiation of idiopathic PD; and the safety to the host when systemically targeting α-synuclein by immunotherapy.
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Encéfalo/metabolismo , Encéfalo/virología , Encefalitis Viral/virología , Mucosa Olfatoria/anatomía & histología , Mucosa Olfatoria/metabolismo , Infecciones por Reoviridae/virología , alfa-Sinucleína/metabolismo , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Encéfalo/diagnóstico por imagen , Encéfalo/patología , Modelos Animales de Enfermedad , Encefalitis Viral/inmunología , Encefalitis Viral/mortalidad , Encefalitis Viral/patología , Femenino , Cabeza , Humanos , Inmunohistoquímica , Masculino , Orthoreovirus Mamífero 3 , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Transgénicos , Vías Nerviosas/anatomía & histología , Vías Nerviosas/diagnóstico por imagen , Vías Nerviosas/metabolismo , Vías Nerviosas/patología , Mucosa Olfatoria/patología , Neuronas Receptoras Olfatorias/metabolismo , Neuronas Receptoras Olfatorias/virología , Infecciones por Reoviridae/inmunología , Infecciones por Salmonella/inmunología , Infecciones por Salmonella/patología , Salmonella typhimurium , Conservación de Tejido/métodos , alfa-Sinucleína/genéticaRESUMEN
The non-structural protein, NS1, is a virulence factor encoded by influenza A viruses (IAVs). In this report, we provide evidence that the conserved residue, tyrosine (Y) 84, in a conserved putative SH2-binding domain in A/Duck/Hubei/2004/L-1 [H5N1] NS1 is critical for limiting an interferon (IFN) response to infection. A phenylalanine (F) substitution of this Y84 residue abolishes NS1-mediated downregulation of IFN-inducible STAT phosphorylation, and surface IFNAR1 expression. Recombinant IAV (rIAV) [H1N1] expressing A/Grey Heron/Hong Kong/837/2004 [H5N1] NS1-Y84F (rWSN-GH-NS1-Y84F) replicates to lower titers in human lung epithelial cells and is more susceptible to the antiviral effects of IFN-ß treatment compared with rIAV expressing the intact H5N1 NS1 (rWSN-GH-NS1-wt). Cells infected with rWSN-GH-NS1-Y84F express higher levels of IFN stimulated genes (ISGs) associated with an antiviral response compared with cells infected with rWSN-GH-NS1-wt. In mice, intranasal infection with rWSN-GH-NS1-Y84F resulted in a delay in onset of weight loss, reduced lung pathology, lower lung viral titers and higher ISG expression, compared with mice infected with rWSN-GH-NS1-wt. IFN-ß treatment of mice infected with rWSN-GH-NS1-Y84F reduced lung viral titers and increased lung ISG expression, but did not alter viral titers and ISG expression in mice infected with rWSN-GH-NS1-wt. Viewed altogether, these data suggest that the virulence associated with this conserved Y84 residue in NS1 is, in part, due to its role in regulating the host IFN response.
Asunto(s)
Subtipo H5N1 del Virus de la Influenza A/metabolismo , Gripe Humana/virología , Interferones/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Proteínas no Estructurales Virales/antagonistas & inhibidores , Virosis/metabolismo , Células A549 , Animales , Antivirales/farmacología , Modelos Animales de Enfermedad , Perros , Células Epiteliales/virología , Fibroblastos , Células HEK293 , Células HeLa , Interacciones Huésped-Patógeno/efectos de los fármacos , Humanos , Subtipo H1N1 del Virus de la Influenza A/genética , Subtipo H1N1 del Virus de la Influenza A/metabolismo , Subtipo H1N1 del Virus de la Influenza A/fisiología , Subtipo H5N1 del Virus de la Influenza A/patogenicidad , Subtipo H5N1 del Virus de la Influenza A/fisiología , Virus de la Influenza A/genética , Virus de la Influenza A/metabolismo , Virus de la Influenza A/fisiología , Interferón beta , Pulmón/patología , Pulmón/virología , Células de Riñón Canino Madin Darby , Masculino , Ratones , Ratones Endogámicos C57BL , Mutagénesis Sitio-Dirigida , Neutrófilos/patología , Neutrófilos/virología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Genética Inversa , Transfección , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/fisiología , Virulencia , Factores de VirulenciaRESUMEN
Fifty-five years after the concept of dopamine replacement therapy was introduced, Parkinson disease (PD) remains an incurable neurological disorder. To date, no disease-modifying therapeutic has been approved. The inability to predict PD incidence risk in healthy adults is seen as a limitation in drug development, because by the time of clinical diagnosis ≥ 60% of dopamine neurons have been lost. We have designed an incidence prediction model founded on the concept that the pathogenesis of PD is similar to that of many disorders observed in ageing humans, i.e. a complex, multifactorial disease. Our model considers five factors to determine cumulative incidence rates for PD in healthy adults: (i) DNA variants that alter susceptibility (D), e.g. carrying a LRRK2 or GBA risk allele; (ii) Exposure history to select environmental factors including xenobiotics (E); (iii) Gene-environment interactions that initiate pathological tissue responses (I), e.g. a rise in ROS levels, misprocessing of amyloidogenic proteins (foremost, α-synuclein) and dysregulated inflammation; (iv) sex (or gender; G); and importantly, (v) time (T) encompassing ageing-related changes, latency of illness and propagation of disease. We propose that cumulative incidence rates for PD (PR ) can be calculated in healthy adults, using the formula: PR (%) = (E + D + I) × G × T. Here, we demonstrate six case scenarios leading to young-onset parkinsonism (n = 3) and late-onset PD (n = 3). Further development and validation of this prediction model and its scoring system promise to improve subject recruitment in future intervention trials. Such efforts will be aimed at disease prevention through targeted selection of healthy individuals with a higher prediction score for developing PD in the future and at disease modification in subjects that already manifest prodromal signs.
Asunto(s)
Interacción Gen-Ambiente , Enfermedad de Parkinson/epidemiología , Enfermedad de Parkinson/genética , Dopamina/metabolismo , Humanos , Incidencia , Mutación/genética , Enfermedad de Parkinson/metabolismo , Proteínas Serina-Treonina Quinasas/genética , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismoRESUMEN
[This corrects the article DOI: 10.1371/journal.ppat.1005446.].
RESUMEN
The immune response to influenza virus infection comprises both innate and adaptive defenses. NK cells play an early role in the destruction of tumors and virally-infected cells. NK cells express a variety of inhibitory receptors, including those of the Ly49 family, which are functional homologs of human killer-cell immunoglobulin-like receptors (KIR). Like human KIR, Ly49 receptors inhibit NK cell-mediated lysis by binding to major histocompatibility complex class I (MHC-I) molecules that are expressed on normal cells. During NK cell maturation, the interaction of NK cell inhibitory Ly49 receptors with their MHC-I ligands results in two types of NK cells: licensed ("functional"), or unlicensed ("hypofunctional"). Despite being completely dysfunctional with regard to rejecting MHC-I-deficient cells, unlicensed NK cells represent up to half of the mature NK cell pool in rodents and humans, suggesting an alternative role for these cells in host defense. Here, we demonstrate that after influenza infection, MHC-I expression on lung epithelial cells is upregulated, and mice bearing unlicensed NK cells (Ly49-deficient NKCKD and MHC-I-deficient B2m-/- mice) survive the infection better than WT mice. Importantly, transgenic expression of an inhibitory self-MHC-I-specific Ly49 receptor in NKCKD mice restores WT influenza susceptibility, confirming a direct role for Ly49. Conversely, F(ab')2-mediated blockade of self-MHC-I-specific Ly49 inhibitory receptors protects WT mice from influenza virus infection. Mechanistically, perforin-deficient NKCKD mice succumb to influenza infection rapidly, indicating that direct cytotoxicity is necessary for unlicensed NK cell-mediated protection. Our findings demonstrate that Ly49:MHC-I interactions play a critical role in influenza virus pathogenesis. We suggest a similar role may be conserved in human KIR, and their blockade may be protective in humans.
Asunto(s)
Antígenos Ly/metabolismo , Evasión Inmune , Virus de la Influenza A/inmunología , Células Asesinas Naturales/inmunología , Subfamilia A de Receptores Similares a Lectina de Células NK/metabolismo , Infecciones por Orthomyxoviridae/inmunología , Receptores KIR/metabolismo , Mucosa Respiratoria/inmunología , Animales , Antígenos Ly/genética , Línea Celular Tumoral , Células Cultivadas , Técnicas de Cocultivo , Cruzamientos Genéticos , Inmunidad Innata , Virus de la Influenza A/fisiología , Células Asesinas Naturales/metabolismo , Células Asesinas Naturales/patología , Células Asesinas Naturales/virología , Pulmón/inmunología , Pulmón/metabolismo , Pulmón/patología , Pulmón/virología , Ratones Noqueados , Ratones Transgénicos , Subfamilia A de Receptores Similares a Lectina de Células NK/agonistas , Subfamilia A de Receptores Similares a Lectina de Células NK/antagonistas & inhibidores , Subfamilia A de Receptores Similares a Lectina de Células NK/genética , Infecciones por Orthomyxoviridae/metabolismo , Infecciones por Orthomyxoviridae/patología , Infecciones por Orthomyxoviridae/virología , Proteínas Citotóxicas Formadoras de Poros/genética , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Receptores KIR/agonistas , Receptores KIR/antagonistas & inhibidores , Receptores KIR/genética , Mucosa Respiratoria/metabolismo , Mucosa Respiratoria/patología , Mucosa Respiratoria/virología , Organismos Libres de Patógenos Específicos , Análisis de Supervivencia , Microglobulina beta-2/genética , Microglobulina beta-2/metabolismoRESUMEN
The influenza A virus RNA polymerase cleaves the 5' end of host pre-mRNAs and uses the capped RNA fragments as primers for viral mRNA synthesis. We performed deep sequencing of the 5' ends of viral mRNAs from all genome segments transcribed in both human (A549) and mouse (M-1) cells infected with the influenza A/HongKong/1/1968 (H3N2) virus. In addition to information on RNA motifs present, our results indicate that the host primers are divergent between the viral transcripts. We observed differences in length distributions, nucleotide motifs and the identity of the host primers between the viral mRNAs. Mapping the reads to known transcription start sites indicates that the virus targets the most abundant host mRNAs, which is likely caused by the higher expression of these genes. Our findings suggest negligible competition amongst RdRp:vRNA complexes for individual host mRNA templates during cap-snatching and provide a better understanding of the molecular mechanism governing the first step of transcription of this influenza strain.
Asunto(s)
Subtipo H3N2 del Virus de la Influenza A/genética , Transcripción Genética , Regiones no Traducidas 5' , Animales , Secuencia de Bases , Línea Celular Tumoral , Mapeo Cromosómico , Secuencia de Consenso , Cartilla de ADN/genética , Regulación Viral de la Expresión Génica , Ontología de Genes , Genes Virales , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Ratones , Datos de Secuencia Molecular , ARN Mensajero/genética , ARN Viral/genética , Análisis de Secuencia de ADN , Sitio de Iniciación de la TranscripciónRESUMEN
All influenza viral neuraminidases (NA) of both type A and B viruses have only one universally conserved sequence located between amino acids 222-230. A monoclonal antibody against this region has been previously reported to provide broad inhibition against all nine subtypes of influenza A NA; yet its inhibitory effect against influenza B viral NA remained unknown. Here, we report that the monoclonal antibody provides a broad inhibition against various strains of influenza B viruses of both Victoria and Yamagata genetic lineage. Moreover, the growth and NA enzymatic activity of two drug resistant influenza B strains (E117D and D197E) are also inhibited by the antibody even though these two mutations are conformationally proximal to the universal epitope. Collectively, these data suggest that this unique, highly-conserved linear sequence in viral NA is exposed sufficiently to allow access by inhibitory antibody during the course of infection; it could represent a potential target for antiviral agents and vaccine-induced immune responses against diverse strains of type B influenza virus.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Secuencia Conservada , Farmacorresistencia Viral/inmunología , Epítopos/inmunología , Virus de la Influenza B/enzimología , Gripe Humana/prevención & control , Neuraminidasa/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/farmacología , Anticuerpos Antivirales/inmunología , Perros , Farmacorresistencia Viral/efectos de los fármacos , Farmacorresistencia Viral/genética , Epítopos/química , Humanos , Virus de la Influenza B/efectos de los fármacos , Virus de la Influenza B/crecimiento & desarrollo , Virus de la Influenza B/inmunología , Gripe Humana/inmunología , Gripe Humana/virología , Células de Riñón Canino Madin Darby , Modelos Moleculares , Datos de Secuencia Molecular , Mutación/genética , Neuraminidasa/antagonistas & inhibidores , Neuraminidasa/químicaRESUMEN
The only universally conserved sequence amongst all influenza A viral neuraminidase (NA) is located between amino acids 222-230 and plays crucial roles in viral replication. However, it remained unclear as to whether this universal epitope is exposed during the course of infection to allow binding and inhibition by antibodies. Using a monoclonal antibody (MAb) targeting this specific epitope, we demonstrated that all nine subtypes of NA were inhibited in vitro by the MAb. Moreover, the antibody also provided heterosubtypic protection in mice challenged with lethal doses of mouse-adapted H1N1 and H3N2, which represent group I and II viruses, respectively. Furthermore, we report amino acid residues I222 and E227, located in close proximity to the active site, are indispensable for inhibition by this antibody. This unique, highly-conserved linear sequence in viral NA could be an attractive immunological target for protection against diverse strains of influenza viruses.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Antivirales/inmunología , Protección Cruzada , Subtipo H1N1 del Virus de la Influenza A/inmunología , Subtipo H3N2 del Virus de la Influenza A/inmunología , Neuraminidasa/inmunología , Infecciones por Orthomyxoviridae/prevención & control , Proteínas Virales/inmunología , Animales , Anticuerpos Monoclonales/aislamiento & purificación , Anticuerpos Antivirales/aislamiento & purificación , Modelos Animales de Enfermedad , Epítopos de Linfocito B/inmunología , Femenino , Ratones , Infecciones por Orthomyxoviridae/inmunología , Infecciones por Orthomyxoviridae/virologíaRESUMEN
BACKGROUND: The genetic basis for avian to mammalian host switching in influenza A virus is largely unknown. The human A/HK/156/1997 (H5N1) virus that transmitted from poultry possesses NS1 gene mutations F103L + M106I that are virulence determinants in the mouse model of pneumonia; however their individual roles have not been determined. The emergent A/Shanghai/patient1/2013(H7N9)-like viruses also possess these mutations which may contribute to their virulence and ability to switch species. METHODS: NS1 mutant viruses were constructed by reverse genetics and site directed mutagenesis on human and mouse-adapted backbones. Mouse infections assessed virulence, virus yield, tissue infection, and IFN induction. NS1 protein properties were assessed for subcellular distribution, IFN antagonism (mouse and human), CPSF30 and RIG-I domain binding, host transcription (microarray); and the natural prevalence of 103L and 106I mutants was assessed. RESULTS: Each of the F103L and M106I mutations contributes additively to virulence to reduce the lethal dose by >800 and >3,200 fold respectively by mediating alveolar tissue infection with >100 fold increased infectious yields. The 106I NS1 mutant lost CPSF binding but the 103L mutant maintained binding that correlated with an increased general decrease in host gene expression in human but not mouse cells. Each mutation positively modulated the inhibition of IFN induction in mouse cells and activation of the IFN-ß promoter in human cells but not in combination in human cells indicating negative epistasis. Each of the F103L and M106I mutations restored a defect in cytoplasmic localization of H5N1 NS1 in mouse cells. Human H1N1 and H3N2 NS1 proteins bound to the CARD, helicase and RD RIG-I domains, whereas the H5N1 NS1 with the same consensus 103F and 106M mutations did not bind these domains, which was totally or partially restored by the M106I or F103L mutations respectively. CONCLUSIONS: The F103L and M106I mutations in the H5N1 NS1 protein each increased IFN antagonism and mediated interstitial pneumonia in mice that was associated with increased cytoplasmic localization and altered host factor binding. These mutations may contribute to the ability of previous HPAI H5N1 and recent LPAI H7N9 and H6N1 (NS1-103L+106M) viruses to switch hosts and cause disease in humans.
Asunto(s)
Factor de Especificidad de Desdoblamiento y Poliadenilación/metabolismo , ARN Helicasas DEAD-box/metabolismo , Subtipo H5N1 del Virus de la Influenza A/inmunología , Subtipo H5N1 del Virus de la Influenza A/patogenicidad , Interferones/antagonistas & inhibidores , Mutación Missense , Proteínas no Estructurales Virales/metabolismo , Sustitución de Aminoácidos , Animales , Proteína 58 DEAD Box , Femenino , Interacciones Huésped-Patógeno , Humanos , Subtipo H5N1 del Virus de la Influenza A/aislamiento & purificación , Pulmón/patología , Pulmón/virología , Enfermedades Pulmonares Intersticiales/patología , Enfermedades Pulmonares Intersticiales/virología , Ratones , Mutagénesis Sitio-Dirigida , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Genética Inversa , Proteínas no Estructurales Virales/genética , Virulencia , Factores de Virulencia/genética , Factores de Virulencia/metabolismoRESUMEN
The only universally conserved sequence among all influenza A viral neuraminidases is located between amino acids 222 and 230. However, the potential roles of these amino acids remain largely unknown. Through an array of experimental approaches including mutagenesis, reverse genetics, and growth kinetics, we found that this sequence could markedly affect viral replication. Additional experiments revealed that enzymes with mutations in this region demonstrated substantially decreased catalytic activity, substrate binding, and thermostability. Consistent with viral replication analyses and enzymatic studies, protein modeling suggests that these amino acids could either directly bind to the substrate or contribute to the formation of the active site in the enzyme. Collectively, these findings reveal the essential role of this unique region in enzyme function and viral growth, which provides the basis for evaluating the validity of this sequence as a potential target for antiviral intervention and vaccine development.
Asunto(s)
Epítopos/metabolismo , Virus de la Influenza A/enzimología , Neuraminidasa/metabolismo , Proteínas Virales/metabolismo , Replicación Viral , Sustitución de Aminoácidos , Animales , Sitios de Unión/genética , Biocatálisis , Dominio Catalítico , Línea Celular , Embrión de Pollo , Estabilidad de Enzimas/genética , Epítopos/química , Epítopos/genética , Células HEK293 , Humanos , Virus de la Influenza A/genética , Cinética , Modelos Moleculares , Mutación , Neuraminidasa/química , Neuraminidasa/genética , Estructura Terciaria de Proteína , Especificidad por Sustrato , Temperatura , Proteínas Virales/química , Proteínas Virales/genéticaRESUMEN
BACKGROUND: Because mammalian reoviruses are isolated from the respiratory tract we modeled the natural history of respiratory infection of adult and suckling mice with T1 Lang (T1L) and T3 Dearing (T3D) reoviruses. METHODS: Adult and suckling Balb/c mice were infected by the intranasal route and were assessed for dose response of disease as well as viral replication in the lung and other organs. Viral antigen was assessed by immunofluorescence and HRP staining of tissue sections and histopathology was assessed on formalin fixed, H + E stained tissue sections. RESULTS: Intranasal infection of adult mice resulted in fatal respiratory distress for high doses (10(7) pfu) of T1L but not T3D. In contrast both T1L and T3D killed suckling mice at moderate viral dosages (10(5) pfu) but differed in clinical symptoms where T1L induced respiratory failure and T3D caused encephalitis. Infections caused transient viremia that resulted in spread to peripheral tissues where disease correlated with virus replication, and pathology. Immunofluorescent staining of viral antigens in the lung showed reovirus infection was primarily associated with alveoli with lesser involvement of bronchiolar epithelium. Immunofluorescent and HRP staining of viral antigens in brain showed infection of neurons by T3D and glial cells by T1L. CONCLUSIONS: These mouse models of reovirus respiratory infection demonstrated age and strain dependent disease that are expected to be relevant to understanding and modulating natural and therapeutic reovirus infections in humans.
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Antígenos Virales/inmunología , Encefalitis Viral/virología , Orthoreovirus de los Mamíferos/fisiología , Neumonía Viral/virología , Infecciones por Reoviridae/virología , Infecciones del Sistema Respiratorio/virología , Factores de Edad , Animales , Animales Lactantes , Encéfalo/patología , Encéfalo/virología , Línea Celular , Modelos Animales de Enfermedad , Encefalitis Viral/patología , Femenino , Humanos , Hígado/patología , Hígado/virología , Pulmón/patología , Pulmón/virología , Ratones , Ratones Endogámicos BALB C , Orthoreovirus de los Mamíferos/crecimiento & desarrollo , Orthoreovirus de los Mamíferos/inmunología , Neumonía Viral/patología , Infecciones por Reoviridae/patología , Infecciones del Sistema Respiratorio/patología , Especificidad de la Especie , Factores de Tiempo , Viremia , Replicación ViralRESUMEN
The NS1 protein of influenza A virus (IAV) is a multifunctional virulence factor. We have previously characterized gain-of-function mutations in the NS1 protein arising from the experimental adaptation of the human isolate A/Hong Kong/1/1968(H3N2) (HK) to the mouse. The majority of these mouse adapted NS1 mutations were demonstrated to increase virulence, viral fitness, and interferon antagonism, but differ in binding to the post-transcriptional processing factor cleavage and polyadenylation specificity factor 30 (CPSF30). Because nuclear trafficking is a major genetic determinant of influenza virus host adaptation, we assessed subcellular localization and host gene expression of NS1 adaptive mutations. Recombinant HK viruses with adaptive mutations in the NS1 gene were assessed for NS1 protein subcellular localization in mouse and human cells using confocal microscopy and cellular fractionation. In human cells the HK wild-type (HK-wt) virus NS1 protein partitioned equivalently between the cytoplasm and nucleus but was defective in cytoplasmic localization in mouse cells. Several adaptive mutations increased the proportion of NS1 in the cytoplasm of mouse cells with the greatest effects for mutations M106I and D125G. The host gene expression profile of the adaptive mutants was determined by microarray analysis of infected mouse cells to show either high or low extents of host-gene regulation (HGR or LGR) phenotypes. While host genes were predominantly down regulated for the HGR group of mutants (D2N, V23A, F103L, M106I+L98S, L98S, M106V, and M106V+M124I), the LGR phenotype mutants (D125G, M106I, V180A, V226I, and R227K) were characterized by a predominant up regulation of host genes. CPSF30 binding affinity of NS1 mutants did not predict effects on host gene expression. To our knowledge this is the first report of roles of adaptive NS1 mutations that impact intracellular localization and regulation of host gene expression.
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Adaptación Biológica/genética , Citoplasma/metabolismo , Regulación de la Expresión Génica/genética , Interacciones Huésped-Patógeno/genética , Subtipo H3N2 del Virus de la Influenza A/genética , Mutación/genética , Proteínas no Estructurales Virales/genética , Animales , Fraccionamiento Celular , Línea Celular , Densitometría , Perros , Perfilación de la Expresión Génica , Humanos , Ratones , Análisis por Micromatrices , Microscopía Confocal , Proteínas no Estructurales Virales/metabolismoRESUMEN
Influenza A virus (IAV) non-structural protein 1 (NS1) has multiple functions, is essential for virus replication and may be a good target for IAV diagnosis. To generate broadly cross-reactive NS1-specific mAbs, mice were immunized with A/Hong Kong/1/1968 (H3N2) 6×His-tagged NS1 and hybridomas were screened with glutathione S-transferase-conjugated NS1 of A/Puerto Rico/8/1934 (H1N1). mAbs were isotyped and numerous IgG-type clones were characterized further. Most clones specifically recognized NS1 from various H1N1 and H3N2 IAV types by both immunoblot and immunofluorescence microscopy in mouse M1, canine Madin-Darby canine kidney and human A549 cells. mAb epitopes were mapped by overlapping peptides and selective reactivity to the newly described viral NS3 protein. These mAbs detected NS1 in both the cytoplasm and nucleus by immunostaining, and some detected NS1 as early as 5 h post-infection, suggesting their potential diagnostic use for tracking productive IAV replication and characterizing NS1 structure and function. It was also demonstrated that the newly identified NS3 protein is localized in the cytoplasm to high levels.
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Anticuerpos Monoclonales/inmunología , Anticuerpos Antivirales/inmunología , Virus de la Influenza A/clasificación , Virus de la Influenza A/inmunología , Proteínas no Estructurales Virales/inmunología , Secuencia de Aminoácidos , Animales , Especificidad de Anticuerpos , Línea Celular , Perros , Mapeo Epitopo , Humanos , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Filogenia , Conformación ProteicaRESUMEN
Influenza viruses from domestic aquatic birds can be transmitted to chickens, resulting in continued prevalence of the disease. H3 viruses are one of the most frequently identified subtypes in domestic ducks. Results from our previous serologic study suggested that H3 virus infections potentially exist in chickens with a wide geographical distribution in China. To better understand their pathogenic potential, two H3N8 influenza viruses isolated from domestic ducks were selected for experimental infections in chickens. We found that viral shedding lasted for at least 14 days postinfection for both viruses; however, one virus caused mortality in the chickens when coinfected with Escherichia coli. Sequencing of the viral HA gene isolated from the inoculated chickens revealed two amino acid mutations within the gene. These findings demonstrate the pathogenicity of the H3N8 domestic duck influenza viruses to chickens, highlighting the need for routine epidemiologic investigations of H3 subtype influenza viruses in chicken populations.
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Pollos , Patos , Infecciones por Escherichia coli/veterinaria , Subtipo H3N8 del Virus de la Influenza A/patogenicidad , Gripe Aviar/virología , Animales , Diarrea/microbiología , Diarrea/veterinaria , Diarrea/virología , Infecciones por Escherichia coli/complicaciones , Heces/virología , Subtipo H3N8 del Virus de la Influenza A/clasificación , Gripe Aviar/complicaciones , Esparcimiento de VirusRESUMEN
Chronic hepatitis C virus (HCV) infection is one of the major causes of chronic liver disease worldwide. In order for HCV to persist, the virus must escape immune recognition or inhibit the host immune response. The NS5A protein contains the interferon sensitivity-determining region (ISDR) and is able to repress dsRNA-dependent protein kinase (PKR) thus influencing the response to interferon (IFN) therapy. Patients who respond to IFN therapy have stronger antibody reactivity against the NS5A compared to IFN non-responders. Therefore, given the possible role for the ISDR in IFN resistance and differential antibody reactivity, it is possible that variation in ISDR may be involved in viral immune escape and development of persistent HCV infection employing aspects of host mimicry. In this study, pre-treatment samples obtained from HCV infected patients were used to investigate the effect of different NS5A ISDR variants on the IFN antiviral response and their involvement in immune evasion. The NS5A was identified as a homologue of the variable region of immunoglobulins (Ig). The IFN resistant genotypes had higher levels of similarity to Ig compared to IFN sensitive genotypes. Expression of NS5A-6003 (HCV genotype 1b) and NS5A-6074 (HCV genotype 2a) was able to rescue vesicular stomatitis virus (VSV) from IFN inhibition and restore luciferase activity. A correlation between Ig-like NS5A structure and also antibody response with the outcome of IFN treatment was observed.