RESUMEN
Clinical Trials Registration: ClinicalTrials.gov [NCT02378753] and Pan African Clinical Trials Registry [PACTR201502001037220].
Asunto(s)
Vacunas contra el Virus del Ébola/administración & dosificación , Vacunas contra el Virus del Ébola/efectos adversos , Epidemias , Monitoreo Epidemiológico , Fiebre Hemorrágica Ebola/diagnóstico , Fiebre Hemorrágica Ebola/prevención & control , Adulto , Femenino , Fiebre Hemorrágica Ebola/epidemiología , Fiebre Hemorrágica Ebola/patología , Humanos , Masculino , Ensayos Clínicos Controlados Aleatorios como Asunto , Sierra Leona/epidemiología , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/efectos adversos , Adulto JovenRESUMEN
Influenza virus encodes only 11 viral proteins but replicates in a broad range of avian and mammalian species by exploiting host cell functions. Genome-wide RNA interference (RNAi) has proven to be a powerful tool for identifying the host molecules that participate in each step of virus replication. Meta-analysis of findings from genome-wide RNAi screens has shown influenza virus to be dependent on functional nodes in host cell pathways, requiring a wide variety of molecules and cellular proteins for replication. Because rapid evolution of the influenza A viruses persistently complicates the effectiveness of vaccines and therapeutics, a further understanding of the complex host cell pathways coopted by influenza virus for replication may provide new targets and strategies for antiviral therapy. RNAi genome screening technologies together with bioinformatics can provide the ability to rapidly identify specific host factors involved in resistance and susceptibility to influenza virus, allowing for novel disease intervention strategies.
Asunto(s)
Ensayos Analíticos de Alto Rendimiento/métodos , Virus de la Influenza A/genética , Gripe Humana/terapia , Interferencia de ARN , Proteínas Virales/genética , Animales , Humanos , Metaanálisis como Asunto , MicroARNs/genética , MicroARNs/metabolismo , FN-kappa B/metabolismo , Proteína Quinasa C/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Transducción de Señal/fisiologíaRESUMEN
The emergence and spread of Zika virus (ZIKV) presented a challenge to the diagnosis of ZIKV infections in areas with transmission of dengue (DENV) and chikungunya (CHIKV) viruses. To facilitate detection of ZIKV infections, and differentiate these infections from DENV and CHIKV, we developed the Trioplex real-time RT-PCR assay (Trioplex assay). Here, we describe the optimization of multiplex and singleplex formats of the assay for a variety of chemistries and instruments to facilitate global standardization and implementation. We evaluated the analytical performance of all Trioplex modalities for detection of these three pathogens in serum and whole blood, and for ZIKV in urine. The limit of detection for the three viruses and in different RNA-extraction modalities is near 103 genome copy equivalents per milliliter (GCE/mL). Simultaneous testing of more than one specimen type from each patient provides a 6.4% additional diagnostic sensitivity. Overall, the high sensitivity of the Trioplex assay demonstrates the utility of this assay ascertaining Zika cases.
Asunto(s)
Fiebre Chikungunya/diagnóstico , Virus Chikungunya/genética , Virus del Dengue/genética , Dengue/diagnóstico , Reacción en Cadena de la Polimerasa Multiplex/métodos , Infección por el Virus Zika/diagnóstico , Virus Zika/genética , Calibración , Fiebre Chikungunya/sangre , Fiebre Chikungunya/virología , Virus Chikungunya/aislamiento & purificación , Dengue/sangre , Dengue/virología , Virus del Dengue/aislamiento & purificación , Humanos , Límite de Detección , Reacción en Cadena de la Polimerasa Multiplex/normas , Juego de Reactivos para Diagnóstico , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/normas , Virus Zika/aislamiento & purificación , Infección por el Virus Zika/orina , Infección por el Virus Zika/virologíaRESUMEN
Human protein kinases (HPKs) have profound effects on cellular responses. To better understand the role of HPKs and the signaling networks that influence influenza virus replication, a small interfering RNA (siRNA) screen of 720 HPKs was performed. From the screen, 17 HPKs (NPR2, MAP3K1, DYRK3, EPHA6, TPK1, PDK2, EXOSC10, NEK8, PLK4, SGK3, NEK3, PANK4, ITPKB, CDC2L5 (CDK13), CALM2, PKN3, and HK2) were validated as essential for A/WSN/33 influenza virus replication, and 6 HPKs (CDK13, HK2, NEK8, PANK4, PLK4 and SGK3) were identified as vital for both A/WSN/33 and A/New Caledonia/20/99 influenza virus replication. These HPKs were found to affect multiple host pathways and regulated by miRNAs induced during infection. Using a panel of miRNA agonists and antagonists, miR-149* was found to regulate NEK8 expression, miR-548d-3p was found to regulate MAPK1 transcript expression, and miRs -1228 and -138 to regulate CDK13 expression. Up-regulation of miR-34c induced PLK4 transcript and protein expression and enhanced influenza virus replication, while miR-34c inhibition reduced viral replication. These findings identify HPKs important for influenza viral replication and show the miRNAs that govern their expression.
Asunto(s)
Virus de la Influenza A/fisiología , Gripe Humana/genética , Gripe Humana/virología , MicroARNs/metabolismo , Proteínas Quinasas/metabolismo , Replicación Viral/genética , Células A549 , Animales , Secuencia de Bases , Perros , Humanos , Virus de la Influenza A/genética , Virus de la Influenza A/aislamiento & purificación , Gripe Humana/enzimología , Gripe Humana/patología , Quinasa 1 de Quinasa de Quinasa MAP/antagonistas & inhibidores , Quinasa 1 de Quinasa de Quinasa MAP/genética , Quinasa 1 de Quinasa de Quinasa MAP/metabolismo , Células de Riñón Canino Madin Darby , MicroARNs/agonistas , MicroARNs/antagonistas & inhibidores , Quinasas Relacionadas con NIMA/antagonistas & inhibidores , Quinasas Relacionadas con NIMA/genética , Quinasas Relacionadas con NIMA/metabolismo , Proteínas de la Nucleocápside , Fenotipo , Proteínas Quinasas/química , Proteínas Quinasas/genética , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Interferencia de ARN , ARN Ribosómico 18S/genética , ARN Ribosómico 18S/metabolismo , Proteínas de Unión al ARN/metabolismo , Alineación de Secuencia , Regulación hacia Arriba , Proteínas del Núcleo Viral/metabolismoRESUMEN
Influenza A virus causes seasonal epidemics and periodic pandemics threatening the health of millions of people each year. Vaccination is an effective strategy for reducing morbidity and mortality, and in the absence of drug resistance, the efficacy of chemoprophylaxis is comparable to that of vaccines. However, the rapid emergence of drug resistance has emphasized the need for new drug targets. Knowledge of the host cell components required for influenza replication has been an area targeted for disease intervention. In this study, the human protease genes required for influenza virus replication were determined and validated using RNA interference approaches. The genes validated as critical for influenza virus replication were ADAMTS7, CPE, DPP3, MST1, and PRSS12, and pathway analysis showed these genes were in global host cell pathways governing inflammation (NF-κB), cAMP/calcium signaling (CRE/CREB), and apoptosis. Analyses of host microRNAs predicted to govern expression of these genes showed that eight miRNAs regulated gene expression during virus replication. These findings identify unique host genes and microRNAs important for influenza replication providing potential new targets for disease intervention strategies.