RESUMO
Clinical Trials Registration: ClinicalTrials.gov [NCT02378753] and Pan African Clinical Trials Registry [PACTR201502001037220].
Assuntos
Vacinas contra Ebola/administração & dosagem , Vacinas contra Ebola/efeitos adversos , Epidemias , Monitoramento Epidemiológico , Doença pelo Vírus Ebola/diagnóstico , Doença pelo Vírus Ebola/prevenção & controle , Adulto , Feminino , Doença pelo Vírus Ebola/epidemiologia , Doença pelo Vírus Ebola/patologia , Humanos , Masculino , Ensaios Clínicos Controlados Aleatórios como Assunto , Serra Leoa/epidemiologia , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/efeitos adversos , Adulto JovemRESUMO
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.
Assuntos
Febre de Chikungunya/diagnóstico , Vírus Chikungunya/genética , Vírus da Dengue/genética , Dengue/diagnóstico , Reação em Cadeia da Polimerase Multiplex/métodos , Infecção por Zika virus/diagnóstico , Zika virus/genética , Calibragem , Febre de Chikungunya/sangue , Febre de Chikungunya/virologia , Vírus Chikungunya/isolamento & purificação , Dengue/sangue , Dengue/virologia , Vírus da Dengue/isolamento & purificação , Humanos , Limite de Detecção , Reação em Cadeia da Polimerase Multiplex/normas , Kit de Reagentes para Diagnóstico , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/normas , Zika virus/isolamento & purificação , Infecção por Zika virus/urina , Infecção por Zika virus/virologiaRESUMO
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.
Assuntos
Vírus da Influenza A/fisiologia , Influenza Humana/genética , Influenza Humana/virologia , MicroRNAs/metabolismo , Proteínas Quinases/metabolismo , Replicação Viral/genética , Células A549 , Animais , Sequência de Bases , Cães , Humanos , Vírus da Influenza A/genética , Vírus da Influenza A/isolamento & purificação , Influenza Humana/enzimologia , Influenza Humana/patologia , MAP Quinase Quinase Quinase 1/antagonistas & inibidores , MAP Quinase Quinase Quinase 1/genética , MAP Quinase Quinase Quinase 1/metabolismo , Células Madin Darby de Rim Canino , MicroRNAs/agonistas , MicroRNAs/antagonistas & inibidores , Quinases Relacionadas a NIMA/antagonistas & inibidores , Quinases Relacionadas a NIMA/genética , Quinases Relacionadas a NIMA/metabolismo , Proteínas do Nucleocapsídeo , Fenótipo , Proteínas Quinases/química , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Interferência de RNA , RNA Ribossômico 18S/genética , RNA Ribossômico 18S/metabolismo , Proteínas de Ligação a RNA/metabolismo , Alinhamento de Sequência , Regulação para Cima , Proteínas do Core Viral/metabolismoRESUMO
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.
Assuntos
Vírus da Influenza A/fisiologia , MicroRNAs/genética , Peptídeo Hidrolases/genética , Replicação Viral/genética , Proteínas ADAM/antagonistas & inibidores , Proteínas ADAM/genética , Proteína ADAMTS7 , Sequência de Bases , Carboxipeptidase H/antagonistas & inibidores , Carboxipeptidase H/genética , Linhagem Celular , Dipeptidil Peptidases e Tripeptidil Peptidases/antagonistas & inibidores , Dipeptidil Peptidases e Tripeptidil Peptidases/genética , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/fisiologia , Humanos , Vírus da Influenza A/patogenicidade , Influenza Humana/enzimologia , Influenza Humana/genética , Influenza Humana/virologia , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Interferência de RNA , RNA Interferente Pequeno/genética , Serina Endopeptidases/genéticaRESUMO
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.
