ABSTRACT
Venezuelan equine encephalitis virus (VEEV) is a neurotropic virus that causes significant disease in both humans and equines. Here we characterized the impact of VEEV on signaling pathways regulating cell death in human primary astrocytes. VEEV productively infected primary astrocytes and caused an upregulation of early growth response 1 (EGR1) gene expression at 9 and 18â¯h post infection. EGR1 induction was dependent on extracellular signal-regulated kinase1/2 (ERK1/2) and protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK), but not on p38 mitogen activated protein kinase (MAPK) or phosphoinositide 3-kinase (PI3K) signaling. Knockdown of EGR1 significantly reduced VEEV-induced apoptosis and impacted viral replication. Knockdown of ERK1/2 or PERK significantly reduced EGR1 gene expression, dramatically reduced viral replication, and increased cell survival as well as rescued cells from VEEV-induced apoptosis. These data indicate that EGR1 activation and subsequent cell death are regulated through ERK and PERK pathways in VEEV infected primary astrocytes.
Subject(s)
Cell Death , Early Growth Response Protein 1/genetics , Encephalitis Virus, Venezuelan Equine/physiology , Encephalomyelitis, Venezuelan Equine/virology , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , eIF-2 Kinase/metabolism , Apoptosis , Astrocytes/metabolism , Astrocytes/pathology , Astrocytes/virology , Cell Survival , Cells, Cultured , Early Growth Response Protein 1/metabolism , Encephalitis Virus, Venezuelan Equine/pathogenicity , Encephalomyelitis, Venezuelan Equine/metabolism , Encephalomyelitis, Venezuelan Equine/pathology , Gene Expression , Gene Knockdown Techniques , Humans , Mitogen-Activated Protein Kinase 1/genetics , Mitogen-Activated Protein Kinase 3/genetics , Signal Transduction , Virus Replication , eIF-2 Kinase/geneticsABSTRACT
Most of the modern techniques used for identification of viral-induced disease are based on identification of viral antigens and/or nucleic acids in patient's blood. Diagnosis in the field or in remote locations can be challenging and alternatively samples are shipped to diagnostic labs for testing. Shipments must occur under controlled temperature conditions to prevent loss of sample integrity. We have tested the ability of magnetic Nanotrap® (NT) particles to improve stability and detection of Venezuelan equine encephalitis virus (VEEV), viral capsid protein, and viral genomic RNA in whole human blood at elevated temperature and prolonged storage conditions. NT particles have previously been shown to capture and enrich multiple pathogens including respiratory syncytial virus, influenza virus, coronavirus, and Rift Valley fever virus. Our study indicates that samples incubated with NT particles had detectable levels of infectious VEEV in blood equal to or greater than samples without NT treatment across all temperatures. Viral RNA detection was increased in the presence of NT particles at later time points (72 h) and higher temperature (40°C) conditions. Likewise, detection of VEEV capsid protein was enhanced in the presence of NT particles up to 72 h at 40°C. Finally, we intranasally infected C3H mice with TC-83, the live attenuated vaccine strain of VEEV, and demonstrated that NT particles could substantially increase the detection of VEEV capsid in infected blood incubated up to 72 h at 40°C. Samples without NT particles had undetectable capsid protein levels. Taken together, our data demonstrate the ability of NT particles to preserve and enable detection of VEEV in human and mouse blood samples over time and at elevated temperatures.