Activation of the 2-5OAS/RNase L pathway in CVB1 or HAV/18f infected FRhK-4 cells does not require induction of OAS1 or OAS2 expression

AbstractThe latent, constitutively expressed protein RNase L is activated in coxsackievirus and HAV strain 18f infected FRhK-4 cells. Endogenous oligoadenylate synthetase (OAS) from uninfected and virus infected cell extracts synthesizes active forms of the triphosphorylated 2-5A oligomer (the only known activator of RNase L) in vitro and endogenous 2-5A is detected in infected cell extracts. However, only the largest OAS isoform, OAS3, is readily detected throughout the time course of infection. While IFNβ treatment results in an increase in the level of all three OAS isoforms in FRhK-4 cells, IFNβ pretreatment does not affect the temporal onset or enhancement of RNase L activity nor inhibit virus replication. Our results indicate that CVB1 and HAV/18f activate the 2-5OAS/RNase L pathway in FRhK-4 cells during permissive infection through endogenous levels of OAS, but contrary to that reported for some picornaviruses, CVB1 and HAV/18f replication is insensitive to this activated antiviral pathway

Molecular mechanisms of neuroprotection by the herpes simplex virus type 2 gene ICP10PK

Recent progress in molecular biology has focused interest on gene therapy as a strategy for the control of chronic and acute neurodegenerative disorders. However, the selection of the appropriate gene and delivery vector is a clinical challenge. Herpes simplex virus type 2 (HSV-2) is a promising gene delivery vector, as it is neurotropic, has a large genome that is amenable to genetic manipulation, and unlike HSV-1, it does not cause encephalitis in adult humans. HSV-2 contains an anti-apoptotic serine/threonine protein kinase (known as ICP10PK), that acts as a constitutively activated growth factor receptor. It activates Ras and its downstream MEK/ERK survival pathway and inhibits apoptosis caused by virus infection of primary hippocampal cultures (Perkins et al. 2003b, Perkins et al. 2002a). The studies described in this report were designed to examine the molecular mechanisms of ICP10PK-mediated neuroprotection, and ensure that it can act independently of other viral proteins. Rat pheochromocytoma (PC12) cells stably transfected with ICP10PK (PC47 and PC70 cells) or its kinase-negative mutant p139™ (PC139 cells), were neuronally differentiated by culture with nerve growth factor (NGF) and examined for cell survival after NGF withdrawal. Apoptosis was seen in PC12 and PC139, but not PC47 and PC70 cells. In PC47 cells, neuroprotection was MEK- and PKA-dependent, associated with stabilization/activation of the transcription factor cAMP-responsive element binding protein (CREB), inhibition (phosphorylation) of the pro-apoptotic protein Bad and stabilization of the anti-apoptotic proteins Bcl-2 and Bag-1. In PC70 cells, neuroprotection occurred downstream of caspase activation, and involved MEK-dependent up-regulation of the anti-apoptotic protein XIAP and down-regulation of the XIAP inhibitor Smac/DIABLO. To examine whether ICP10PK is also neuroprotective in other paradigms, we examined its effect in an in vitro model of Parkinson's disease, using the neurotoxin MPP+. ICP10PK, but not p139™, inhibited MPP +-induced programmed cell death through inhibition of calpain-dependent Bax translocation to the mitochondria, AIF nuclear translocation, and caspase activation, indicating that the actions of ICP10PK are kinase-dependent. Collectively, the data indicate that ICP10PK has broad-spectrum neuroprotective activity that extends beyond apoptotic cellular programs. Further study of its use as a gene therapy strategy is warranted

Challenges for estimating human norovirus infectivity by viability RT-qPCR as compared to replication in human intestinal enteroids

Viability RT-qPCR, a molecular detection method combining viability marker pre-treatment with RT-qPCR, has been proposed to infer infectivity of viruses which is particularly relevant for non-culturable viruses or sophisticated cell culture systems. Being human noroviruses (HuNoV) most frequently associated with foodborne outbreaks, this study compared different viability techniques and infectivity in human intestinal enteroids (HIE) to ultimately determine whether the molecular approaches could serve as rapid assays to predict HuNoV inac-tivation in high-risk food. To this end, the performance of three viability RT-qPCR assays with different inter-calating markers ((Viability PCR Crosslinker Kit (CL), propidium monoazide (PMAxxTM), and platinum chloride (PtCl4)) in estimating survival of HuNoV exposed to thermal and high pressure (HPP) treatments was compared to replication tested in the HIE cell culture model. A nearly full-length genomic molecular assay coupled with PMAxxTM to infer HuNoV thermal inactivation was also assessed. The experimental design included HuNoV genogroup I.3 [P13], GII.4 Sydney [P16], GII.6 [P7], along with Tulane virus (TV) serving as surrogate. Finally, viability RT-qPCR was tested in HPP-treated strawberry puree, selected as a food matrix with high viral contamination risk. PMAxxTM and CL performed evenly, while PtCl4 affected HuNoV infectivity. Taking all experimental data together, viability RT-qPCR was demonstrated to be an improved method over direct RT-qPCR to estimate viral inactivation at extreme thermal (95 degrees C) and HPP (450 MPa) exposures, but not under milder conditions as amplification signals were detected. Despite its complexity and limitations, the HIE demonstrated a more robust model than viability RT-qPCR to assess HuNoV infectivity

Human Gut-On-A-Chip Supports Polarized Infection of Coxsackie B1 Virus In Vitro

Analysis of enterovirus infection is difficult in animals because they express different virus receptors than humans, and static cell culture systems do not reproduce the physical complexity of the human intestinal epithelium. Here, using coxsackievirus B1 (CVB1) as a prototype enterovirus strain, we demonstrate that human enterovirus infection, replication and infectious virus production can be analyzed in vitro in a human Gut-on-a-Chip microfluidic device that supports culture of highly differentiated human villus intestinal epithelium under conditions of fluid flow and peristalsis-like motions. When CVB1 was introduced into the epithelium-lined intestinal lumen of the device, virions entered the epithelium, replicated inside the cells producing detectable cytopathic effects (CPEs), and both infectious virions and inflammatory cytokines were released in a polarized manner from the cell apex, as they could be detected in the effluent from the epithelial microchannel. When the virus was introduced via a basal route of infection (by inoculating virus into fluid flowing through a parallel lower ‘vascular’ channel separated from the epithelial channel by a porous membrane), significantly lower viral titers, decreased CPEs, and delayed caspase-3 activation were observed; however, cytokines continued to be secreted apically. The presence of continuous fluid flow through the epithelial lumen also resulted in production of a gradient of CPEs consistent with the flow direction. Thus, the human Gut-on-a-Chip may provide a suitable in vitro model for enteric virus infection and for investigating mechanisms of enterovirus pathogenesis

Cytokine secretion and caspase-3 expression in infected gut chips.

<p>IP-10 (<b>A</b>) and IL-8 (<b>B</b>) were quantified in effluents from the apical (gray bars) and basal (white bars) microchannels in gut chips that were either uninfected or infected apically or basally (n = 4 chips/condition; *p < 0.05, **p < 0.001) at 24 hpi. (<b>C</b>) Epithelium lining gut chips that were uninfected (controls C1 and C2), or infected apically (A1, A2, A3) or basally (B1, B2, B3, B4), or infected apically or basally, were lysed and pro-caspase-3 and cleaved caspase-3 levels were visualized on the gel as indicated.</p

Apical fluid flow generates a gradient of CPE.

<p>Phase contrast micrographs of apically and basally CVB1-infected Gut-on-a-Chips at 24hpi. The flow direction (from the inlet to the outlet) is from left to right (the arrow). Note the increasing CPE intensity towards the downstream outlet; bar, 100 μm.</p

Coxsackie virus B1 readily infects the human Gut-on-a-Chip.

<p>(<b>A</b>) Phase contrast micrographs of the villus epithelium within the Gut-on-a-Chip apically infected with CVB1 at 6, 24 and 48h post infection (hpi); bar, 100 μm. (<b>B</b>) Confocal immunofluorescence micrographs of uninfected and apically infected villus epithelium 24hpi that were stained for CVB1 (green), F-actin (red) and nuclei (blue). Note the destruction of villi in infected samples; bar, 100 μm. (<b>C</b>) Graph showing quantitation of viral loads in the effluents of the apical (epithelial) versus basal (vascular) microchannels after apical infection of the gut chips with CVB1 at an MOI of 0.2 (*p < 0.05 compared to 0–6 hpi).</p

CVB1 release is polarized towards the epithelial lumen.

<p>(<b>A</b>) Graph showing quantitation of viral loads in the effluents of the apical (epithelial) versus basal (vascular) microchannels after basal infection of the gut chips with CVB1 at an MOI of 0.2 (*p < 0.05 compared to 0–6 hpi). (<b>B</b>) Confocal fluorescence micrographs of apically and basally infected gut chips at 6 and 24 hpi, showing horizontal sections at the base, middle and top of the villi (left to right columns). Infected chips were stained for CVB1 (green) and nuclei (blue); bar, 100 μm.</p

Human Gut-on-a-Chip microfluidic culture device.

<p>Photograph (<b>A</b>) and schematic diagram (<b>B</b>) of the human Gut-on-a-Chip device. (<b>C</b>) Phase contrast micrograph of human Caco-2 intestinal epithelial cells cultured for 6 days in a Gut-on-a-Chip under apical flow (30μl/hr; 0.02 dyne.cm^-2) and cyclic mechanical strain (10% at 0.15 Hz); bar, 100 μm. (<b>D</b>) Apparent permeability (P_app) of the epithelium assessed by adding fluorescent inulin-FITC daily to the upper channel for 6 days after seeding (n = 3 chips). Note that an ECM-coated Gut-on-a-Chip without cells used as a control exhibited a very high permeability. (<b>E</b>) Confocal immunofluorescence micrograph of human villus intestinal epithelium formed inside the Gut-on-a-Chip and stained for villin (yellow) to visualize the apical brush border and nuclei (blue); bar, 10 μm. (<b>F</b>) Scanning electron micrograph of the apical surface of the villus epithelium cultured for 6 days in the Gut-on-a-Chip under flow and mechanical strain. Note the microvilli at the surface of the cells (bar, 10 μm).</p