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

Human norovirus culture in B cells

Human noroviruses (HuNoVs) are a leading cause of foodborne disease and severe childhood diarrhea, and they cause a majority of the gastroenteritis outbreaks worldwide. However, the development of effective and long-lasting HuNoV vaccines and therapeutics has been greatly hindered by their uncultivability. We recently demonstrated that a HuNoV replicates in human B cells, and that commensal bacteria serve as a cofactor for this infection. In this protocol, we provide detailed methods for culturing the GII.4-Sydney HuNoV strain directly in human B cells, and in a coculture system in which the virus must cross a confluent epithelial barrier to access underlying B cells. We also describe methods for bacterial stimulation of HuNoV B cell infection and for measuring viral attachment to the surface of B cells. Finally, we highlight variables that contribute to the efficiency of viral replication in this system. Infection assays require 3 d and attachment assays require 3 h. Analysis of infection or attachment samples, including RNA extraction and RT-qPCR, requires ∼6 h