Abstract
Human noroviruses are the most common cause of acute gastroenteritis worldwide. Recently, cell culture systems have been described using either human embryonic intestinal epithelial cells (Int-407) or human epithelial colorectal adenocarcinoma cells (Caco-2) growing on collagen-I porous micro carrier beads in a rotating bioreactor under conditions of physiological fluid shear. Here, we describe the efforts from two independent laboratories to implement this three dimensional (3D) cell culture system for the replication of norovirus. Int-407 and Caco-2 were grown in a rotating bioreactor for up to 28 days. Prior to infection, cells were screened for the presence of microvilli by electron microscopy and stained for junction proteins (zonula occludens-1, claudin-1, and β-catenin). Differentiated 3D cells were transferred to 24-well plates and infected with bacteria-free filtrates of various norovirus genotypes (GI.1, GI.3, GI.8, GII.2, GII.4, GII.7, and GII.8). At 12 h, 24 h, and 48 h post inoculation, viral RNA from both cells and supernatants were collected and analyzed for norovirus RNA by real-time reverse transcription PCR. Despite observations of high expression of junction proteins and microvilli development in stained thin sections, our data suggest no significant increase in viral titer based on norovirus RNA copy number during the first 48 h after inoculation for the different samples and virus culture conditions tested. Our combined efforts demonstrate that 3D cell culture models using Int-407 or Caco-2 cells do not support norovirus replication and highlight the complexity and difficulty of developing a reproducible in vitro cell culture system for human norovirus.
Highlights
Human noroviruses are recognized as the most common cause of outbreaks of acute gastroenteritis worldwide [1]
Norovirus infection In all conditions tested during infection, the 3D cells remained attached to the beads and viable throughout the experiments
None of the Int-407 or Caco-2 cell culture supernatants collected at 12 h, 24 h, and 48 h after infection showed an increase in the viral RNA titer compared with 1h post inoculation (Table 2 and Figure 5)
Summary
Human noroviruses are recognized as the most common cause of outbreaks of acute gastroenteritis worldwide [1]. The majority of the outbreaks are caused by GII.. Since the detection of Norwalk virus, the prototype strain for norovirus, in stool samples of patients with acute gastroenteritis in 1968, many investigators have attempted to develop a cell culture system to better study the virus and assess the effectiveness of control measures such as disinfectants [3]. Freshly collected adult human duodenal tissues were infected successfully with a GII. strain as demonstrated by virus RNA production and immunohistochemical staining; when a human intestinal epithelial cell line from fetal ileum tissue was infected, only limited virus replication was detected [8]. More recent findings suggested that human norovirus may have a tropism for non-epithelial cells of the human duodenum [9]
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