EXPRESSION OF THE MICROFOLD CELLS IN THREE-DIMENSIONAL COCULTURE SYSTEM FOR IN VITRO CULTIVATION OF HUMAN NOROVIRUS

Mizanurfakhri Ghazali,Sharaniza Ab-Rahim,Mudiana Muhamad

doi:10.22159/ijap.2019.v11s5.t0052

Mizanurfakhri Ghazali, Sharaniza Ab-Rahim + Show 1 more

Open Access

https://doi.org/10.22159/ijap.2019.v11s5.t0052

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Abstract

Introduction: Human Norovirus (HuNoV), a food-borne virus is the leading cause for acute gastroenteritis. However, its inability to propagate in vitropersists as major challenge in understanding HuNoV biology.Objective: This study aims to determine an effective culture system for HuNoV.Methods: The Caco-2 cells were cocultured with Raji B cells on alginate hydrogel beads. Scanning electron microscopy (SEM) was performed to confirmthe three-dimensional (3D) cells morphology. Western blot (WB) analysis was performed to detect protein markers expressed by Microfold (M) cells.Results: Optimization of Caco-2 cells monoculture in the alginate hydrogel beads showed optimum number of cells of 1 × 106 cells/ml, indicatedby the intact structure of the beads. Result of SEM showed clear structure of monoculture in the alginate hydrogel beads indicated by the presenceof smooth and regular apical surface while the coculture showed reduced apical surface of M cells. The result of WB showed downregulation ofUlex europaeus antibody expression.Conclusion: It is evident that the expression of M cells grown in 3D alginate hydrogel beads was successful, indicated by the structural morphologyseen under SEM as well as expression of protein marker by M cells. This established in vitro system is highly potential for cultivation of HuNoV.

Highlights

Human Norovirus (HuNoV), a food-borne virus is the leading cause for acute gastroenteritis
It is evident that the expression of M cells grown in 3D alginate hydrogel beads was successful, indicated by the structural morphology seen under Scanning electron microscopy (SEM) as well as expression of protein marker by M cells
Coculture of Caco-2 and Raji B cells in alginate hydrogel beads The adherent Caco-2 cells presented as monolayer of epitheliallike cells while the suspension Raji B cells were observed to have lymphoblast-like morphology (Fig. 1)

Summary

Introduction

Human Norovirus (HuNoV), a food-borne virus is the leading cause for acute gastroenteritis. Human Norovirus (HuNoV) is currently the primary cause for nonbacterial endemic acute gastroenteritis This food-borne virus belongs to the family Caliciviridae [1], having a single-stranded positive-sense RNA as its genome. It is estimated that HuNoV infection leads to 200,000 deaths annually worldwide, HuNoV remains as the most poorly characterized virus, mainly due to lack of a reliable and reproducible cell culture system for its propagation. This major setback is hindering studies toward virus-host interactions as well as its pathogenesis. The role of M cells includes induction of immune responses within mucosa-associated lymphoid tissue and transcytosis; a process of particles such as antigen and bacteria is transported from the apical side into lymphocytes dome-like structure at the basolateral side of the intestinal epithelium [10,11]

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