Tick-borne encephalitis virus replication, intracellular trafficking, and pathogenicity in human intestinal Caco-2 cell monolayers.

Chao Yu,Joerg D Schulzke,Roland Bücker,Katharina Achazi,Lars Möller,Matthias Niedrig,Tian Wang

doi:10.1371/journal.pone.0096957

Abstract

Tick-borne encephalitis virus (TBEV) is one of the most important vector-borne viruses in Europe and Asia. Its transmission mainly occurs by the bite of an infected tick. However, consuming milk products from infected livestock animals caused TBEV cases. To better understand TBEV transmission via the alimentary route, we studied viral infection of human intestinal epithelial cells. Caco-2 cells were used to investigate pathological effects of TBEV infection. TBEV-infected Caco-2 monolayers showed morphological changes including cytoskeleton rearrangements and cytoplasmic vacuolization. Ultrastructural analysis revealed dilatation of the rough endoplasmic reticulum and further enlargement to TBEV containing caverns. Caco-2 monolayers maintained an intact epithelial barrier with stable transepithelial electrical resistance (TER) during early stage of infection. Concomitantly, viruses were detected in the basolateral medium, implying a transcytosis pathway. When Caco-2 cells were pre-treated with inhibitors of cellular pathways of endocytosis TBEV cell entry was efficiently blocked, suggesting that actin filaments (Cytochalasin) and microtubules (Nocodazole) are important for PI3K-dependent (LY294002) virus endocytosis. Moreover, experimental fluid uptake assay showed increased intracellular accumulation of FITC-dextran containing vesicles. Immunofluorescence microscopy revealed co-localization of TBEV with early endosome antigen-1 (EEA1) as well as with sorting nexin-5 (SNX5), pointing to macropinocytosis as trafficking mechanism. In the late phase of infection, further evidence was found for translocation of virus via the paracellular pathway. Five days after infection TER was slightly decreased. Epithelial barrier integrity was impaired due to increased epithelial apoptosis, leading to passive viral translocation. These findings illuminate pathomechanisms in TBEV infection of human intestinal epithelial cells and viral transmission via the alimentary route.

Highlights

Tick-borne encephalitis virus (TBEV) belongs to the genus flavivirus, family Flaviviridae, mainly distributed in Europe and Asia
Nearly 100% of the cells were found TBEV-positive at 48 h p.i., while only few cells were positive at 24 h p.i. This rapid virus spread between cells confirmed that TBEV replication is efficient in human intestinal Caco-2 monolayers and that the cells in general are susceptible to TBEV infection
TUNEL assays were performed to assess the apoptosis ratio in TBEV infected Caco-2 cells (Figure S1). 2 days p.i. the percentage of apoptotic cells was close to 0 and not different from untreated controls, but 5 days p.i. around 5% apoptotic cells were found (Figure 6D). These results suggest that TBEV significantly accelerated apoptosis in Caco-2 cells in the late phase of infection

Summary

Introduction

Tick-borne encephalitis virus (TBEV) belongs to the genus flavivirus, family Flaviviridae, mainly distributed in Europe and Asia. More than 10,000 cases are reported annually [3,4,5]. TBEV is mainly transmitted by the bite of an infected tick [6]. Alimentary transmission of the virus by consumption of raw milk products from infected animals (mainly goats, sheep and cows) is described [7,8]. Since milk-borne epidemics or single cases where reported from Eastern Europe and from Austria and Germany. The number of TBE cases caused by consuming non-pasteurized milk or dairy products decreased until the early 1980s but in recent years the number of reports has increased again [5]

Methods

Results

Conclusion