title: Tick-borne encephalitis virus replication, intracellular trafficking, and pathogenicity in human intestinal Caco-2 cell monolayers.
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.
authors.affiliation: Centre for Biological Threats and Special Pathogens, ZBS 1: Highly Pathogenic Viruses, Robert Koch Institute, Berlin, Germany.
authors.forename: Chao
authors.initials: C
authors.lastname: Yu
authors.affiliation: Institute of Chemistry and Biochemistry, Freie Universität Berlin, Berlin, Germany.
authors.forename: Katharina
authors.initials: K
authors.lastname: Achazi
authors.affiliation: Centre for Biological Threats and Special Pathogens, ZBS 4: Advanced Light and Electron Microscopy, Robert Koch Institute, Berlin, Germany.
authors.forename: Lars
authors.initials: L
authors.lastname: Möller
authors.affiliation: Department of Gastroenterology, Infectious Diseases and Rheumatology, Division of Nutritional Medicine, Charité - Universitätsmedizin Berlin, Campus Benjamin Franklin, Berlin, German.
authors.forename: Joerg D
authors.initials: JD
authors.lastname: Schulzke
authors.affiliation: Centre for Biological Threats and Special Pathogens, ZBS 1: Highly Pathogenic Viruses, Robert Koch Institute, Berlin, Germany.
authors.forename: Matthias
authors.initials: M
authors.lastname: Niedrig
authors.affiliation: Department of Gastroenterology, Infectious Diseases and Rheumatology, Division of Nutritional Medicine, Charité - Universitätsmedizin Berlin, Campus Benjamin Franklin, Berlin, German.
authors.forename: Roland
authors.initials: R
authors.lastname: Bücker
mesh_terms: D001692:Biological Transport; D018938:Caco-2 Cells; D003599:Cytoskeleton / Q000821:virology; D004669:Encephalitis Viruses, Tick-Borne / Q000378:metabolism / Q000472:pathogenicity* / Q000502:physiology*; D006801:Humans; D007422:Intestines / Q000166:cytology* / Q000648:ultrastructure / Q000821:virology*; D042541:Intracellular Space / Q000821:virology*; D010873:Pinocytosis; D014774:Virulence; D053586:Virus Internalization; D014779:Virus Replication