Co-infection experimental design Vero cells, an African green monkey kidney cell line (ATCC CRL 1587), were used for all infection experiments. They were propagated in GM without gentamycin Foretinib nmr at 37°C in an atmosphere of 5% CO2. Vero cells were divided into four groups: for mock infection, chlamydial infection, ca-PEDV infection, and both Chlamydia and ca-PEDV double infection. Host cells were infected with a MOI of 1 for Chlamydia and an infective dose of 1 × 105,5 TCID50/ml for ca-PEDV, respectively. For ca-PEDV monoinfections and negative controls, infection medium was used. All co-infection experiments were done three times and monoinfections with Chlamydia and ca-PEDV

were performed simultaneously. The optimal experimental protocol (adding the virus several hours after chlamydial infection) for co-infection procedure was developed before (data not shown). For dual infections, cell monolayers were first

infected with Chlamydia at a MOI of 1. All coverslips were centrifuged at 1000 × g for 1 h at 25°C. Timepoint 0 (T0) was defined after centrifugation and supernatant was replaced subsequently LY2874455 by incubation medium. Infected monolayers were then incubated for 14 h at 37°C (T0 – T14). All cell layers for dual infections or ca-PEDV monoinfection were exposed to a ca-PEDV suspension (1 × 105,5 TCID50), the samples were centrifuged again for 1000 × g for 1 h at 25°C and incubated for 24 h at 37°C. After this incubation period, all monolayers were fixed and further investigated by FK506 in vitro Indirect immunofluorescence and transmission electron microscopy. Re-infection experiments were performed to compare the production of infectious chlamydial elementary bodies (EBs) between monoinfections and mixed infections. Indirect Immunofluorescence For indirect immunofluorescence analyses, infected cells were fixed in absolute methanol (-20°C) for 10 min. and IF labeling

of cell cultures was performed immediately Morin Hydrate after fixation. For viral antigen detection, a mouse monoclonal antibody against the M protein of PEDV (mcAb 204, kindly provided by Prof. Dr. M. Ackermann, Institute of Virology, University of Zurich), diluted 1:4 in PBS supplemented with BSA, and an Alexa Fluor 594-conjugated secondary antibody (goat anti-mouse, 1:500, Molecular Probes, Eugene, USA) were used. Chlamydial inclusions were labeled with a Chlamydiaceae family-specific mouse monoclonal antibody directed against the chlamydial lipopolysaccharide (mLPS; Clone ACI-P, Progen, Heidelberg, Germany) and a secondary Alexa Fluor 488-conjugated secondary antibody (goat anti-mouse, 1:500, Molecular Probes). DNA was labeled with 1 μg/ml 4′, 6-Diamidin-2′-phenylindoldihydrochlorid (DAPI, Molecular Probes). All staining procedures were conducted at room temperature.