Cell-to-cell transmission of DENV in C6/36 cells demonstrated in transwell and co-culture systems.

<p>A) Diagram showing transwell (0.4 μm pore size) or co-culture systems used to investigate virus spread between donor (D) and recipient (R) cells, either with or without the presence of neutralizing antiserum (NeuAb). For more detailed information, please see the description in the Materials and Methods. Cells were observed with an immunofluorescence assay (IFA) after treatment with respective antibodies. B) Mock-infection donor cells were used as controls while recipient cells were transfected with rhodamine fluorescent protein or RFP (red) prior to the investigation. In the transwell system, donor cells infected by DENV for 24 h at MOI = 1 were identified by polyclonal antibodies against the viral NS3 protein (green). Meanwhile, some recipient cells were actually infected by the virus, indicating the possibility of diffuse movement by viruses released by donor cells (white arrow). As no infection occurred in recipient cells in the presence of NeuAb, it seemed that intercellular virus spread may have been blocked by adding NeuAb. Results from co-cultures revealed that a number of recipient cells were infected and thus positive to NS3 antibodies even NeuAb has been added to the culture. This suggested an intercellular spreading mode by DENV, most likely by cell-to-cell transmission. C) Recipient cells sorted by flow cytometry were collected for viral RNA analysis by reverse transcriptase (RT)-PCR. No viral RBA can be detected in mock-infected cells (mock). A small amount of viral RNA was detected in cells collected from the transwell system without NeuAb (transwell), but not in the presence of antiserum (transwell+antiserum). However, a large amount of viral RNA was amplified in the co-culture group even in the presence of NeuAb (co-culture+antiserum), further implying the possibility of cell-to-cell transmission of DENV. The RNA level of 18S was used as the internal control in this experiment.