Evidence for a biphasic mode of respiratory syncytial virus transmission in permissive HEp2 cell monolayers

Tra Nguyen Huong, Laxmi Iyer Ravi, Boon Huan Tan, Richard J. Sugrue

During respiratory syncytial virus (RSV) infection filamentous virus particles are formed on the cell surface. Although the virus infectivity remains cell-associated, low levels of cell-free virus is detected during advanced infection. It is currently unclear if this cell-free virus infectivity is due to a low-efficiency specific cell-release mechanism, or if it arises due to mechanical breakage following virus-induced cell damage at the advanced stage of infection. Understanding the origin of this cell-free virus is a prerequisite for understanding the mechanism of RSV transmission in permissive cells. In this study we describe a detailed examination of RSV transmission in permissive HEp2 cell monolayers. HEp2 cell monolayers were infected with RSV using a multiplicity of infection of 0.0002, and the course of infection monitored over 5 days. The progression of the virus infection within the cell monolayers was performed using bright-field microscopy to visualise the cell monolayer and immunofluorescence microscopy to detect virus-infected cells. The cell-associated and cell-free virus infectivity were determined by virus plaque assay, and the virus-induced cell cytotoxicity determined by measuring cell membrane permeability and cellular DNA fragmentation. At 2 days-post infection (dpi), large clusters of virus-infected cells could be detected indicating localised transmission in the cell monolayer, and during this stage we failed to detect either cell-free virus or cell cytotoxicity. At 3 dpi the presence of much larger infected cell clusters correlated with the begining of virus-induced changes in cell permeability. The presence of cell-free virus correlated with continued increase in cell permeability and cytotoxicity at 4 and 5 dpi. At 5 dpi extensive cell damage, syncytial formation, and increased cellular DNA fragmentation was noted. However, even at 5 dpi the cell-free virus constituted less than 1 % of the total virus infectivity. Our data supports a model of RSV transmission that initially involves the localised cell-to-cell spread of virus particles within the HEp2 cell monolayer. However, low levels of cell free-virus infectivity was observed at the advanced stages of infection, which correlated with a general loss in cell monolayer integrity due to virus-induced cytotoxicity.