Evidence for a biphasic mechanism of virus transmission of the human metapneumovirus in LLC-MK2 cell monolayers.

We examined transmission of the human metapneumovirus (HMPV) in LLC-MK2 cell monolayers using a low multiplicity of infection (moi). In this low moi infection model HMPV transmission initially occurred by localised cell-to-cell transmission, and the virus infectivity remained largely cell associated. At the later stages of infection more widespread virus transmission occurred and was associated with the presence of cell-free virus. The appearance of the cell-free virus correlated with changes in plasma membrane integrity and increased membrane permeability in the cell monolayers. Imaging analysis of HMPV infected cells at the early stages of infection showed the presence of numerous virus filaments attached to the surface of HMPV-infected cells. At the later stages of infection both virus filaments and virus particles with a spherical morphology that was attached to the distal ends of the virus filaments was noted. A proportion of these spherical particles detached from the virus filaments and attached to adjacent non-infected cells at the later stages of infection. The activation of the JNK and MAPKp38 signalling pathways in HMPV-infected cells correlated with increased HMPV replication and appearance of the cell-free virus infectivity. In addition, after the initial phase of STAT1 activation in HMPV-infected cells, both reduced expression of the STAT1 protein and the activated STAT1 protein occurred as the infection proceeded. Collectively, these data provide evidence for a biphasic mode of HMPV transmission involving different virus particle morphologies, a localised virus transmission by virus filaments followed by widespread virus transmission involving cell-free virus particles.