Furthermore, molecular dynamics simulations were performed for the inhibitor-complexed system HA1-Neo6 and the inhibitoruncomplexed system HA1, respectively. The root mean square deviation (RMSD) from initial conformation is a central criterion used to evaluate the difference of the protein system. The stability of a simulation system was evaluated based on its RMSD. The RMSD values for both Neo6-HA1 (green curve) and HA1 (red curve) versus the simulation time were illustrated in Fig. 5A , in which the RMSD for Neo6-HA1 system is a little smaller than that of HA1 system, indicating that the flexibility of HA1 was decreased after the Neo6 binding to HA1. In order to investigate the motions about the important residues interacted with the inhibitor in the binding site defined as loops (Loop1-Loop4) in Fig. 1 , the root mean square fluctuations (RMSF) for all the sidechain atoms of protein were calculated, as shown in Fig. 5B . The curves of RMSF associated with Loop1, Loop2, Loop3, and Loop4 are colored orange, light blue, dark blue, and maroon, respectively. It can be clearly seen from Fig. 5 that the fluctuating magnitudes of the four loops in HA1 are much larger than those in Neo6-HA1, clearly indicating that the receptor HA1 is more stable after binding with the ligand Neo6. The RMSD for all backbone atoms of the Neo6-HA1 system (green) and the HA1 system (red). (B) The RMSF for side-chain atoms of the Neo6-HA1 system (green) and the HA1 system (red). The curves associated with Loop1, Loop2, Loop3, and Loop4 are colored orange, light blue, dark blue, and maroon, respectively. doi:10.1371/journal.pone.0028111.g005 Accordingly, among the series of Neo compound candidates, Neo6 is anticipated to be a promising drug candidate for further experimental investigation to develop new and effective drug against influenza viruses. Tamiflu-Resistant but HA-Mediated Cell-to-Cell Transmission through Apical Membranes of Cell-Associated Influenza Viruses The infection of viruses to a neighboring cell is considered to be beneficial in terms of evasion from host anti-virus defense systems. There are two pathways for viral infection to “right next door”: one is the virus transmission through cell-cell fusion by forming syncytium without production of progeny virions, and the other is mediated by virions without virus diffusion, generally designated cell-to-cell transmission. Influenza viruses are believed to be transmitted as cell-free virus from infected cells to uninfected cells. Here, we demonstrated that influenza virus can utilize cell-to-cell transmission pathway through apical membranes, by handover of virions on the surface of an infected cell to adjacent host cells. Live cell imaging techniques showed that a recombinant influenza virus, in which the neuraminidase gene was replaced with the green fluorescence protein gene, spreads from an infected cell to adjacent cells forming infected cell clusters. This type of virus spreading requires HA activation by protease treatment. The cell-to-cell transmission was also blocked by amantadine, which inhibits the acidification of endosomes required for uncoating of influenza virus particles in endosomes, indicating that functional hemagglutinin and endosome acidification by M2 ion channel were essential for the cell-to-cell influenza virus transmission. Furthermore, in the cell-to-cell transmission of influenza virus, progeny virions could remain associated with the surface of infected cell even after budding, for the progeny virions to be passed on to adjacent uninfected cells. The evidence that cell-to-cell transmission occurs in influenza virus lead to the caution that local infection proceeds even when treated with neuraminidase inhibitors. It is generally accepted that viruses, released as cell-free virions from an infected cell, transmit to distant cells and tissues. This spreading pathway contributes to wide-ranged diffusion of cell-free viruses. However, in this spreading pathway, viruses are exposed to host anti-virus defense systems. In contrast, direct infection to a neighboring cell is considered to be beneficial for the virus in terms of evasion from the host anti-virus defense. There are two typical manners in infection to ''right next door'': one is the virus transmission through cell-cell fusion by forming syncytium without production of progeny virions, and the other is mediated by virions without virus diffusion, generally designated cell-to-cell transmission [1, 2] .