Excel (Microsoft) was used to determine the percentage of plaque inhibition and IC50. MD simulations of NA. Taken together, these data suggest that H5N1 viruses isolated from wild birds have already acquired OTV-resistant point mutations without any exposure to a drug. Introduction Influenza A viruses are highly communicable and are responsible for seasonal epidemics and the occasional pandemics (Horimoto & Kawaoka, 2005; Palese, 2004). However, the emergence and re-emergence of the highly pathogenic avian influenza (HPAI) H5N1 virus with the potential of causing a pandemic has raised the need for meticulous study of the virus worldwide. HPAI virus of the subtype H5N1, discovered for the first time in 1996 in Guangdong province in China, was the precursor of the H5N1 virus that spread to humans in Hong Kong (Xu experiments proceeded only for the WT and S236F populations. The rescued recombinant WT and S236F variant viruses replicated well in MDCK (MadinCDarby canine kidney) cells and peaked at 1.2??0.03??107 p.f.u. ml?1 and 1.5??0.13??106 p.f.u. ml?1 for WT and S236F variant, respectively. A plaque reduction assay was then employed to determine the IC50 values EGT1442 of OTV when infected with either the WT or S236F population. The results showed the percentage plaque inhibition of the WT and S236F variant that occurred at varying concentrations of OTV (Table 1). The IC50 values calculated were 18.30??3.08 and 104.74??32.55?nM for the WT and S236F population, respectively. This illustrates that the IC50 value of the S236F mutant is approximately sixfold higher than that of the WT virus. An IC50 value of greater than 50?nM can be considered as having reduced effect on the virus while an IC50 value of less than 50?nM can be considered as susceptible (Stoner (2011). These data support that mutations of the residues located at the back of the active site can affect the conformation of the 150- and 270-loops, such as driving the 150-loop from closed configuration into open form. Open in a separate window Fig. 4. Distance between the various atoms of the 150-loop was measured to determine the open and closed formation of the 150-loop during the 100?ns simulation time. The position of Asp151 is considered the median point for the 150-loop residues and 3C4?? or 5?? was indicated as closed or open conformation, respectively. (a) Plot of the 150-cavity between the CG1 atom of V149 and CA atom of P431. (b) Distance between the N2 atom of OTV and the CG carbon of Asp151. (c) Distance between the CZ atom of R152 and the O3 atom of OTV. Here, the WT virus, the S236F population, the double mutant population, the quadruple mutant population and the C278Y population are represented in black, red, blue, green and grey, respectively. The dynamic change of the 150-loop was further monitored to discriminate the closed/open conformation by plotting the distance between two binding residues belonging to the 150-loop (D151 and R152) and heteroatoms of their interaction side chain (Fig. 4b, c). A distance between the CG atom belonging to the D151 catalytic residue and the ammonium N2 atom of OTV of 3C4?? or 5?? was indicated as closed or open conformation, respectively (Udommaneethanakit 2010). The reduced susceptibility to OTV in variants conferring the S236F mutation was discovered by plaque reduction assay as described above. To seek for the effect of drug binding corresponding to the natural quasispecies in H5N1 NA, OTV binding towards NA for both WT and quasispecies was evaluated in terms of hydrogen bond interactions (Fig. 6) and change in dihedral angles of bulky group (Fig. S2). The percentage occupation of hydrogen bonds between residues in the NA binding pocket and three side chains of OTV was determined over the stable MD structures (50C100?ns) based on the criteria of a maximum distance of 3.5?? UPK1B between hydrogen donor and acceptor, and a minimum angle of 120 for donor-H-acceptor. In addition, the conformation of NA and OTV complexes taken every 20?ns from 0 to 100?ns is shown in Fig. S3. Open in EGT1442 a EGT1442 separate EGT1442 window Fig. 6. The percentage occupation of hydrogen bonds formed between OTV and the active-site residues for each of the protein systems was determined using the Ptraj suite of.