Moreover, the optimal activation pH may change upon introduction of an influenza virus into a new host species or environment. Aquatic birds are a natural reservoir of influenza viruses, but surprisingly little is known about the molecular basis of influenza virus propagation high throughput screening in these species. To test the hypothesis that the pH of activation of the HA protein contributes to the pathogenicity and transmissibility of H5N1 influenza viruses in the mallard, a prototypic aquatic bird, we previously generated four recombinant H5N1 viruses containing mutations that altered the acid stability of the HA protein without changing its level of expression, cleavage, receptor binding, or membrane fusion efficiency (36).

Two of the mutations increased the pH of membrane fusion of the H5N1 HA protein (Y231H and N1142K), and the other two mutations reduced the pH of fusion (H241Q and K582I). HA1 mutations Y231H and H241Q (H5 numbering with subscripts denoting HA1 and HA2 subunits) are located in the fusion peptide pocket and were originally chosen because of their presence in H1 and H9 subtypes, respectively. The K582I mutation in the A-helix of HA2 was chosen because it decreases the pH of membrane fusion of the H3 HA protein by 0.7 unit (44). The N1142K mutation in the fusion peptide pocket was chosen because it increases the pH of membrane fusion by approximately 0.5 unit in H3 and H7 subtypes (6). Here we measured the effects of the H5 HA protein mutations on virus replication in vitro, on genetic stability after repeated passage in eggs, and on environmental stability.

Mallards were inoculated with the recombinant viruses and were housed with contact ducks in order to determine the effects of the mutations on virus shedding, pathogenesis, and transmissibility. An H241Q mutation in the HA protein was found to decrease the pH of activation by 0.3 pH unit, to increase the titers of infectious virus recovered from ducks’ water dishes, and to prolong the persistence of infectious virus in the environment. In general, changes in the acid stability of the HA protein were found to alter H5N1 influenza virus replication, pathogenicity, and transmissibility. MATERIALS AND METHODS Viruses, plasmids, and cell culture. Recombinant viruses and plasmids containing HA protein mutations Y231H, H241Q, K582I, and N1142K were generated previously (36).

All experiments using H5N1 influenza viruses were performed in a USDA-approved biosafety level 3+ containment facility. Monolayer cultures of Vero cells (ATCC CCL-81) Dacomitinib were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, 1% glutamine, 1% penicillin, and 1% streptomycin. Monolayers of Madin-Darby canine kidney (MDCK) cells (ATCC CCL-34) were grown in minimum essential medium (MEM) supplemented with 10% fetal bovine serum, 1% glutamine, 1% penicillin, and 1% streptomycin. Virus growth kinetics.