1 unit. The highest pH at which cell-cell mostly membrane fusion was induced in cells infected with wild-type virus was 5.9 (Fig. (Fig.2).2). The H241Q and K582I mutations reduced the pH of membrane fusion to 5.6 and 5.4, respectively, while the Y231H and N1142K mutations increased the pH of membrane fusion to 6.3 and 6.4, respectively. The N1142K mutation, which increased the pH of HA activation by 0.5 pH unit, resulted in decreased virus fitness over several cycles of replication in vitro, while the other mutations did not alter in vitro replication kinetics. FIG. 2. The pH of HA-mediated membrane fusion by wild-type and mutant H5N1 influenza viruses in Vero cells was measured at 0.1 pH increments and is expressed as the highest pH value at which syncytium formation was observed.

The N1142K mutation is genetically unstable over multiple passages in eggs. To test the genetic stability of the HA protein mutations, wild-type and mutant viruses were passaged 10 times in 10-day-old embryonated chicken eggs. The sequence identity of each of the passage 1 (P1) recombinant viruses had been confirmed previously (36). Purified viral RNA sampled from allantoic fluid at P5 and P10 was sequenced. In parallel, syncytium formation assays were performed using Vero cells infected with P10 viruses to determine whether repeated passage in eggs resulted in any mutations that might alter the acid stability of the viral HA proteins (Table (Table1).1). Wild-type virus and viruses containing the HA protein mutation H241Q or K582I showed no additional mutations over the course of 10 passages and no change in the pH of membrane fusion.

The virus containing the Y231H mutation maintained the mutation for at least 5 passages in eggs and acquired an additional HA protein mutation, R2281I, between P5 and P10. Residue R228 (H5 numbering) is located in the receptor-binding pocket of the HA1 subunit with its side chain facing away from the pocket (46, 57) such that the R2281I mutation may enhance receptor binding in eggs (56). Despite the extra R2281I mutation, the P10 virus caused membrane fusion at a pH of 6.3, as did P1 Y231H virus without the additional R2281I mutation. The virus containing the N1142K mutation in the HA protein was the only recombinant virus whose pH of membrane fusion was altered at P10 from that for the P1 stock virus, a decrease from pH 6.4 to 6.1 (Table (Table1).

1). Both P5 and P10 K1142N viruses showed reversion mutations, demonstrating that the N1142K mutation was not genetically stable and was selected against within 5 passages. TABLE 1. Genetic stability of recombinant H5N1 influenza viruses containing HA protein mutations after serial passages Dacomitinib in embryonated chicken eggs Changes in the pH of activation of the HA protein can alter the environmental stability of H5N1 influenza viruses.