The study of highly pathogenic avian influenza (HPAI) seeks to understand the pathogenic nature of these viruses. Belonging to the H5 or H7 subtype, HPAI causes significant economic loss in poultry and in recent years has also led to die offs in wild birds. In addition, the zoonotic nature of H5N1 has caused human infection associated with high mortality, although these viruses have not yet evolved to cause a pandemic. Mechanisms of virulence by HPAI have previously been attributed to a multi-basic HA cleavage site (MBCS) which facilitates systemic dissemination of the virus. Yet more recently, other factors have been described as necessary for a highly pathogenic phenotype. This study utilises reverse genetics to investigate the importance of three mutations in the HA of A/turkey/England/50-92/91 (50-92) H5N1 virus which are crucial for virulence. A novel mutation in the fusion peptide appeared to alter the pH of fusion by the HA. The importance of HA fusion for pathogenesis and host adaptation is discussed. Eurasian lineage H5N1 highly pathogenic avian influenza viruses (HPAIV), were first detected in Qinghai Lake, China in 2005, and subsequently spread through Asia, Europe and Africa. Importantly, these viruses carried a lysine at position 627 of the PB2 protein (PB2 627K), a known mammalian adaptation motif. Previous avian influenza isolates have carried glutamic acid in this position (PB2 627E), commonly described to restrict virus polymerase function in the mammalian host. We sought to examine the effect of PB2 627K on viral maintenance in the avian reservoir. Reverse genetics viruses were engineered to contain converse PB2 627K/E mutations in a Eurasian H5N1 virus (A/turkey/Turkey/5/2005; Ty/05) and, for comparison, a historical pre-Asian HPAI H5N1 virus that naturally bears PB2 627E (A/turkey/England/50-92/1991; 50-92). Our observations suggest PB2 627K is supported in Eurasian lineage viruses; in contrast PB2 627K carries a significant fitness cost in the historical pre-Asian 50-92 virus. These data have important implications for understanding the restriction of HPAI for humans and for the maintenance of a key genetic motif associated with host range in the avian reservoir.