Characterization of mutations associated with the adaptation of a low-pathogenic H5N1 avian influenza virus to chicken embryos.

Migratory waterfowls are the most common reservoir for avian influenza virus (AIV), thus viral adaptation is required for efficient replication in land fowls. To date, low pathogenic (LP) H5 subtype AIVs have been isolated from migratory waterfowls, and the adaptation of these viruses to land fowls might lead to the generation of highly pathogenic AIVs. Thus, A/wild duck/Korea/50-5/2009 (H5N1) LPAIV was passaged 20 times through embryonated chicken eggs (ECEs), and the resulting genetic and phenotypic changes were investigated. The pathogenicities of the early (50-5-E2) and final passage (50-5-E20) strains to chicken embryos were similarly high, but the 50-5-E20 titer was 100 times higher than that of 50-5-E2. 50-5-E20 showed 8 amino acid changes in PA (1), HA (4), NA (1), M1 (1) and M2 (1), with different frequencies among influenza A viruses (0-99.6%). The relevance of these changes, except H103Y in HA, to viral replication remains unknown. To investigate the roles of internal genes and mutations in HA and NA in viral replication, four recombinant viruses possessing combinations of HA and NA genes of 50-5-E2 and 50-5-E20 with 6 internal genes of PR8 were generated through reverse genetics. The embryo pathogenicities of the H5N1 recombinant viruses carrying internal PR8 genes were reduced, and the titers of the recombinant viruses with 50-5-E20 HA were higher than those with 50-5-E2 HA. Therefore, the identified mutations might be useful as chicken adaptation markers for the generation of high growth H5N1 recombinant viruses in ECEs.