Highly pathogenic
avian influenza viruses (HPAIVs) with H5 and
H7 hemagglutinin (HA) subtypes are derived from their low pathogenic counterparts following the acquisition of multiple
basic amino acids in their HA cleavage site. It has been suggested that consecutive
adenine residues and a stem-loop structure in the
viral RNA region that encodes the cleavage site are essential for the acquisition of the polybasic cleavage site. By using a reporter assay to detect non-templated
nucleotide insertions, we found that insertions more frequently occurred in the
RNA region (29 nucleotide-length) encoding the cleavage site of an H5 HA gene that was predicted to have a stem-loop structure containing consecutive adenines than in a mutated corresponding
RNA region that had a disrupted loop structure with fewer adenines. In virus particles generated by using reverse genetics,
nucleotide insertions that created additional
codons for
basic amino acids were found in the
RNA region encoding the cleavage site of an H5 HA gene but not in the mutated
RNA region. We confirmed the presence of virus clones with the ability to replicate without
trypsin in a plaque assay and to cause lethal
infection in chicks. These results demonstrate that the stem-loop structure containing consecutive adenines in HA genes is a key molecular determinant for the emergence of H5 HPAIVs.