Characterization of Low Virulent Strains of Highly Pathogenic A/Hong Kong/156/97 (H5N1) Virus in Mice After Passage in Embryonated Hens' Eggs

Avian influenza A H5N1 viruses were isolated from humans for the first time in Hong Kong in 1997. The virulence of A/Hong Kong/156/97 (HK156) strain in mice was found to change significantly depending on the passage history of the virus. Madin-Darby canine kidney (MDCK) cell-grown parental virus and three of its clones derived from mouse brain showed high pathogenicity in mice after intranasal or intracerebral infection. In contrast, the egg-derived parental virus HK156-E3 and its cloned viruses were markedly less pathogenic in mice. It appeared that differences in pathogenicity among viruses derived from MDCK cells and eggs were due to their ability or inability to disseminate from the lungs to the brain. Sequence analysis of the entire protein coding regions of all eight RNA genome segments revealed a total of six conserved amino acid differences in the HA1 domain (residue 211) of the HA protein, as well as the PB1 (residues 456 and 712), PA (residue 631), NP (residue 127), and NS1 (residue 101) proteins that correlated with observed changes in virulence and neurovirulence of HK156 virus in mice. Thus it was evident that the passaging of HK156 in embryonated eggs led to the adaptation and selection of variants demonstrating markedly decreased pathogenicity and neurovirulence in mice that appeared to be attributable to specific amino acid changes in the HA and internal proteins.