Glycosylation of the HA protein of H5N1 virus increases its virulence in mice by exacerbating the host immune response. Mus musculus

The highly pathogenic avian influenza (HPAI) H5N1 viruses continue to circulate in nature and threaten public health. Although several viral determinants and host factors that influence the virulence of HPAI H5N1 viruses in mammals have been identified, the detailed molecular mechanism remains poorly defined and requires further clarification. In our previous studies, we characterized two naturally isolated HPAI H5N1 viruses that had similar viral genomes but differed substantially in their lethality in mice. Here, we explored the molecular determinants and potential mechanism for this difference in virulence. By using reverse genetics, we found that a single amino acid at position 158 of the hemagglutinin (HA) protein substantially affected the systemic replication and pathogenicity of these H5N1 influenza viruses in mice. We further found that the G158N mutation introduced an N-linked glycosylation at sites 158–160 of the HA protein and that this N-linked glycosylation enhanced viral productivity in infected mammalian cells and induced stronger host immune and inflammatory responses to viral infection. These findings further our understanding of the determinants of pathogenicity of H5N1 viruses in mammals. Overall design: For all gene expression analyses, groups of three mice were inoculated with PBS or 106 EID50 of CK/1180 or CK/1180-1214HA. Total RNA was extracted from lung tissues on day 3 p.i. by using a QIAGEN RNeasy kit. The microarray assay was performed by using a Low RNA Input Linear Amplification Kit (Agilent Technologies, Santa Clara, CA) and Agilent’s Whole Mouse Genome Microarray Kit, 4×44 K (G4122F)