[Impact of mutations in the hemagglutinin gene of H5N1 influenza virus on antigenicity and virulence as revealed by site-specific mutagenesis].

Abstract In our earlier studies, we have shown that amino acid changes in the hemagglutinin (HA) of influenza H5N1 virus escape mutants conferring resistance to monoclonal antibodies (MAbs) may correlate with a decrease of virus virulence for mice and that the virulence can be restored to the initial level by serial passages. In the present study, the mutations identical to those observed in the HA of a low-virulent escape mutant and its readapted variant were introduced into the HA gene by site-specific mutagenesis. The viruses produced by plasmid transfection and containing the HA gene either of A/Vietnam/1203/2004 (H5N1) virus with a deletion at the cleavage site, or of a low-virulent escape mutants, or of its readapted variant, in the presence of 6 genome segments of A/Puerto Rico/8/34 (H1N1) virus and the NA gene of A/Vietnam/1203/2004 (H5N1) virus, were assayed for virulence. Determination of virulence for mice indicated that amino acid substitution in the HA gene of a low-virulent escape mutant produced a decrease of virulence whereas the additional mutation identical to that acquired by the escape mutant in the course of readaptation restored the virulence to initial level. The findings are the first strong evidence for lower H5N1 virus virulence resulting from the amino acid substitution changing the antigenic specificity of HA and for restored virulence arising from compensating mutation in the HA gene.