Antigenic drift in H5N1 avian influenza virus in poultry is driven by mutations in major antigenic sites of the hemagglutinin molecule analogous to those for human influenza virus

Abstract

H5N1 highly pathogenic avian influenza virus has been endemic in poultry in Egypt since 2008, notwithstanding the implementation of mass vaccination and culling of infected birds. Extensive circulation of the virus has resulted in a progressive genetic evolution and an antigenic drift. In poultry, the occurrence of antigenic drift in avian influenza viruses is less well documented and the mechanisms remain to be clarified. To test the hypothesis that H5N1 antigenic drift is driven by mechanisms similar to type A influenza viruses in humans, we generated reassortant viruses, by reverse genetics, that harbored molecular changes identified in genetically divergent viruses circulating in the vaccinated population. Parental and reassortant phenotype viruses were antigenically analyzed by hemagglutination inhibition (HI) test and microneutralization (MN) assay. The results of the study indicate that the antigenic drift of H5N1 in poultry is driven by multiple mutations primarily occurring in major antigenic sites at the receptor binding subdomain, similarly to what has been described for human influenza H1 and H3 subtype viruses.

Fig. 1.

Amino acid sequence alignment between HA proteins of A/chicken/Egypt/1709-6/2008, A/chicken/Egypt/1709-1/2007, A/chicken/Mexico/232/94, and A/Viet-Nam/1203/04 strains. The figure shows the receptor binding subdomain (9) as a dashed line, the major 130 and 220 loops and 190 α-helix as shaded squares, and amino acid differences between A/chicken/Egypt/1709-6/2008 and A/chicken/Egypt/1709-1/2007 as smaller, unshaded squares.

Fig. 2.

A/chicken/Egypt/1709-6/2008 HA monomer. Amino acid differences in the HA monomer between A/chicken/Egypt/1709-6/2008 and A/chicken/Egypt/1709-1/2007 are shown in white; the major 130 and 220 loops and 190 helix are shown in magenta (image drawn with Pymol software). The template protein was obtained by submitting the amino acid sequence of the HA protein to the structure homology modeling server http://swissmodel.expasy.org. The Protein Data Bank accession codes obtained refer to influenza A virus (24) for the full monomer (Protein Data Bank identification 2wr1A) and to strain A/Vietnam/1194/04 (41) for the portion from amino acid residue 1 to 340 (Protein Data Bank identification 2ibX).

Fig. 3.

A/chicken/Egypt/1709-6/2008 HA monomer. Amino acid differences in the HA monomer between A/chicken/Egypt/1709-6/2008 and A/chicken/Egypt/1709-1/2007. In red are amino acid substitutions in antigenic site A (positions 140 and 141); in yellow are amino acid substitutions in antigenic site B (positions 162 and 184). All the positions in white are the differences not included in the antigenic sites.