Effect of vaccine use in the evolution of Mexican lineage H5N2 avian influenza virus

Abstract

An outbreak of avian influenza (AI) caused by a low-pathogenic H5N2 type A influenza virus began in Mexico in 1993 and several highly pathogenic strains of the virus emerged in 1994-1995. The highly pathogenic virus has not been reported since 1996, but the low-pathogenic virus remains endemic in Mexico and has spread to two adjacent countries, Guatemala and El Salvador. Measures implemented to control the outbreak and eradicate the virus in Mexico have included a widespread vaccination program in effect since 1995. Because this is the first case of long-term use of AI vaccines in poultry, the Mexican lineage virus presented us with a unique opportunity to examine the evolution of type A influenza virus circulating in poultry populations where there was elevated herd immunity due to maternal and active immunity. We analyzed the coding sequence of the HA1 subunit and the NS gene of 52 Mexican lineage viruses that were isolated between 1993 and 2002. Phylogenetic analysis indicated the presence of multiple sublineages of Mexican lineage isolates at the time vaccine was introduced. Further, most of the viruses isolated after the introduction of vaccine belonged to sublineages separate from the vaccine's sublineage. Serologic analysis using hemagglutination inhibition and virus neutralization tests showed major antigenic differences among isolates belonging to the different sublineages. Vaccine protection studies further confirmed the in vitro serologic results indicating that commercial vaccine was not able to prevent virus shedding when chickens were challenged with antigenically different isolates. These findings indicate that multilineage antigenic drift, which has not been observed in AI virus, is occurring in the Mexican lineage AI viruses and the persistence of the virus in the field is likely aided by its large antigenic difference from the vaccine strain.

FIG. 1.

Phylogenetic tree based on nucleotide sequences of the HA1 and NS genes from Mexican lineage isolates. The tree, generated by the maximum parsimony method with PAUP 4.0b10, is the result of heuristic search and midpoint rooting.

FIG. 2.

Alignment of HA1 amino acid sequences of Mexican lineage isolates with that of the consensus sequence made with 18 early isolates. The underlined residues are potential glycosylation sites, and the residues in the open boxes are previously identified antigenic sites with H1 and H3 molecules. ▾, substitution sites demonstrated in an H5 escape mutant; ↓, amino acid substitution sites in sublineage A and B viruses analyzed in this study. Overlapping sites are circled.

FIG. 3.

Ribbon diagram of the monomer of H5 (A/Duck/Singapore/3/97) HA. Front (a) and back (b) views are shown. Location of amino acid changes in HA1 and potential glycosylation sites of Mexican lineage isolates are labeled. The color scheme is as follows: green, amino acid changes in sublineage A virus; blue, amino acid changes in sublineage B virus; red, overlapping amino acid changes in sublineage A and B viruses; pink, potential glycosylation sites; purple, termini of HA1 (N1 and C1) and HA2 (N2 and C2).