Antigenic analysis of H5N1 highly pathogenic avian influenza viruses circulating in Egypt (2006-2012)

The highly pathogenic avian influenza (HPAI) H5N1 in Egypt circulated continuously after its introduction in February 2006 with substantial economic losses and frequent human infections. Phylogenetic analysis of the available HA sequences revealed the presence of two main sublineages; the classic 2.2.1 and the variant 2.2.1.1. The classic 2.2.1 had subdivided into two clusters of viruses; cluster C1 contained the originally introduced virus and isolates from 2006 to 2009 and cluster C2 emerged in 2007 and continues to circulate. The variant 2.2.1.1 represents the isolates mainly from chickens and subdivided into two clusters; cluster V1 contains isolates from 2007 to 2009 and cluster V2 contains isolates from 2008 to 2011. Sequence analysis revealed 28 amino acid mutations in the previously reported antigenic sites and high evolution rate which may be due to selective pressure from vaccination and/or natural infection. Antigenic analysis of 18 H5N1 isolates from 2006 to 2012 that represent different clusters was conducted using hemagglutination inhibition (HI) and virus neutralization (VN) assays using hyperimmune sera produced by immunizing SPF chickens with inactivated whole-virus. Antigenic relatedness of ancestral Egyptian H5N1 isolate (459-3/06) with other isolates ranged from 30.7% to 79.1% indicating significant antigenic drift of the H5N1 viruses from the ancestral strains. The antigenic relatedness between C2 and V2 clusters ranged from 28.9% to 68% supporting the need for vaccine seed strains from both clusters. Interestingly, A/CK/EG/1709-6/2008 H5N1 strain showed a broad cross reactivity against viruses in different H5N1 clusters (antigenic relatedness ranged from 63.9% to 85.8%) demonstrating a potential candidate as a vaccine strain. Antigenic cartography which facilitates a quantitative interpretation and easy visualization of serological data was constructed based on HI results and further demonstrated the several antigenic groups among Egyptian H5N1 viruses. In conclusion, the cross reactivity between the co-circulating H5N1 strains may not be adequate for protection against each other and it is recommended to test vaccines that contain isolates from different antigenic groups in experimental infection trials for the selection of vaccine seed strain. Furthermore, the continuous monitoring for detecting the emerging variants followed by detailed antigenic analysis for updating vaccines is warranted.