Abstract
The current study investigated the prevalence and genetic diversity of highly pathogenic avian influenza (HPAI) viruses in non-vaccinated backyard chicken flocks in Qalubia governorate during the period 2012-2014. Results revealed the emergence of two novel H5N1 HPAI viruses, designated A/Chicken/Egypt/D2296E/2012 (CH/E12) and A/Chicken/Egypt/D2296H/2014 (CH/H14). Phylogenetic analysis of the hemagglutinin (HA) genes revealed that CH/E12 belongs to the predominant subclade 2.2.1.2, meanwhile CH/H14 provides an empiric evidence for the ongoing circulation of the 2.2.1.1 viruses, which were thought to have disappeared from Egypt since 2011. In addition, genetic analysis revealed that CH/E12 and CH/H14 retained the amino acid substitutions which characterize the subclade 2.2.1.2 and 2.2.1.1, respectively. However, the HA-receptor binding residues of the two viruses carried numerous substitutions, suggesting a preferable binding to human type receptors. Vaccination-challenge studies were conducted under standard laboratory conditions using three commercially available vaccines. Results revealed that the hemagglutination inhibition (HI) titers as well as the levels of protection conferred by each vaccine were positively correlated with the HA identity between the vaccine and challenge viruses. The antigenically related (90.8–96.2%) A/swan/Hungary/4999/2006 and A/chicken/Egypt/18-H/09 vaccine strains provided the highest (86.6–93.3%) protection, meanwhile the antigenically distinct (78.3–81%) A/chicken/Mexico/232/1994 H5N2 vaccine strain provided the lowest level of protection (80–83.3%) against lethal challenge. These findings highlight the significance of backyard chickens as a constant reservoir of HPAI H5N1 viruses in Egypt and raise the need for monitoring the prevalence of H5N1 viruses in backyard chicken flocks in order to understand the virus evolution and to select the proper vaccination strategy.

Key words: Avian influenza; H5N1; Backyard chickens; Vaccine; Egypt