Genetic and antigenic evolution of H9N2 avian influenza viruses circulating in Egypt between 2011 and 2013

Abstract

Avian influenza virus subtype H9N2 has been circulating in the Middle East since the 1990s. For uncertain reasons, H9N2 was not detected in Egyptian farms until the end of 2010. Circulation of H9N2 viruses in Egyptian poultry in the presence of the enzootic highly pathogenic H5N1 subtype adds a huge risk factor to the Egyptian poultry industry. In this study, 22 H9N2 viruses collected from 2011 to 2013 in Egypt were isolated and sequenced. The genomic signatures and protein sequences of these isolates were analyzed. Multiple mammalian-host-associated mutations were detected that favor transmission from avian to mammalian hosts. Other mutations related to virulence were also identified. Phylogenetic data showed that Egyptian H9N2 viruses were closely related to viruses isolated from neighboring Middle Eastern countries, and their HA gene resembled those of viruses of the G1-like lineage. No reassortment was detected with H5N1 subtypes. Serological analysis of H9N2 virus revealed antigenic conservation among Egyptian isolates. Accordingly, continuous surveillance that results in genetic and antigenic characterization of H9N2 in Egypt is warranted.

Fig. 1

Phylogenetic trees of the nucleotide sequences of PB2, PB1 and PA of H9N2 viruses. Isolates sequenced specifically for this study are indicated by a red rhomboid. Non-H9N2 subtypes are indicated in blue. The percentage of replicate trees in which the associated sequences clustered together in the bootstrap test (1000 replicates) is shown at the dendrogram nodes. The phylogenetic analysis was performed using MEGA version 5.2.

Fig. 2

Virulence and host specificity determinants in Egyptian H9N2 viruses isolated from Egypt in the period 2011–2013. The numbers in parentheses indicate the number of H9N2 viruses that contain the specific residues. Red indicates that the residue is critical for virulence, host range determinants, antiviral resistance, or enzyme activity. Blue indicates the 12 viral proteins that were analyzed.

Fig. 3

Phylogenetic trees of the nucleotide sequences of HA and NA of H9N2 viruses. Isolates sequenced specifically for this study are indicated by a red rhomboid.

Fig. 4

Phylogenetic tree of the nucleotide sequences of NP, M, and NS of H9N2 viruses. Isolates sequenced specifically for this study are indicated by a red rhomboid. Non-H9N2 subtypes are indicated in blue.

Fig. 5

Antigenic cartography representation of the hemagglutination inhibition data generated using a panel of polyclonal (A) and monoclonal (B) antibodies. The map was produced using AntigenMap (http://sysbio.cvm.msstate.edu/AntigenMap). One unit (grid) represents a twofold change in the HI results. Isolates of each year are indicated by symbols and colors.