Genetic Characterization of Highly Pathogenic Avian Influenza A(H5N8) Virus in Pakistani Live Bird Markets Reveals Rapid Diversification of Clade 2.3.4.4b Viruses

The highly pathogenic (HPAI) avian influenza A(H5N1) viruses have undergone reassortment with multiple non-N1-subtype neuraminidase genes since 2008, leading to the emergence of H5Nx viruses. H5Nx viruses established themselves quickly in birds and disseminated from China to Africa, the Middle East, Europe and North America. Multiple genetic clades have successively evolved through frequent mutations and reassortment, posing a continuous threat to domestic poultry and causing substantial economic losses. Live bird markets are recognized as major sources of avian-to-human infection and for the emergence of zoonotic influenza. In Pakistan, the A(H5N1) virus was first reported in domestic birds in 2007; however, avian influenza surveillance is limited and there is a lack of knowledge on the evolution and transmission of the A(H5) virus in the country. We collected oropharyngeal swabs from domestic poultry and environmental samples from six different live bird markets during 2018–2019. We detected and sequenced HPAI A(H5N8) viruses from two chickens, one quail and one environmental sample in two markets. Temporal phylogenetics indicated that all novel HPAI A(H5N8) viruses belonged to clade 2.3.4.4b, with all eight genes of Pakistan A(H5N8) viruses most closely related to 2017 Saudi Arabia A(H5N8) viruses, which were likely introduced via cross-border transmission from neighboring regions approximately three months prior to virus detection into domestic poultry. Our data further revealed that clade 2.3.4.4b viruses underwent rapid lineage expansion in 2017 and acquired significant amino acid mutations, including mutations associated with increased haemagglutinin affinity to human α-2,6 receptors, prior to the first human A(H5N8) infection in Russian poultry workers in 2020. These results highlight the need for systematic avian influenza surveillance in live bird markets in Pakistan to monitor for potential A(H5Nx) variants that may arise from poultry populations