Surveillance of H9N2 is currently focused on areas central to the commercial poultry industry. This study determined the prevalence of H9N2 virus in commercial and backyard poultry flocks in Punjab Province, Pakistan. Oral and tracheal swabs were collected from commercial and backyard poultry from January 2015 through June 2016. Antisera against H5, H7, H9, and Newcastle disease viruses were used for virus identification. Molecular confirmation was made by reverse transcription PCR. Avian influenza virus subtypes H5 and H7 were not detected. The H9N2 virus was isolated in 5.7% of 905 tested flocks (5–10 birds/flock). Prevalence in commercial and backyard poultry was 6.7% of 687 flocks and 2.7% of 218 flocks, respectively. Hemagglutinin and neuraminidase-gene-based phylogenetic analysis of commercial and backyard poultry isolates showed 100% homology. Within sublineage B2 of Pakistan, identity among most recent isolates (2015) was 100%, compared to 75%–99% identity with previously isolated viruses (2010–12), indicating continued virus evolution. Most of the previously reported and currently studied viruses were isolated near the Pakistan-India border. Phylogenetic analysis showed that Pakistani and Indian isolates were closely related, indicating that avian influenza virus transmission may occur across this border.

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