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A (H9N2) avian influenza A viruses were first detected in Uganda in 2017 and have since established themselves in live bird markets. The aim of this study was to establish the subsequent genetic evolution of H9N2 viruses in Uganda. Cloacal samples collected from live bird market stalls in Kampala from 2017 to 2019 were screened by RT-PCR for influenza A virus and H9N2 viruses were isolated in embryonated eggs. One hundred and fifty H9N2 isolates were subjected to whole genome sequencing on the Illumina MiSeq platform. The sequence data analysis and comparison with contemporary isolates revealed that the virus was first introduced into Uganda in 2014 from ancestors in the Middle East. There has since been an increase in nucleotide substitutions and reassortments among the viruses within and between live bird markets, leading to variations in phylogeny of the different segments, although overall diversity remained low. The isolates had several mutations such as HA-Q226L and NS-I106M that enable mammalian host adaptation, NP-M105V, PB1-D3V, and M1-T215A known for increased virulence/pathogenicity and replication, and PA-E199D, NS-P42S, and M2-S31N that promote drug resistance. The PA-E199D substitution in particular confers resistance to the endonuclease inhibitor Baloxavir acid, which is one of the new anti-influenza drugs. Higher EC50 was observed in isolates with a double F105L+E199D substitution that may suggest a possible synergistic effect. These H9N2 viruses have established an endemic situation in live bird markets in Uganda because of poor biosecurity practices and therefore pose a zoonotic threat. Regular surveillance is necessary to further generate the needed evidence for effective control strategies and to minimize the threats.

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Genetic Evolution of Avian Influenza A (H9N2) Viruses Isolated from Domestic Poultry in Uganda Reveals Evidence of Mammalian Host Adaptation, Increased Virulence and Reduced Sensitivity to Baloxavir