Molecular Surveillance of Avian Influenza Virus Based on HA Gene Isolated from Commercial Poultry in Pakistan

Avian influenza virus (AIV) continues to pose a major risk to the global poultry industry and human health on account of its high mutation rate, segmented genome, and its ability to undergo genetic reassortment. H9N2 is among the low-pathogenic types that are particularly important due to being highly endemic in the poultry, causing severe economic losses, and is a possible source of internal genes of the highly pathogenic and zoonotic influenza viruses. Continuous molecular surveillance of circulating H9N2 strains is essential to monitor the evolution of the virus, its reassortment potential, and its effectiveness in vaccines. The aim of the study was to examine the molecular prevalence and partial genetic characterization of low-pathogenic subtype of AIV H9N2 in commercial poultry in Punjab, Pakistan, in 2024. One hundred pooled oropharyngeal, tracheal, lungs, and cloacal samples were collected from symptomatic flocks with respiratory symptoms and low egg production and 40 samples were processed to isolate the virus in specific antibody-negative embryonated chicken eggs and tested with hemagglutination (HA) and hemagglutination inhibition (HI) assays. Molecular confirmation was done through SYBR Green based real-time RT-PCR targeting a 765bp fragment of the hemagglutinin (HA) gene. PCR-positive samples were sequenced and analyzed through BLAST, multiple sequence alignment, and phylogenetic analysis. 10 isolates were classified as H9N2, showing nucleotide similarity of 87.8% to 98.3% with previously reported Pakistani isolates. Mutation analysis revealed various deletions and nucleotide substitutions, indicating that there has been continuous genetic evolution. All indigenous isolates were clustered within the G1-like lineage of Eurasian H9N2 viruses in phylogenetic analysis. In conclusion, the study confirms the continuation of H9N2 AIV circulation and genetic diversification in Pakistani Poultry and highlights the importance of continued molecular surveillance to support effective control and vaccination strategies.