Genomic surveillance of influenza A virus in live bird markets during the COVID-19 pandemic

Background and Aim: Despite the global focus on coronavirus disease 2019 (COVID-19), the avian influenza virus (AIV) continues to circulate in Indonesia, particularly in traditional live bird markets (LBMs), which serve as critical nodes for virus amplification and interspecies transmission. This study aimed to investigate the co-circulation and genetic features of AIV, particularly the H9N2 subtype, and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in LBMs in East Java during the COVID-19 pandemic. Materials and Methods: Environmental surveillance was conducted in seven traditional markets across four districts in East Java Province in 2021. Surface swabs were collected from high-risk areas, including poultry display tables, knives, cutting boards, and napkins. Samples were tested using reverse transcriptase polymerase chain reaction for influenza A and SARS-CoV-2. Positive AIV samples were further subtyped, sequenced, and analyzed for genetic markers associated with virulence, reassortment, and mammalian adaptation. Results: Of 156 samples tested, 17 (10.9%) were positive for influenza A, with 3 (1.9%) confirmed as the H9 subtype. These H9-positive samples were collected from a knife, cutting board, and napkin in the same market location in Lamongan Regency. Phylogenetic and molecular analyses revealed that two isolates (LSJ/Env/83 and LSJ/Env/84) were H9N2 reassortants, harboring key molecular markers such as Q226L, T160A, and S138A in the hemagglutinin protein, indicative of increased affinity for human-type receptors. Additional substitutions in PB2, MP, and NS1 proteins were associated with enhanced replication and virulence in mammalian and avian hosts. All samples tested negative for SARS-CoV-2. Conclusion: This study demonstrates the continued environmental circulation of reassortant H9N2 AIVs in traditional markets during the COVID-19 pandemic, with isolates displaying genetic features indicative of zoonotic potential. These findings underscore the necessity for sustained genomic surveillance and stricter biosecurity interventions in LBMs to prevent cross-species transmission and mitigate pandemic risk.