Background: Avian influenza viruses (AIVs) cause significant harm to the poultry industry due to mortality as well as high morbidity along with the risk of potential zoonotic transmission to humans.

Aims: The study aimed to investigate the prevalence of influenza H5, H7, and H9 viruses and their co-infections in layers having respiratory distress such as sneezing, coughing, and tracheal rales.

Methods: Totally, 960 tracheal swabs (240 swabs in each season) were collected from 120 poultry flocks, including 10 farms per month and 8 samples per flock, located in Karachi where the outbreaks were reported. The samples were confirmed through antigen ELISA and subtyped by RT-PCR.

Results: Antigen ELISA revealed that the prevalence of avian influenza viruses was 26.45%; however, seasonal differences were not significant (P<0.05). RT-PCR subtyping of hemagglutinin (HA) gene revealed the higher prevalence of H9 virus (40.16%) as compared to H7 virus (5.51%) and H5 virus (4.73%). The co-infections comprised H5/H7/H9 (37.0%) and H5/H9 (12.6%).

Conclusion: This study shows that AI is endemic in layer farms in Karachi where the H9 subtype is predominant along with co-infections of H5/H7/H9 subtypes.