Background: Influenza viruses continuously threaten avian and mammalian species. After the circulation of highly pathogenic avian influenza (HPAI) H5N1 in 2015 in Iraq, an H5N4 influenza virus emerged in domestic chickens.

Aim: This study aimed to identify and characterize the presence of avian influenza H5N4 viruses in domestic poultry during the HPAI H5N1 circulation in 2015 in Iraq.

Methods: Polymerase chain reaction, Sanger sequencing, and bioinformatics analysis were performed on the hemagglutinin and neuraminidase (NA) genotypes isolated from chickens in Iraq in 2015.

Results: Out of 13 samples, eight (62%) were positive for the H5 gene, nine (69%) were positive for the NA gene, eight for N1, and one for N4. One sample, KCVL15-013, initially amplified using N1-specific primers, was later confirmed by sequencing to contain the N4 gene, likely due to partial primer homology. Sequence annotation confirmed the subtype as H5N4. Cleavage site analysis revealed a multibasic motif (RRRKR/GLF), indicating high pathogenicity. Phylogenetic analysis of the N4 gene showed that KCVL15-013 formed a distinct branch, clustering only with another Iraqi chicken-origin H5N4 strain, with weak bootstrap support (37%), and diverging from globally clustered wild bird-origin N4 viruses.

Conclusion: This study concludes that H5N4 was co-detected during an H5N1 outbreak, indicating the concurrent circulation of multiple influenza A virus subtypes in poultry. Multiple strain genotypes of the influenza virus A could cause a co-infection in domestic chickens, particularly during a pandemic. Incorporating DNA sequencing and annotation into routine surveillance is essential for accurate detection of strain genotype.