Abstract

Background:
Influenza viruses continuously threaten avian and mammalian species. After the circulation of 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 (PCR), Sanger sequencing, and bioinformatics analysis were performed on the HA and NA genotypes isolated from chickens in Iraq in 2015.

Results:
Out of 13 samples, eight (62%) were positive for the H5 gene and nine (69%) were positive for the neuraminidase 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, suggesting 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.

Key words: Avian influenza; H5N4; HPAI; Sanger sequencing; NCBI.