Molecular Detection and Phylogenetic Analysis of Fowl Adenovirus-4 from Broiler Chickens in Diyala, Iraq

Hydropericardium Hepatitis Syndrome (HHS) is a highly infectious disease of broiler chickens caused by fowl adenovirus serotype 4 (FAdV-4) from the Adenoviridae family. It primarily affects chickens between 3 to 5 weeks of age and causes significant economic losses, with mortality rates reaching up to 50%. The disease is characterized by the accumulation of amber-colored fluid in the pericardial sac, anemia and necrotic hepatitis. This study aimed to detect and characterize FAdV-4 strains associated with HHS and confirm their presence in Diyala Governorate. Tissue samples (liver, spleen, kidney, heart, and intestine) were collected from clinically infected broiler chickens from five regions: Baqubah, Al-Galibia, Al-Khalis, Kanaan, and Baladrose. Infected birds exhibited depression, reduced feed intake, and yellow to greenish droppings. Post-mortem findings revealed pericardial effusion with amber fluid, enlarged necrotic liver, congested kidneys with urate deposits, and splenomegaly. A total of 125 samples were analyzed by PCR, with 80 (64%) positive and 45 (36%) negative for HHS. In this study, the hexon gene was amplified, yielding an 882 bp amplicon showing 99% sequence similarity to reference sequences in NCBI. Seven local strains (S1–S7) were identified and deposited in GenBank under accession numbers PQ621406–PQ621412. Sequence alignment revealed four nucleotide substitutions (325T>C, 440G>C, 595A>G, and 667C>T), including three silent (p.N181=, p.G271=, p.D275=) and one missense mutation (p.E220Q), where Glutamic acid is replaced by Glutamine at position 220, indicating genetic diversity among the local strains.