Complete genome sequencing and evolutionary analyses of duck hepatitis a viruses in Egyptian duck farms

Duck hepatitis virus (DHV) continues to pose a substantial threat to duck production worldwide, causing acute hepatitis, neurological manifestations, and elevated mortality rates, even in vaccinated flocks. During 2022-2023, a significant outbreak involving DHV-1 and DHV-3 was reported in duck farms across five governorates in North Egypt. The outbreaks primarily affected Pekin ducklings aged 4-15 days, with mortality rates ranging from 50% to 70%. Affected ducklings exhibited characteristic pathological lesions, including hepatomegaly with haemorrhages, splenomegaly, and renal enlargement.

A total of 30 liver and spleen samples collected from affected farms were analysed using reverse transcription PCR (RT-PCR) targeting the 3' untranslated region (3'UTR) and the VP1 gene. DHV was detected in 56.7% (17/30) of the samples. Among the positive cases (n = 17), DHV-1 was identified in 29.4% (5/17), whereas DHV-3 accounted for 70.6% (12/17). Phylogenetic analysis based on whole-genome sequencing revealed that DHV-1 strains clustered within sub-clade 1a, demonstrating high genetic similarity (99.2-99.8%) with previously reported Egyptian isolates and Chinese reference strains (e.g., DHAV-1-CH-2012). In contrast, DHV-3 strains grouped within sub-clade 3a and showed close genetic relatedness to Chinese strains (97.1-98.7%), while showing marked divergence (75.9-76.3% nucleotide identity) from currently used vaccine strains. Genetic analysis identified multiple mutations in both viral types. DHV-1 exhibited amino acid substitutions within VP0, VP3, and non-structural protein regions, alongside five mutations in hypervariable region 1 (HVR1) and one mutation in HVR2 of the VP1. DHV-3 demonstrated distinct mutations within the VP1, including a unique E681K substitution identified in the Egyptian strain ND3.

The findings demonstrate ongoing genetic evolution of DHV circulating in Egyptian duck farms, characterized by the predominance of genetically distinct DHAV-3 strains, with significant divergence from currently used vaccine strains. This genetic divergence may contribute to altered vaccine performance and continued disease outbreaks; however, its impact on vaccine-induced protection requires further experimental validation. Continuous genomic surveillance and the development of updated, strain-matched or multivalent vaccines are therefore essential to control DHV infections and minimise economic losses in Egypt's duck industry.