Lethality and Molecular Characterization of an HPAI H5N1 Virus Isolated from Eagles Smuggled from Thailand into Europe

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Abstract
On October 18, 2004, two crested hawk eagles, Spizaetus nipalensis, smuggled into Europe from Thailand were seized at Brussels International Airport. A highly pathogenic avian influenza virus, denominated A/crested eagle/Belgium/01/2004, was isolated from these birds and antigenically characterized as H5N1. Here we report on the molecular characterization of A/crested eagle/Belgium/01/2004 (H5N1). We completely sequenced all eight genome segments. The hemagglutinin (HA) and neuraminidase (NA) sequences clustered within the Z genotype and were closely related to strains circulating in Thailand during 2004, although some mutations in the HA were evident, notably a unique arginine (R) > lysine (K) replacement in the cleaving site. The HA cleavage site contained six basic amino acids, confirming its high pathogenicity (intravenous pathogenicity index = 2.94). The 20-amino acid deletion in the NA stalk region is consistent with its Thai-Viet origin. We further discuss the assembled genetic information in the light of currently known host adaptation, virulence, and antiviral resistance factors. Using infection experiments, we show that pathogenicity in chickens depends on breed, inoculation route (oculonasal vs. intramuscular), and dose. Additionally, in Muscovy ducks, pathogenicity proved to be age dependent.