Characterization of an avian influenza virus H5N1 Egyptian isolate

The highly pathogenic influenza virus H5N1 that infected chickens in Egypt in 2006 was characterized at immunologic and molecular levels. Cloacal swabs from chicken were analyzed by rapid antigen detection and RT-PCR using H5- and N1-specific primers, which confirmed the presence of an H5N1 influenza virus in infected chickens. Sequencing results revealed 100% homology of both genes with previously published sequences of H5N1 isolates from Egypt and the Middle East. The virus was isolated and propagated in MDBK cells in culture. Host cells showed a substantial cytopathic effect within 2 days of infection, which increased dramatically by the fourth day. Plaque infectivity titers of virus harvested from cell culture were initially 105 PFUs/ml and increased to 108 PFUs/ml after two additional passages and ultrafiltration. Formaldehyde treatment completely inactivated the virus, and MDBK cells inoculated with the killed virus showed no cytopathic effect. Two days after chickens were immunized with the killed virus, their sera showed that the killed Egyptian isolate was highly immunogenic. Western blot analysis showed that sera had antibodies reacting to four viral peptides: hemagglutinin (61.5 kDa), RNA-binding protein (56 kDa), neuraminidase (50 kDa), and 45-kDa protein. In a challenge infection, the vaccine protected immunized chickens from death and reduced viral shedding.