Highly pathogenic avian influenza A (H5N1) virus

Lung samples were collected from dead birds (chickens, ducks, pigeons, and partridges) from farms in 3 affected regions (Greater Accra, Volta and Ashanti regions) in Ghana. Samples were frozen at 80°C in virus transport medium containing 2.5% veal infusion broth (Becton Dickinson, Franklin Lakes, NJ, USA), 0.5% bovine serum albumin (Sigma, St. Louis, MO, USA), 100mg/mL gentamicin (Gibco, Fisher Scientific, Pittsburgh, PA, USA), and 2 mg/mL fungizone (Hyclone Laboratory Inc., South Logan, UT, USA) and were shipped to the Heinrich Pette Institute, Leibniz Institute for Experimental Virology in Hamburg, Germany. Three lung tissue samples from chickens (layers >21 weeks of age) were randomly selected in each of the 3 affected regions and homogenized in phosphate-buffered saline. Virus-containing supernatants were used to inoculate 11-day-old embryonated specific-pathogen–free chicken embryos that were then incubated at 37°C for 48 hours. Infected chicken embryos were incubated at 4°C overnight and harvested the next day (1). Embryos were not alive at this point. Allantoic fluids were tested by using a standard hemagglutination assay, as previously described (2). Viral RNA isolated from positive allantoic fluids were subjected to Sanger sequencing (Seqlab Laboratories, Göttingen, Germany). Sequences were obtained for the hemagglutinin, basic polymerase protein 2, nucleoprotein, and neuraminidase genes. Sequences were assembled and analyzed by using Clone Manager 9 Professional Edition (Scientific and Educational Software, Denver, CO, USA). Phylogenetic analyses were performed by using sequences downloaded from the Global Initiative on Sharing All Influenza Data (http://platform.gisaid.org) and GenBank databases.