Characterization, Variation and Wild Bird Surveillance of Avian Influenza Viruses in Taiwan

This study is divided into three parts; the first is monitoring of AIV in migratory birds. The study has been performed in Taiwan since 1998. From 1998 to 2007, 29,287 samples were collected from wild ducks, shorebirds, and other wild birds in the four wetlands around Taiwan and two outside islets, Penghu and Kinmen. Virus isolation was performed for all collected samples by inoculating chicken embryos, and the AIV in the allantoic fluid was identified by reverse transcription polymerase chain reaction and hemagglutination. The pathogenicity of isolated AIV H5 and H7 subtypes was determined by intravenous pathogenicity index (IVPI) test in specific-pathogen-free chickens and hemagglutinin (HA) cleaveage sequencing. The AIV prevalence from those samples was 0.81% (237/29,287). The peak prevalence reached 1.06% (186/17,493) from September to December, in which migrating ducks came from the north; the prevalence during January-April was 0.51; however, no virus was isolated during May-August. The partial HA genes of 28 H4 AIVs were sequenced and analyzed. The phlylogenetic tree showed that a correlation existed between isolation years and evolutional distances. All of the H5 or H7 AIVs isolated were determined to be low pathogenic AIVs by IVPI test and the HA cleavage sequences.The second part is isolation and characterization of H5N2 influenza virus from a chicken in Taiwan in 2008. During the surveillance of avian influenza, an H5N2 influenza A virus was isolated from a cloacal swab sample of apparently healthy chicken in Taiwan in October 2008. It was found that the heamagglutinin (HA) of the virus had a pair of dibasic amino acid residues at the cleavage site, which is a marker of highly pathogenic avian influenza virus. Intravenous pathogenicity index of the isolate was 0.89, indicating that the virus was on the way of acquisition of high pathogenicity in chicken. Virus isolation was negative in 2,916 birds of 146 farms in the circle of 3-km radius around the farm where the virus was isolated. Genetic analysis of the eight segments of the isolate indicate that the isolated virus was a reassortant whose HA and neuraminidase gene segments belonged to the American lineage and the others to Eurasian lineage.The third part is in order to understand the virulence tendency of H5N2 low pathogenic avian influenza virus, the virus strain of v1209 isolated in Taiwan 2003 was used for this study. After serial passages in fourteen-day-old chicken embryonic eggs, the pathogenicity of different passages were compared with the original strain before passage. The plaque formed at the 10th passage but not at 5th passage. The intracerebral pathogenicity index (ICPI) in the 10th passage was 1.64, higher than that of the 5th passage of 0.71. The intravenous pathogenicity index (IVPI) of the 40th passage was 1.45, while the original strain was 0.0. All the tests indicated that the pathogenicity of v1209 strain was higher in high passage than in low passage after serial passages in 14-day-old chicken embryo. However, it is noteworthy that, the number of basic amino acid in haemagglutinin cleavage site in this test was not correlated to pathogenic change. The amino acid sequence of cleavage site was changed from -REKR- in original to -RRKKR-at the 20th passage and the 30th passage, and then changed back to -REKR- at the 40th and the 50th passages. It indicates that the number of basic amino acid in haemagglutinin cleavage site is not related completely with pathogenicity.