| Background and Objective:In 1997, an avian influenza epidemic in domestic poultries caused by H5N1 took place in Hong Kong. The mortality in affected chickens approached to 75-100%. It not only affected the industry of poultry breeding, but also caused 18 human cases infected and 6 died. This is the first record of the pure avian influenza viruses infected humans in the history. After the depopulating all the poultries to stop the epidemic in Hong Kong, the H5Nl/97-like viruses disappeared. While the reasserting events recovered after 2001 and many new genotypes of H5N1 were detected. Outbreaks of bird flu caused by H5N1 were reported in 2001 and 2002 in Hong Kong. No more human cases involved until early 2003 that two people were confirmed to have been infected by this lethal pathogen. One of them died. From late 2003 to early 2004, 9 countries and regions in Southeastern Asia were overwhelmed by a highly pathogenic H5N1. Millions of chickens were killed and 25 people infected. Whether the H5N1 would turn to be the next pandemic strain became the hot topic all over the world. The survey of the influenza pandemic showed that the southern China played an important role in the last three global outbreaks. This region had been regarded asthe "influenza epicenter" by many influenza researchers. Considering that most of the poultries sold in Hong Kong market were imported from the adjacent provinces, that is, from the southern China, the study of the H5N1 viruses isolated in this region may help to unveil how the highly pathogenic H5N1 generated and how it would evolve. Ever since 1997, after the Hong Kong government slaughtered all the poultries to stop the epidemic, the H5N1/97 like viruses had never been detected again. 7 years after that accident, what happened to the H5N1 viruses in southern China? What new genotypes generated by gene reassortment between different viruses in their natural reservoir (aquatic birds)? Are these new genotypes the same as the H5N1/97? We tried to get the answers to these questions through our research.Materials and Methods:Fecal, cloacal and tracheal swabs from different types of poultries were collected in live-bird retail markets once a week. They were inoculated into 9-11 days embryonated chicken eggs and incubated in 35 癈 for 72 hours. Hemagglutinin inhibition test (HI test) and Neuraminidase inhibition test (NI test) were performed to detect the subtypes if HA test was positive(AD 16) and contamination test was negative. 27 H5N1 influenza viruses were selected for further antigenic and genetic analysis. Viral RNAs were extracted from virus-infected allantoic fluids using QIAamp Mini-extraction Kits. After reverse transcription, cDNA was amplified using specific primers for each gene segment. PCR products were purified with Qiaquick PCR Purification Kit, followed by sequencing using the Big Dye Terminator Cycle Sequencing Ready Reaction Kits used with AmpliTaq DNA polymerase FS. Samples were electrophoresised and analyzed on a Perkin-Elmer model 377XL DNA sequencer. All sequence data were assembled and edited using Wisconsin Package, version 10.2. Phylogeneticanalyses were carried out using PAUP, version 4.1. Alignment and homology analyses were performed with Mega, version 2.1 and Bioedit version 5.0.9, to quantitate the nucleotide and amino acid substitutions.Results1. Varieties of H subtypes were co-circulating in aquatic birds in South of China, including H5. This region provided a suitable environment for the H5N1 to evolve; the isolation rate of the H5 subtype has been increasing dramatically during the past two years.2. Antigenic characterization showed on the basis of time and genotype, two groups were devided. One group had high liter with the monoclonal antibody, while the other had low. The Z genotype, the present dominated one, had great change in the antigenic characterization when compared with others.3. The phylogenetic analysis showed the HA gene of the H5 viruses circulating in the southern China was d... |
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