The Differential Expression Of The Human Lung Carcinoma Cells (A549) Infected With Highly Pathogenic Avian Influenza Virus A/Anhui/1/2005 (H5N1)

Influenza A virus are enveloped RNA viruses with an eight-segmented, negative-strand RNA belonging to Orthomyxoviridae. According to their antigenicity of Hemagglutinin and Neuraminidase on the surface of the virus, there are 16H and 9N subtypes of influenza A virus. Every subtype can be epidemic in birds and H1,H3 are major subtypes in humans. The segmented genome determines "antigen shift" and "antigen drift" in influenza virus, making new pandemic strains emerging. The first human infection of H5N1 avian influenza virus was reported in Hongkong in 1997. This H5N1 virus A/Hongkong/156/97 was in fact a reassortant virus with the HA(H5) derived from A Goose/Guangdong/1/96 and the other genes being derived from H9N2 and H6N1 viruses prevalent in quail. Since then, there were increasing cases of human infection with H5N1 avian influenza virus and most of the cases were associated with direct handling of infected poultry. As highly pathogenic avian influenza virus mutated continually, with broader hosts and higher pathogenecity, the risk of pandemic influenza makes the study of the mechanism of the disease urgently for most researchers devoting to influenza research. Based on the first isolate of human H5N1 influenza virus strain A/Anhui/1/2005, we try to study the differential expression of the cells and apoptosis effect to the cells infected with H5N1 virus, and then to provide significant information for the clarification of pathogenesis mechanism of the disease.The main contents of this study include three parts: First, to detect the gene expression of human lung carcinoma cells (A549) with chips after infected by the highly pathogenic avian influenza virus H5N1 at 0h,4h and 24h. Second, to obtain the genes expression profiles of cells infected with human H5N1 virus and select the candidate genes possibly associated with the disease after analyzing the chip data. Third, to confirm the different expression of the candidate genes by quantitative Real-time PCR.1. Detecting the gene expression of A549 cells infected with H5N1 virus by human genome microarray.The cells were harvested and lysed at 0h,4h and 24h after infected with human H5N1 virus. The total RNA was extracted and the first-strand cDNA was synthesized, then the second-strand cDNA was synthesized and transcribed to cRNA. cRNA was then fragmented and biotin-labeled. The fluorescence signal was harvested after the hybridization.2. Analyzing the data of the fluorescence signal and selecting the candidate genes possibly associated with disease.The data of fluorescence signal was normalized by the software. The folds change of every human gene at different time-point of infection was calculated in EXCEL and analyzed by ANOVA. According the folds changes, the human genes were divided into DataSetI～IV.The 238 genes in DataSet I were treated by K-mean clustering and Gene Ontology analysis. The gene BIRC3 was selected as the candidate gene and testified by the Real-time PCR. We found that the expression of the BIRC3 in the cells infected with H5N1 was weaker than that in the cells infected with H1N1.The genes in DataSet III～IV were taken the Gene Ontology analysis and selected with the folds change. We found some genes in the cellular apoptosis pathway and the mTOR pathway had the different expression. 16 genes became the candidates, which join in the two cellular pathways and associate with cellular immunity.3. Confirming the differential expression of the candidate genes by quantitative Real-time PCRAccording the sequences provided from PubMed, primers were designed by the Vector NTI. The target size was between 70～300bp. The different expression of 16 candidate genes in the cells infected with H5N1 virus was confirmed by the quantitative Real-time PCR kit. The expression of 6 genes increased and 6 genes decreased in cells infected with H5N1 virus at time-point 4h comparing with that in the control cells, and 12 genes increased and 1 gene decreased comparing with that in the cells infected with H1N1.In conclusion, the differential expression profiles of genes was obtained in the cells infected with H5N1, of which 16 candidate genes were identified in the cellular apoptosis pathway, mTOR pathway, and the cellular immunity as well. Our results suggest that H5N1 exerts a stronger impact on eliciting apoptosis of infected cells than the common influenza virus H1N1, which is consistent with the findings in clinical studying. This study provides a theoretical basis to clarify the pathogenesis of highly pathogenic avian influenza virus.