.丙型肝炎病毒的发现.ppt

丙型肝炎病毒的发现 丙型肝炎病毒的发现 1.传染性肝炎(HA) 血清型肝炎(HB) 2.动物模型（黑猩猩） 3.基因文库 4.放射免疫标记 5.全基因测序 丙型肝炎病毒的发现 1970’s: Hepatitis A and B diagnostics Most transfusion associated hepatitis not caused by HAV and HBV or other known viral agents (NEJM 292: 767; Gastro. 69:1278; JID 139:511; Vox Sang. 44:231) non-A, non-B hepatitis (NANBH) Might be a small, enveloped virus transmissible to chimpanzees (Gastro 88:773; JID 156:636) Standard immunological methods failed to identify viral antibodies or antigens Chiron group Thought that there was not enough viral antigen in NANBH So they decided to try and concentrate the virus High titer NANBH chimpanzee plasma Ultracentrifugation at 104,000g for 5 hours Extract nuclei acid, random primed cDNA into ?gt11 phage Immunoscreened with NANBH patient serum Clone 5-1-1 Larger overlapping Clone 81 丙型肝炎病毒的发现 ? Phage with portions of HCV genome Infect E.coli Phage kills E.coli and leaves plaques Screen with HCV Patient serum Library Screening 丙型肝炎病毒的发现 Western blot Clone 5-1-1 expressed as SOD fusion protein called PS5 PS5 SOD NANBH patient serum Experimentally infected chimp serum Protein from clone 5-1-1 closely associated with NANBH infections HCV was the first virus to be isolated and characterized solely by molecular methods, ie without culture methods 黑猩猩易感 HCV病原学 黄病毒科（Flaviviridae）丙型肝炎病毒属 包膜 糖蛋白 包膜 +ssRNA 核衣壳 Genomic RNA is infectious HCV Life Cycle Typical virus life cycle: Binding, internalization, uncoating, protein expression/replication, assembly, release HCV Replication----- 病毒的入侵需要细胞膜表面的受体 HCV Replication Assembly and Release Capsid protein interacts with 5’ NCR Homotypic capsid protein interaction E1 and E2 on ER membrane: Nucleocapsid precursors bud into ER to aquire envelope Pass through host secretory pathway, released Nature Review Microbiology -HCV core protein drives assembly at the lipid droplet （LD） -LD is bound by core, then NS5A and other NS protein