title: Characterization and E protein expression of mutant strains during persistent infection of KN73 cells with Japanese encephalitis virus.
abstract: OBJECTIVE
  To study the character of mutants originating from Japanese encephalitis viruses and the relationship between the characterization of mutant strains and E protein expression.
  
  
  METHODS
  Persistent infection was established with standard strains of Japanese encephalitis viruse, known as parental viruse, in a human hepatoma cell line, KN73. Cells were subcultured weekly using trypsinization techniques. Cell-associated viruses of persistently infected cells were collected by a freeze and thaw method. Virus titers were examined by plaque method using baby hamster kidney (BHK) cells. Indirect immunofluorescence assays were used to examine E and NS3 protein antigens. Western blot analysis was used to test expression of E and NS3 proteins.
  
  
  RESULTS
  In the early phase (24 - 36 h) post-infection, virus titer in culture fluid from KN73 cells infected with parental viruses were 10(6) PFU/ml. They were 10(3 - 4) PFU/ml in the late phase (3 years) post-infection. The titer of cell-associated viruse was 10(2 - 3) PFU/ml. A virus super-infection assay found that virus titers in culture fluid from persistently infected KN73 cells acutely super- infected with parental viruses were much lower than that of culture fluids in acutely infected normal KN73 at the same phase. Indirect immunoflurescence assay revealed that the quantity of viral antigens in persistently infected KN73 cells was lower than that in acutely infected KN73 cells with parental viruses. Western blot analyses indicated that the molecular weights of E and NS3 proteins were 53 kD and 73 kD, respectively. Expression of NS3 protein in persistently infected KN73 cells was stable but expression of E protein was markedly suppressed.
  
  
  CONCLUSIONS
  The virulence and reproduction of viruses obtained from persistently infected KN73 cells, which have some features of DI viruses and were involved in persistent infection, was lower than that of parental viruses. These mutants may have be related to the decrease in E protein expression.
authors.affiliation: Department of Infectious Diseases, Second Clinical College of China Medical University, Shenyang 110004, China.
authors.forename: Guohe
authors.initials: G
authors.lastname: Feng
authors.forename: Tsutomu
authors.initials: T
authors.lastname: Takegami
authors.forename: Guizhen
authors.initials: G
authors.lastname: Zhao
mesh_terms: D006528:Carcinoma, Hepatocellular / Q000821:virology; D003673:Defective Viruses / Q000502:physiology; D004664:Encephalitis Virus, Japanese / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D006801:Humans; D008562:Membrane Glycoproteins / Q000032:analysis*; D009154:Mutation; D020365:RNA Helicases; D012697:Serine Endopeptidases; D014407:Tumor Cells, Cultured; D014759:Viral Envelope Proteins / Q000032:analysis*; D017361:Viral Nonstructural Proteins / Q000032:analysis; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
chemical_list: D008562:Membrane Glycoproteins; D020365:RNA Helicases; D012697:Serine Endopeptidases; D014759:Viral Envelope Proteins; D017361:Viral Nonstructural Proteins; C060028:NS3 protein, flavivirus; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; C080628:glycoprotein E, Japanese encephalitis virus