The fusogenic capacity in AP-61 cell monolayers of 10 strains of Japanese encephalitis (JE) virus from different geographic locations was compared. One strain, isolated from Beijing (JE-Bei), did not fuse AP-61 cells after replication (fusion from within; FFWI), whereas all other strains fused these cells by 72 h post-infection. JE-Bei also readily established a non-cytolytic persistent infection in AP-61 cells. Differences in the envelope proteins of fusogenic and non-fusogenic virus were detected by haemagglutination-inhibition tests and by antigenic analysis using monoclonal antibodies. Yields of infectious virus in either AP-61 or Vero cell cultures were similar if JE-Bei was compared with the fusogenic strain (JE-Sar) but yields of haemagglutinin were 50-100 fold higher with the non-fusogenic virus, implying excessive generation of non-infectious particles. When added directly to AP-61 cell monolayers at pH6, only JE-Bei produced significant fusion from without (FFWO) presumably reflecting the larger quantity of antigen. Cell monolayers persistently infected with JE-Bei or monolayers treated with UV-inactivated JE-Bei, were resistant to superinfection with JE, West Nile and dengue 2 viruses but were susceptible to infection with the alphavirus Sindbis. When administered intracerebrally (I/C) to newborn and weanling mice, the viruses were equally neurovirulent. However, fusogenic JE-Sar was significantly more neurovirulent than JE-Bei for weanling mice after intraperitoneal (I/P) or subcutaneous (S/C) inoculation. Mice given non-fusogenic JE-Bei, resisted the peritoneal challenge with fusogenic JE-Sar, and West Nile but not Semliki Forest virus when given 6 h after the first virus. The potential significance of cell fusion by JE virus and interference through over production of non-infectious virus, is discussed in the context of JE virus virulence.