title: Persistent, triple-virus co-infections in mosquito cells.
abstract: BACKGROUND
  It is known that insects and crustaceans can carry simultaneous, active infections of two or more viruses without showing signs of disease, but it was not clear whether co-infecting viruses occupied the same cells or different cells in common target tissues. Our previous work showed that successive challenge of mosquito cell cultures followed by serial, split-passage resulted in stabilized cultures with 100% of the cells co-infected with Dengue virus (DEN) and an insect parvovirus (densovirus) (DNV). By addition of Japanese encephalitis virus (JE), we tested our hypothesis that stable, persistent, triple-virus co-infections could be obtained by the same process.
  
  
  RESULTS
  Using immunocytochemistry by confocal microscopy, we found that JE super-challenge of cells dually infected with DEN and DNV resulted in stable cultures without signs of cytopathology, and with 99% of the cells producing antigens of the 3 viruses. Location of antigens for all 3 viruses in the triple co-infections was dominant in the cell nuclei. Except for DNV, this differed from the distribution in cells persistently infected with the individual viruses or co-infected with DNV and DEN. The dependence of viral antigen distribution on single infection or co-infection status suggested that host cells underwent an adaptive process to accommodate 2 or more viruses.
  
  
  CONCLUSIONS
  Individual mosquito cells can accommodate at least 3 viruses simultaneously in an adaptive manner. The phenomenon provides an opportunity for genetic exchange between diverse viruses and it may have important medical and veterinary implications for arboviruses.
authors.affiliation: Department of Biotechnology, Faculty of Science, Mahidol University, Rama 6 Road, Bangkok 10400, Thailand.
authors.forename: Nipaporn
authors.initials: N
authors.lastname: Kanthong
authors.forename: Nuanpan
authors.initials: N
authors.lastname: Khemnu
authors.forename: Sa-Nga
authors.initials: SN
authors.lastname: Pattanakitsakul
authors.forename: Prida
authors.initials: P
authors.lastname: Malasit
authors.forename: Timothy W
authors.initials: TW
authors.lastname: Flegel
mesh_terms: D000818:Animals; D002460:Cell Line; D009033:Culicidae / Q000166:cytology / Q000821:virology*; D003716:Dengue Virus / Q000472:pathogenicity*; D017990:Densovirus / Q000472:pathogenicity*; D004664:Encephalitis Virus, Japanese / Q000472:pathogenicity*; D005434:Flow Cytometry; D007303:Insect Vectors / Q000821:virology; D018613:Microscopy, Confocal; D014777:Virus Diseases / Q000821:virology