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Persistent, triple-virus co-infections in mosquito cells

Nipaporn Kanthong1, Nuanpan Khemnu2, Sa-Nga Pattanakitsakul2, Prida Malasit23 and Timothy W Flegel456*

Author Affiliations

1 Department of Biotechnology, Faculty of Science and Technology, Rajamangala University of Technology Tawan-ok, Sriracha, Chonburi 20110, Thailand

2 Division of Medical Molecular Biology, Office for Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand

3 Medical Biotechnology Unit, National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Science Park Pathumthani, 12120, Thailand

4 Department of Biotechnology, Faculty of Science, Mahidol University, Rama 6 Road, Bangkok 10400, Thailand

5 Centex Shrimp, Faculty of Science, Mahidol University, Rama 6 Road, Bangkok 10400, Thailand

6 National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency, Klong 1, Klong Luang, Pratum Thani 12120, Thailand

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BMC Microbiology 2010, 10:14  doi:10.1186/1471-2180-10-14

Published: 20 January 2010



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.


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.


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.