Interference Between Dengue Virus Types In Cultured Mosquito Cells Demonstrated Using Monoclonal Antibody Probes

Date of Award




Degree Name

Doctor of Philosophy (Ph.D.)


Microbiology and Immunology


Coinfection of cultured mosquito cells (Aedes albopictus, clone C6/36) with two dengue virus types was investigated using anti-dengue virus monoclonal antibodies to detect cell-associated viral antigens. These antibodies were obtained from hybridomas, which were prepared by fusion of a cultured line of mouse plasmacytoma cells with splenocytes from mice immunized with dengue type-1 or type-3 viral antigen. The majority of hybridomas isolated secreted antibody against antigenic determinants found on all four dengue virus types. Several hybridomas (9 total) were also found that secreted antibody directed against dengue virus type-specific antigenic determinants. When these antibodies were used with an indirect immunofluorescent staining technique to stain mosquito cell cultures coinfected with two dengue virus types, individual cells containing each of the dengue virus type-specific antigens could be identified.The mosquito cell cultures were used because infection with a single dengue virus type was demonstrated to have an outcome analogous to that in live mosquitoes. In the mosquito, it has previously been established that dengue virus infection is life-long, and without detectable effect upon the insects health. In the present investigation, dengue infection of cultured mosquito cells did not damage individual cells, and resulted in the establishment of persistently infected cell lines.Mosquito cell cultures that were persistently infected with dengue-1 virus were found to be resistant to superinfection with dengue-3 virus. This was demonstrated when the dengue-1 infected cultures, which were stained with dengue-3 type-specific monoclonal antibody 3 days after superinfection with dengue-3 virus(MOI=2.0), had dengue-3 antigen in 0.1-1.0% of the cells. Control cultures infected with dengue-3 virus at the same multiplicity contained dengue-3 antigen in greater than 90% of the cells. The resistance to superinfection was not interferon-mediated, and occured within 20 hours after primary dengue virus infection. In cultures simultaneously infected with two dengue virus types, one virus type was excluded from replication in most cells. A small population of cells was also found (about 1%) that contained type-specific antigen of both virus types.These results demonstrated a form of interference between dengue virus types in cultured mosquito cells(heterotypic interference) that has not previously been described. This interference is unique because it is rapidly induced, and can be demonstrated without the use of virus strains that have been repeatedly passed in cultured mosquito cells. Studies on the mechanism of dengue virus heterotypic interference indicated that it has a viral directed, intracellular mechanism.


Biology, Microbiology

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