| dengue virus(DEN) is a subgroup of one serotype in flavivirus of flaviviridae, and diffusion through medium of insects such as stegomyia and Aedes albopictus,which caused dengue fever(DF), dengue hemorrhagic fever(DHF) and dengue shock syndrome (DSS). Especially rescent years,because of many factors such as global warming and frequent population mobility, the morbidity of dengue fever increased about30times through worldwide. The quantity of people infected with dengue fever increased constantly in the world in last two years, in the middle and south area of Southeast Asia、America and south pacific region,80million people infected with dengue virus, estimately24thousand people died and3billion people of more than100contries were threatened, so it becomes a world wide public health problem.It still has no dengue vaccine been approved to sell in all the world, so the research of dengue vaccine was urgent. The most potential traditional attenuated live vaccine was the alternative vaccine invented by WRAIR which using embryonic lung diploid cells of rhesus as host.Vero cells was also considered as a host in the research of vaccine, but the alternative vaccine which using human cells as host was unperceived. So it’s very meaningful to dengue vaccine development on research of the adaptability of dengue virus in human cells.Human embryonic lung diploid cells KMB17was from human embryo,which was separated by institute of medical biology and has already been approved to produce hepatitis A vaccine on a large scale. It’s safety and stablity has widely been indentified.In our research,100samples collected from the border region of China and Burma were numbered and record the messages. IgM and IgG were detected by Ig M/Ig G detecting Kit of dengue antibody. Stat and analysis result shows that Ig M/Ig G negtive samples just have5%, Ig M positive/Ig G negtive samples have26%, and Ig G positive samples have69%, After amplification in C6/36cells, all the samples were detected by blood clotting efficacity detection. None of them could agglutinate the fresh erythrocyte of goose. Because Ig M/Ig G positive samples were so much and get95%high, so it’s very difficult to separate dengue virus from the samples。Then Ⅰ～Ⅳ serotype of Dengue virus were abtained through cooperation with Guangdong CDC. After dengue virus replication inC6/36cells,determination of the titer,and amplified the typical gene segment of dengue virus by RT-PCR as MOHC publicized as well, the Ⅰ-Ⅳ serotype of Dengue virus Chinese strain was subcultured in KMB17cells with4.0MOI till the virus completely adapted to multiply in cell, then the culture condition was optimized. Continued subculturing in KMB17cells for10passages. The adapted strain was screened out, and it was purified through plaque purification. Virus titer of10passages was detected by microtitrimetry, the antigenicity was detected by IFA,and the proliferation kinetics was detected by Real Time-PCR. Virus culture fluid was purified by sucrose gradient centrifugation and ultracentrifugation.After that,the virus morphology was observed under electronic speculum. Results showes that after replication in C6/36cells, the RNA of Ⅰ-Ⅳ serotype of dengue virus Chinese strain was extracted as templet, the typical511bp gene segment of dengue virus and specific482bp、119bp、290bp and392bp gene segment of Ⅰ-Ⅳ serotype of dengue virus were amplified by RT-PCR. The CPE of KMB17cells was appeared earlier after continuous subculture,their titer increased with the increasing passages and get the highest on passage10. Purified virus strain was filtered through three cycles of plaque purification.Antigenicity of passage10strain was positive detecting by IFA, while morphology of viru particles were regular observed through electronic speculum after purification by sucrose gradient centrifugation and ultracentrifugation. Real-time PCR show that replication of dengue virus D9964strain in KMB17cells have already begun on the3rd day,then became fastest during the5th-6th day and get to top on the7th day. In the research, dengue adapted strains were screened out that could replication stably in KMB17cells with high titers, which lay the foundation of the exploration of dengue inactivated vaccine and live attenuated vaccine. |
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