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| Adaptability of Dengue-Ⅱ Virus D01090 Strain in KMB17 Cells and Its Preliminary Identification |
| ZHAO Yu-jiao, PAN Yue, YAN Ling-mei, YUE Yao-fei, YANG Li-juan, SUN Qiang-ming |
| Yunan Key Labratory of Vaccine Research Development on Severe Infectious Diseases, Institute of Medical Biology, Chinese Academy of Medical Sciences & Peking Union Medical College, Kunming 650118, China |
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Abstract Objective: To select the adaptive strain of Dengue-Ⅱ virus D01090 strain in KMB17 cells, which lay the foundation of the exploration of dengue vaccine using human cells as host. Methods: After extraction of dengue-Ⅱ D01090 strain genome and identification of the serotype of dengue virus through RT-PCR, the virus was replicated an detected for the titer; The Dengue-Ⅱ D01090 strain was subcultured in KMB17 cells with 4.0 MOI till the virus completely adapted to multiply in cells, then continued subculturing in KMB17 cells for 10 passages. The adapted strain was screened out, Virus culture fluid was purified by sucrose gradient centrifugation and ultracentrifugation, KMB17 cells was ultrathinsected and observed the pathology under transmission electron microscope after infected with virus; Adapted strain was purified through plaque assay, and the antigenicity was detected by IFA. Results: After dengue-Ⅱ virus D01090 strain RNA was extracted as templete, a typical 511bp gene segment of dengue virus and a specific 119bp gene segment of dengue-Ⅱ virus were amplified by RT-PCR. After replication in C6/36 cells, the virus titer could reach 4.5 CCID50/ml, The CPE of KMB17 cells was appeared earlier after continuous subculture, their titer increased with the increasing passages and get the highest 5.0 CCID50/ml on passage 10.KMB17 cells was ultrathinsected and observed the pathology under transmission electron microscope after 6 days infected with virus and CPE get +++, the new packaging virus particles were observed in the endoplasmic reticulum, many small fragment were generated around the cell, with the virus drifting out of the cell. Purified virus strain was screened through three cycles of plaque purification, while antigenicity of purified strain was positive detecting by IFA. Conclusion: Dengue-Ⅱvirus D01090(China) adapted strain was screened out which could stably proliferated in KMB17 cells and keep a high virulence, also maintained good antigenicity through plaque purification.
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Received: 12 July 2012
Published: 25 November 2012
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