High genetic stability of dengue virus propagated in MRC-5 cells as compared to the virus propagated in vero cells.
This work investigated the replication kinetics of the four dengue virus serotypes (DEN-1 to DEN-4), including dengue virus type 4 (DEN-4) recovered from an infectious cDNA clone, in Vero cells and in MRC-5 cells grown on Cytodex 1 microcarriers. DEN-1 strain Hawaii, DEN-2 strain NGC, DEN-3 strain H-87, and DEN-4 strain H-241 , and DEN-4 strain 814669 derived from cloned DNA, were used to infect Vero cells and MRC-5 cells grown in serum-free or serum-containing microcarrier cultures. Serum-free and serum-containing cultures were found to yield comparable titers of these viruses. The cloned DNA-derived DEN-4 started genetically more homogeneous was used to investigate the genetic stability of the virus propagated in Vero cells and MRC-5 cells. Sequence analysis revealed that the DEN-4 propagated in MRC-5 cells maintained a high genetic stability, compared to the virus propagated in Vero cells. Amino acid substitutions of Gly(104)Cys and Phe(108)Ile were detected at 70%, 60%, respectively, in the envelope (E) protein of DEN-4 propagated in Vero cells, whereas a single mutation of Glu(345)Lys was detected at 50% in E of the virus propagated in MRC-5 cells. Sequencing of multiple clones of three separate DNA fragments spanning 40% of the genome also indicated that DEN-4 propagated in Vero cells contained a higher number of mutations than the virus growing in MRC-5 cells. Although Vero cells yielded a peak virus titer approximately 1 to 17 folds higher than MRC-5 cells, cloned DEN-4 from MRC-5 cells maintained a greater stability than the virus from Vero cells. Serum-free microcarrier cultures of MRC-5 cells offer a potentially valuable system for the large-scale production of live-attenuated DEN vaccines.
Liu
Chia-Chyi
CC
Institute of Biotechnology, Department of Life Science, National Tsing Hua University, Hsinchu, Taiwan.
Lee
Shiang-Chi
SC
Butler
Michael
M
Wu
Suh-Chin
SC
D019943:Amino Acid Substitution; D000818:Animals; D001769:Blood; D002460:Cell Line; D002522:Chlorocebus aethiops; D006224:Cricetinae; D016895:Culture Media, Serum-Free; D003716:Dengue Virus / Q000235:genetics* / Q000254:growth & development / Q000502:physiology; D009154:Mutation; D013045:Species Specificity
D016895:Culture Media, Serum-Free