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High Genetic Stability of Dengue Virus Propagated in MRC-5 Cells as Compared to the Virus Propagated in Vero Cells

19 Mar 2008

Valero

Citation: Liu C-C, Lee S-C, Butler M, Wu S-C (2008) High Genetic Stability of Dengue Virus Propagated in MRC-5 Cells as Compared to the Virus Propagated in Vero Cells. PLoS ONE 3(3): e1810. doi:10.1371/journal.pone.0001810

This study investigate the replication kinetics of four dengue virus serotypes (DEN-1 to DEN-4) and discusses from these findings the potential value for the large-scale production of live-attenuated DEN vaccines. The clone DNA-derived DEN viruses in Vero (African green monkey kidney) and MRC-5 cells (human fetal lung fibroblast cells. Both cells line are diploids and are currently used to develop human vaccines (1).

The findings indicate some genomic stability of cloned DNA-derived DEN-4 in virus propagated in MRC-5 cells versus to virus propagated in Vero cells. However the sequences of multiple clones of three genomic fragments increased genetic mutations of cloned DEN4 in Vero cells. They were probably caused by passage number or high-density microcarrier culture, rather than the increased virus yield.

This work represents both high priority and relevance for public health, because there is currently no available, safe and effective dengue vaccine for prevention of infection or disease severity. According to World Health Organization statistics, 50–100 million people are infected with the dengue virus and there are over 250,000 cases of dengue haemorrhagic fever (DHF) annually, with fatality rates from 1% to 5%. Most cases are in tropical and subtropical areas. Dengue is endemic in Southeast Asia, Latin America and Caribbean islands and Africa (2).

The high mortality rate and the most recent explosive epidemics affecting people from urban areas in capitals and tourist regions in different countries, requires increased efforts to develop a DEN vaccine that can be delivered effectively to those at risk through the Expanded Programme of Immunization (3,4). The control strategy must consider how the vaccine will be delivered. A single-dose vaccine with long-lasting protection is ideally needed. A cocktail of antigens against the four serotypes may also be a possibility. These features would help to minimize the risk of severe dengue infections and could also reduce the mortality rate. However, as the authors mention, it is important to continue further studies to compare viral yield and genetic stability of DEN viruses between Vero and MRC-5 cell lines.

References

1. World Health Organization (2000). Strengthening implementation of the global strategy for dengue fever/dengue hemorrhagic fever prevention and control: report of the informal consultation; 18-20 October 1999; World Health Organization, , Geneva. Available from: http://whqlibdoc.who.int/hq/2000/WHO_CDS_(DEN)_IC_2000.1.pdf.

2. Aunins JG (2000) Viral vaccine production in cell culture. In: Spier RE, ed. Encyclopedia of Cell Technology John Wiley & Sons 2: 1182–1217.

3. Simasathien S, Thomas SJ, Watanaveeradej V, Nisalak A, Barberousse C, Innis BL, Sun W, Putnak JR, Eckels KH, Hutagalung Y, Gibbons RV, Zhang C, De La Barrera R, Jarman RG, Chawachalasai W, Mammen MP Jr (2008). Safety and immunogenicity of a tetravalent live-attenuated dengue vaccine in flavivirus naive children. Am J Trop Med Hyg. 78(3):426-33.

4. Sim AC, Lin W, Tan GK, Sim MS, Chow VT, Alonso S (2008). Induction of neutralizing antibodies against dengue virus type 2 upon mucosal administration of a ecombinant Lactococcus lactis strain expressing envelope domain III antigen. Vaccine. 26;26(9):1145-54

© 2008 Liu et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Source

Title:
High genetic stability of dengue virus propagated in MRC-5 cells as compared to the virus propagated in vero cells.

Authors:
Liu CC, Lee SC, Butler M, Wu SC

References:
PLoS ONE 2008: Vol. 3 no. 3, pp. e1810

Abstract:
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.

PMID: 18350148
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