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Updated: Jul 16, 2026
Measuring Dengue Virus RNA in the Culture Supernatant of Infected Cells by Real-time Quantitative Polymerase Chain Reaction
Published on: November 1, 2018
Attenuating characteristics of DEN-2 PDK53 in flavivirus-naïve peripheral blood mononuclear cells
Jundee Rabablert1, Chantapong Wasi, Richard Kinney
1Center for Vaccine Development, Institute of Science and Technology, Mahidol University, Nakhon Pathom, and Department of Microbiology, Faculty of Medicine Siriraj Hospital, Bangkok, Thailand.
Abstract:
A live-attenuated DEN-2 virus, DEN-2 strain 16681-PDK53, has been found to be attenuated for both humans and mice with an unknown mechanism. To partially answer this question, responses of flavivirus-naïve primary human PBMC to infection with attenuated DEN-2 PDK53 (D2/IC-VV45R) virus and its parental, virulent DEN-2 16681 virus (D2/IC-30P-A) were investigated at the cellular and genetic levels using cDNA array analysis. Both DEN-2 viruses produced similar replication kinetics in flavivirus-naïve PBMC. In contrast, virulent DEN-2 virus caused a higher percentage of apoptotic death. A macro-array analysis showed that the virulent D2/IC-30P-A virus induced changes in the expression of a greater number of genes than did the attenuated D2/IC-VV45R virus, 31 genes versus 19 genes, respectively, by 24 h post-infection. Interestingly, both viruses stimulated cytokines known to be virulence factors for DEN virus infection, such as IL-1beta, IL-6, IL-8, IL-10, MIP-1beta, and MIP-1alpha. The virulent virus additionally up-regulates immune suppression factors and down-regulates immune activator and growth factors. In conclusion, our data demonstrated that D2-PDK53 effected less change in PBMC than D2-16681 in terms of observable cellular effect and expression of cytokine and chemokine related genes.
Insights
The attenuated Dengue virus type 2 (DEN-2 PDK53) caused fewer changes in human peripheral blood mononuclear cells (PBMC) compared to the virulent DEN-2 16681 strain. This suggests a mechanism for DEN-2 attenuation at the cellular and genetic levels.
Area of Science:
- Virology
- Immunology
- Molecular Biology
Background:
- Dengue virus (DEN) infection poses a significant global health threat.
- The mechanism of attenuation for live-attenuated DEN-2 strain 16681-PDK53 remains unclear.
- Understanding DEN-2 attenuation is crucial for vaccine development.
Purpose of the Study:
- To investigate the cellular and genetic responses of human peripheral blood mononuclear cells (PBMC) to infection with attenuated DEN-2 PDK53 and virulent DEN-2 16681 viruses.
- To elucidate the molecular mechanisms underlying DEN-2 virus attenuation.
Main Methods:
- Primary human PBMC were infected with attenuated DEN-2 PDK53 (D2/IC-VV45R) and virulent DEN-2 16681 (D2/IC-30P-A) viruses.
- Cellular responses, including apoptosis, were assessed.
- Gene expression changes were analyzed using cDNA macro-array analysis.
Main Results:
- Both DEN-2 viruses exhibited similar replication kinetics in PBMC.
- The virulent DEN-2 virus induced a higher rate of apoptotic cell death.
- Virulent DEN-2 altered the expression of 31 genes, while attenuated DEN-2 affected 19 genes within 24 hours.
- Both viruses stimulated pro-inflammatory cytokines, but the virulent strain also upregulated immune suppressors and downregulated immune activators.
Conclusions:
- The attenuated DEN-2 strain (D2-PDK53) induced less cellular and genetic alteration in PBMC compared to the virulent strain (D2-16681).
- Differential gene expression, particularly in cytokine and chemokine pathways, contributes to the attenuation of DEN-2 virus.
- These findings provide insights into the molecular basis of DEN-2 virus attenuation.
