Dengue Virus Research: Impact of Genetic Mutations on Virus Transmission and Discoveries

Introduction

This study was conducted by Allyson N. X. Choi et al. and published in Science Translational Medicine on July 31, 2024. The research aims to reveal the impact of genetic changes in dengue virus on its transmission ability and disease outbreaks during the dengue fever epidemic in the South Pacific in the 1970s. It is currently known that different dengue virus (DENV) genotypes affect its transmission potential in human populations, but the specific mechanisms are not yet clear.

Research Background and Objectives

Dengue virus is divided into four serotypes (DENV-1 to DENV-4), causing frequent and explosive epidemics in tropical and some subtropical regions. These epidemics are usually attributed to low immunity in populations, but viral genetic differences also play an important role. Like other viruses, dengue virus evolves during transmission to adapt to hosts, thereby enhancing its transmission potential. However, like SARS-CoV-2 and its variants, dengue virus is also constantly evolving, adapting to epidemiology, and gradually forming more adaptable variants.

The dengue virus type 2 (DENV-2) epidemic in the South Pacific in the 1970s provided a unique opportunity to understand the impact of viral genetics on epidemiological outcomes. Except in Tonga, the virus caused severe epidemics on other South Pacific islands. The study found that genetic changes in the DENV-2 virus strain in Tonga, especially an amino acid mutation at position 86 of the premembrane protein (prM), may explain the attenuated nature of this strain and mild cases.

Research Methods and Process

This study was jointly conducted by several scientific teams from Duke-NUS Medical School and published in Science Translational Medicine.

1. Genomics and Molecular Biology

The research team first conducted a whole-genome phylogenetic analysis of DENV-2 virus strains isolated from different South Pacific islands, finding that the Tongan virus strain was phylogenetically separated from other island strains. Using reverse genetics methods, infectious clones of DENV-2 were constructed to further explore the effects of specific genetic mutations on viral phenotypes.

2. Cell Culture and Viral Phenotype Analysis

South Pacific wild-type DENV-2 infectious clones were constructed using the Gibson assembly method. Plaque assays and human hepatocellular carcinoma cells (Huh-7 cells) and African green monkey kidney epithelial cells (Vero cells) were used to determine viral plaque size, replication rate, and the virus’s effect on interferon (IFN) response.

3. In Vivo and In Vitro Experiments

To verify the impact of genetic mutations on viral phenotypes, the research team injected virus strains with different genetic mutations into AG129 mice (mice lacking type I and II interferon receptors) and Aedes aegypti mosquitoes to determine the replication efficiency and transmission ability of the virus in different hosts.

4. Molecular Modeling and Bioinformatics Analysis

Western blot and immunofluorescence methods were used to analyze the effect of mutations on viral protein expression. Additionally, the team conducted protein molecular modeling and charge distribution analysis to explore the potential impact of mutations on viral protein structure and function.

Research Results

1. Viral Genetics and Phylogeny

Through whole-genome phylogenetic analysis, the research team identified three specific mutations: homologous isotype I78M at position 78 of non-structural protein 4a (NS4a), histidine-arginine (H86R) at position 86 of premembrane protein (prM), and serine-glycine (S115G) at position 115 of non-structural protein 2a (NS2a). Among these, only the prM H86R mutation was associated with virus attenuation.

2. Viral Replication and Protein Translation

In Huh-7 cells, the Tongan DENV-2 virus strain showed lower replication rates and lower expression levels of infection-related proteins (such as NS3, E, and prM). These phenotypes were also significant in virus strains with specific mutations. When the prM H86R mutation was restored through reverse genetics, the viral plaque area increased, and the replication rate improved. This indicates that the mutation reduced the translation efficiency of the virus in mammalian cells.

3. Viral Infection Advantage

In Aedes aegypti, the prM H86R mutation significantly improved the infection efficiency of the virus in the mosquito’s intestine. This mutation may promote virus transmission in the alkaline environment of the mosquito gut (pH 8.5 to 9.5) by increasing the binding efficiency of the virus to mosquito cell membrane glycans.

4. Recombination and Detection

After transfecting cells with mutant viruses, whole-genome sequencing revealed that gene replacement using DENV-4 genes was not stable. This suggests that the impact of this mutation may differ in different genetic backgrounds, further confirming the complexity of gene interactions (repulsion/synergy) between different virus strains.

Research Conclusions and Significance

Through this study, scientists discovered the H86R mutation at position 86 of the prM protein, which leads to reduced translation efficiency of the virus in mammalian cells but enhances viral transmission ability in Aedes aegypti. This finding suggests that dengue virus may exhibit different adaptation mechanisms and molecular strategies in different hosts, thereby maximizing its transmission efficiency while reducing pathological effects on the host.

The research not only provides new insights into the epidemiology and viral evolution of dengue virus but also has important implications for vaccine design and infectious disease prevention and control. In the future, in-depth research on how viral genes affect cross-host transmission and clinical manifestations will better help scientists design effective prevention and control strategies.

Research Highlights

1. Identification of a Single Gene Mutation: Through genome sequencing and reverse genetics methods, the research team successfully identified the correspondence between the prM H86R mutation and viral phenotypes.
2. Cross-Host Study: Revealed different phenotypes exhibited by the virus in different hosts, providing new ideas for further understanding viral adaptive evolution.
3. Molecular Mechanism Analysis: Through Western blot and molecular modeling, the impact of the prM H86R mutation on viral protein expression and structure was revealed, laying the foundation for understanding virus-host interaction mechanisms.

This study provides new insights into the evolution and transmission mechanisms of dengue virus and provides scientific basis for future prevention and control of dengue fever epidemics. The research demonstrates the organic combination of molecular biology, phylogenetics, and cross-host transmission studies, showcasing the broad prospects of modern virology research.