Spread by virus-carrying Aedes mosquitoes, dengue induces a flu-like fever and body aches, and sometimes leads to dengue hemorrhagic fever, which is potentially fatal. The disease is limited to tropical and sub-tropical regions, but is increasingly becoming a larger threat on the back of climate change, urbanization and the increased mobility of people across national borders.
Dengue is endemic in over 100 countries with some 50 million infections every year, according to the World Health Organization. About 500,000 of those affected require hospitalization each year, and about 2.5% of those patients die. Dengue hemorrhagic fever is a leading cause of death among children in some Asian countries, but recent reports from Singapore also warn that the number of adults being affected is rising.
Yet despite the growing concern about dengue fever, there are still no antiviral vaccines or medicines specific for the disease. Reducing exposure to mosquitoes can help, but even in Singapore, which is renowned for its success in minimizing mosquito numbers, there has been a major resurgence of dengue fever in recent years, with thousands of infections reported annually.
Scientists at A*STAR in Singapore are now accelerating their efforts to contain the ever-growing threat of dengue. “What we really need is a treatment or vaccine. Only then will we be able to tackle this disease effectively,” says Martin Lloyd Hibberd, senior group leader and associate director for infectious diseases at the A*STAR Genome Institute of Singapore (GIS) (Fig. 1).
Singapore is at the forefront of vector control for a range of tropical diseases, and A*STAR scientists such as Hibberd contribute significantly to therapy development for dengue fever by unveiling new insights into the disease’s viral mechanisms. At the same time, other scientists are engaged in building novel systems for the rapid and easy diagnosis of dengue. Singapore itself acts as a hub for accumulating resources and scientific and clinical findings through the Singapore Dengue Consortium, which consists of 11 Singapore-based institutions including the GIS, the A*STAR Singapore Immunology Network (SIgN) and the Novartis Institute for Tropical Diseases.
There are four serotypes of the Flavivirus virus responsible for dengue fever, and infection with one of the serotypes may increase the risk for severe disease following infection with the other forms (Fig. 2).
Antibodies for the original infection actually help the entry of other strains into host cells, meaning that patients could be at real risk of enhanced infection and an escalation of symptoms. This ‘antibody-dependent enhancement’ can lead to dengue hemorrhagic fever and dengue shock syndrome.
However, severe forms of dengue can also occur after primary infection, and conversely, only a small fraction of patients with pre-existing antibodies develop severe disease. The real problem clinicians face is that it has so far been impossible to determine which patients will develop more severe symptoms.
In addition to the lack of markers to identify severity, there are also many obstacles surrounding vaccine development. All of the present vaccine candidates aim to produce immunity against all four serotypes, but the virus mutates rapidly, and even one amino-acid change in a protein could render the vaccine ineffective.
“We often underestimate the mutation rate of dengue,” says Katja Fink, a principal investigator at the SIgN. Furthermore, it is difficult to test the vaccine in vitro and in mice because the virus is human-specific and there are no suitable commercial assays.
“Much has been accomplished over the past few years, but dengue results from a complex interplay between virus, humans and mosquitoes that is really challenging to understand and many problems remain unsolved,” says Hibberd.
Hibberd’s group is investigating viral and human sequences and host pathogen interactions in an effort to develop a specific prognosis and treatment. The team’s work became the backbone of the consortium’s study, called Early DENgue (EDEN), which has led to the discovery of several new findings about the disease. The researchers revealed that outbreaks of disease are not due to viral evolution, and also that changes in viral type may signal an imminent outbreak.
On the clinical side, the study has involved the first major investigation of adult dengue disease. Singapore was the first Asian country in which the disease shifted demographically from children to include adults. “The study has also suggested that the early host response may be a marker for the later disease, opening the possibility of prognostic markers for early and better treatment of the disease,” Hibberd says.
Hibberd’s group is currently investigating how the type of the virus determines the clinical outcome of patients. The researchers also plan to analyze the data from dengue patients in Vietnam by taking advantage of their pioneering genome-wide association study for Kawasaki disease, a pediatric infectious disease that damages the coronary arteries. “We hope the dengue analysis will have equally revealing results,” says Hibberd.
At the SIgN, Fink has found that dengue patients have stronger inflammation compared with patients suffering from other fever-causing diseases.
The inflammation stimulates the activation of immune cells by polyclonal activation, resulting in the production of a pool of cross-reactive antibodies and T cells. The antibodies are cross-protective against all four serotypes for the first few months following natural infection with the dengue virus, but become serotype-specific thereafter.
“We could learn from this natural strategy of the immune system to create many different, yet not too specific antibodies and T cells when developing a vaccine,” Fink says (Fig. 3).
For vaccination, it will also be important to understand both the positive and negative impacts of pre-existing dengue-specific antibodies and T cells. Almost all adults in dengue-endemic countries like Thailand, Vietnam or Cambodia have been infected at least once in their lifetime and have the corresponding immune memory.
Fink says that her team aims to characterize and understand the molecular and cellular requirements for polyclonal activation. Understanding the inflammatory response mechanism is absolutely critical, she adds, because “Nobody wants a vaccine that creates dengue disease,” she says.
“We’re analyzing patient samples at different time points after infection to identify dengue-specific immunological patterns. We hope that this knowledge will help in the development of safe and effective vaccines.”
All-in-one diagnostic system
At the same time as genetic and molecular researchers are elucidating the viral mechanisms of dengue, researchers at the A*STAR Institute of Microelectronics have been developing a compact, all-in-one diagnostic system for the disease.
“A lot of patients with fever go to hospital just after symptoms become apparent. We want to differentiate dengue patients from others as quickly and simply as possible,” says Yu Chen, a principal investigator involved in the Point-of-Care Diagnostic Section of the Bio-Electronics Program. The system was developed in collaboration with Ooi Eng Eong, associate professor of Duke-NUS Graduate Medical School in Singapore.
In March, Chen and her colleagues achieved the integration of the various parts and obtained full system testing results. They are now working on optimizing the overall system, with the aim of developing a prototype within the next year. The core technologies are a dedicated chip to extract and amplify samples and a proprietary silicon-based nano-wire biosensor module, both developed by the Institute of Microelectronics (Fig. 4).
With a tiny drop of blood, just 80 microliters, the system performs the entire diagnosis process, from sample extraction and RNA amplification to virus detection and identification of its serotype—all in less than four hours. Apart from significantly reducing the time required for diagnosis (currently 24 hours), the device could also ease reliance on skilled laboratory technicians. Chen and her team are now trying to halve the time for diagnosis to just two hours.
“For the next-generation version, we aim to develop a highly effective miniature system. We would also like to make it mobile for field diagnosis,” Chen says.