A biomarker that detects dengue hemorrhagic fever is discovered

Elucidating the mechanism by which dengue virus infection develops into its hemorrhagic form could lead to new treatments and vaccines. The research was done in Brazil with support from FAPESP.

By Maria Fernanda Ziegler  |  Agência FAPESP – Patients infected by dengue virus will soon be able to take a new blood test capable of predicting whether the disease will progress to severe dengue or dengue hemorrhagic fever, a more serious and potentially more fatal condition. In addition to the classical symptoms, which include high temperature and myalgia (muscle pain), severe dengue involves bleeding from the skin and other organs.

A study performed in São Paulo State, Brazil, with FAPESP’s support by researchers at the University of Campinas’s School of Pharmaceutical Sciences (FCF) and Biology Institute (IB-UNICAMP), with colleagues at São José do Rio Preto Medical School (FAMERP), identified lipids that serve as biological markers of hemorrhagic dengue. The findings were based on mass spectrometry screening of more than 4,000 molecules in human plasma.

In particular, the researchers elucidated the key role played by phosphatidylcholine, the most common phospholipid in human tissue, in disturbing the balance of the coagulation cascade. This process stops bleeding by combining platelets and fibrinogen, among other molecules, to form blood clots, and an imbalance in the cascade may result in hemorrhagic dengue.

In an article published in Scientific Reports, the group described the evolution of the disease in blood samples from 20 patients treated for dengue hemorrhagic fever at FAMERP’s teaching hospital. The control group comprised ten healthy adults with no signs of infection by dengue virus.

“We succeeded in verifying for the first time that dengue virus assists phosphorylation [addition of phosphate groups to proteins], leading to increased levels of phosphatidylcholine in the blood and creating a natural imbalance in the coagulation pathway. This lipid impairs coagulation and therefore contributes to the development of hemorrhagic fever,” said Rodrigo Ramos Catharino, a professor at FCF-UNICAMP and a co-author of the article.

The study resulted from the PhD research of Carlos Fernando Odir Rodrigues Melo, conducted with a scholarship from FAPESP and supervised by Catharino.

Coagulation cascade imbalance can be caused by other factors, such as hemophilia. “But we don’t know whether the intermediate molecules in these other processes are the same as in hemorrhagic dengue,” Catharino said. “They may be different molecules from the lipids identified in our study, which are specific markers produced by dengue virus.”

The researchers’ analysis of the blood taken from these patients showed how dengue hemorrhagic fever develops and enabled them to understand the mechanism of the disease. According to the study, alterations in lipids in the cells infected by the virus become evident when the virus takes over control of cellular metabolism, modulating the process of autophagy [degradation and recycling of cellular components] to meet the needs of viral replication.

The scientists also observed the action of intermediate molecules, such as diacylglycerol (DAG) and inositol triphosphate (IP₃). They hypothesized that progression to hemorrhagic fever was mainly due to the virus’s contribution to an imbalance in the coagulation cascade via phosphorylation, especially in kinases—proteins that play a major role in cellular activation by facilitating phosphate transfer to other molecules.

“We were able to observe the mechanism by which the disease developed in the patients’ blood and identify markers of this progression precisely at the time when there was a drop in platelet levels,” Catharino explained. “The rise in phosphatidylcholine, therefore, is an indicator that hemorrhagic fever will occur. Coagulation cascade balance is altered by the virus when it acts on several intermediate molecules or lipids to prevent coagulation.”

How dengue fever progresses

In addition to identifying biomarkers for hemorrhagic dengue as the basis for a blood test to diagnose the disease, the study also enables scientists to more deeply understand the mechanisms by which the virus acts to produce severe dengue.

Although the structure and composition of dengue virus and its four serotypes have been widely studied, the infection phenotype and the role of small molecules such as lipids have not yet been clearly established.

Dengue is an acute sudden-onset viral disease that produces fever, headache, and muscle and joint pain, among other symptoms. According to the World Health Organization (WHO), recent estimates indicate 390 million dengue infections per year on a global scale. A small proportion of cases develop into severe dengue or dengue hemorrhagic fever, a far more lethal complication. These patients are usually children or adults infected for a second time by a different serotype, although the reason is unknown.

“We know that the sooner hemorrhagic dengue is identified the greater the patient’s chances of survival,” Catharino said. “However, because it isn’t yet possible to know when the disease will progress to severe dengue, this often happens after patients are sent home.”

According to the researchers, their description of the mechanisms that underlie the progression to severe dengue could contribute to the development of new treatments and vaccines.

“Our main goals are to develop tests that predict the occurrence of severe dengue and to understand the process of the disease more profoundly,” he added. “Does it take three, four or more days to manifest itself? We don’t know even this for sure. Another interesting line of research entails correlating the mechanism we discovered with vaccines. Today, one of the difficulties of immunization is precisely hemorrhagic dengue.”

According to what is known from the scientific literature, the outcome of infection by dengue virus depends on many factors linked to the onset of the infection, such as viral load, whether non-neutralizing antibodies are present, cell recruitment, and production of immune mediators.

“These factors determine whether the environment is favorable or unfavorable to progression of the disease, controlling the viral infection or impairing the inflammatory reaction associated with vascular permeability. Nevertheless, the lack of reliable immunological and other metabolic markers for either protective or pathological responses is still an important gap that hinders the development of new diagnostic or prognostic tests and vaccine candidates,” the authors of the article conclude.

The article “The role of lipids in the inception, maintenance and complications of dengue virus infection” (doi: 10.1038/s41598-018-30385-x) by Carlos Fernando Odir Rodrigues Melo, Jeany Delafiori, Mohamad Ziad Dabaja, Diogo Noin de Oliveira, Tatiane Melina Guerreiro, Tatiana Elias Colombo, Maurício Lacerda Nogueira, Jose Luiz Proenca-Modena and Rodrigo Ramos Catharino can be read at nature.com/articles/s41598-018-30385-x.