New test identifies prior infection by zika virus and all four dengue virus serotypes

Brazilian researchers have developed a simple test that almost instantly tells whether a person has been exposed to any of the four dengue virus serotypes, as well as zika virus. The test is an enzyme immunoassay similar to ELISA, a platform widely used in laboratory testing throughout Brazil. The technology can easily be adapted for use in automated systems and rapid point-of-care tests.

The study was conducted at the University of São Paulo’s Biomedical Sciences Institute (ICB-USP) in partnership with researchers at the same university’s medical school (FM-USP), and collaborators at the Federal University of West Bahia (UFOB) led by Jaime Henrique Amorim. An article reporting the findings is published in the Journal of Medical Virology.

Dengue is the most common arboviral disease worldwide. It is caused by a flavivirus with four antigenically distinct serotypes (DENV-1, -2, -3 and -4) and co-circulates with at least nine other flaviviruses, including zika (ZIKV). Similarities between DENV and ZIKV antigens lead to cross-reactivity, hindering specific serological diagnosis.

Increasing assay detection specificity

To enhance the assay’s accuracy in detecting antibodies in the blood of previously infected people (even those without apparent symptoms), the researchers used fragments of viral proteins, especially a part of DENV’s envelope protein (the outermost layer of the virus) known as EDIII, which is involved in viral binding to human cell receptors.

During the study, which was funded by FAPESP via six projects (16/20045-7, 22/10408-6, 23/02345-7, 21/05661-1, 20/08943-5 and 17/24769-2), and conducted during the PhD research of Samuel Santos Pereira at ICB-USP, the test was initially validated with blood samples from mice experimentally infected with DENV and ZIKV.

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This stage of the study confirmed the test’s capacity to distinguish between specific antibodies against each virus, as well as the antibodies produced in response to all four DENV serotypes. 

Monitoring serological immunity for dengue and zika virus

Next, the test was validated with around 650 blood samples donated by people in São Paulo during the Brazilian zika epidemic (2015-17). The results showed 87.8% sensitivity (avoidance of false negatives) and 91.4% specificity (avoidance of false positives).

“We also tested 318 blood samples donated by healthy people with no prior diagnosis of dengue in Barreiras (a city in the western region of Bahia state, where DENV and other arboviruses are endemic), to assess the assay’s capacity to monitor specific antibodies produced in response to exposure to the viruses,” said Luís Carlos de Souza Ferreira, full professor at ICB-USP and principal investigator for the study.

According to Ferreira, about 65% of the samples reacted to at least one dengue antigen (88 to DENV-1 alone, and 90 to more than one). Curiously, only three samples were reactive for ZIKV, suggesting either that this virus has not circulated in the region for some years or that the specific antibodies produced in response to infection by the virus do not last long.

“All told, these results show that we have a powerful tool for monitoring the serological immunity of anyone exposed to these viruses, especially people who live in areas endemic for DENV and ZIKV, or who have taken or plan to take one of the available dengue vaccines,” he said.

Outbreak control and applying vaccines

Officially reported deaths from dengue in Brazil reached 21 in the first four weeks of 2025, with two-thirds occurring in São Paulo state. The reappearance of DENV-3 after a gap of 17 years has alarmed specialists, who fear it may lead to increased severity in outbreaks of the disease. 

In future outbreaks, Ferreira added, the test developed at ICB-USP will serve to detect individuals and populational groups without immunity against the circulating DENV serotypes, as a basis for action to combat the vector and apply vaccines.

“The antibodies measured in the assay [mainly immunoglobulin G, or IgG] are largely responsible for the immunity that protects people against the disease, as they stop the virus from entering our cells. This characteristic means the test can be used to evaluate the immunity of healthy people who have been exposed to the viruses in the past, as well as the immunity of people who have been vaccinated,” Ferreira  said.

“In the case of ZIKV, it will be possible to test the immunity of people of reproductive age and pregnant women, the main at-risk groups, without having to worry about the possibility of cross-reactivity with antibodies produced after infection by the four DENV serotypes. This will serve as a basis for measures to prevent infection when ZIKV reemerges in Brazil,” he said.