Targeted mass spectrometry can be used to obtain detailed information about the NS1 peptide of flaviviruses, a boon for diagnosing diseases such as dengue and yellow fever.

© 2018 A*STAR Institute of Molecular and Cell Biology

One test to determine them all

14 Jun 2019

A*STAR researchers have devised a method that can detect and distinguish closely related flaviviruses with 100 percent accuracy.

When disease outbreaks occur, identifying the microbe responsible for causing the infection is critical for the correct treatment to be administered. Health authorities also rely on accurate and rapid diagnostics to coordinate containment strategies.

However, some infectious organisms such as those belonging to the flavivirus family are difficult to distinguish from one another using conventional antibody methods, and remain undetectable by RNA-based diagnostics even at a late stage of infection. Examples of viruses in this family include four strains of the dengue virus, Zika virus, yellow fever virus and Kunjin virus.

“Each flavivirus diagnosis also requires a specific test, and combining multiple tests within a single diagnostic reaction often compromises detection sensitivity,” said A*STAR’s Jayantha Gunaratne.

Seeking to improve flavivirus identification, Gunaratne and his team at the Institute of Molecular and Cell Biology (IMCB), in collaboration with the National Environment Agency’s Environmental Health Institute and the National Centre for Infectious Diseases, developed a proteomic mass spectrometry (MS)-based assay that can simultaneously detect all seven of the above-mentioned flaviviruses, with high sensitivity and specificity.

Gunaratne’s team first focused on the virus non-structural protein 1 (NS1), a protein not found in humans but expressed by flaviviruses. Using their technique, they determined that each flavivirus, including the four strains of the dengue virus, has a unique NS1 peptide sequence that could be used in a way similar to fingerprints for diagnosis.

The researchers then proceeded to validate their approach in serum samples from infected patients, demonstrating that they could distinguish the four strains of dengue virus with 100 percent accuracy. The assay could also be used to pinpoint exactly which flavivirus species or strains were culpable for co-infected samples.

Importantly, the assay remained sensitive and specific when applied to samples from patients with secondary flavivirus infections, during which the virus’ NS1 peptide may be bound to immune proteins. The researchers noted that a step in their protocol likely resulted in the dissociation of immune complexes from the NS1 peptide, thereby preventing the immune proteins from interfering with the assay.

“Proteomic MS offers single amino acid resolution when analyzing peptide sequences, so I was confident that the technology could be exploited in the diagnosis and typing of flaviviruses,” Gunaratne said. “Our findings could pave the way for a more efficient response towards outbreaks, especially for new emerging flaviviruses such as the Zika virus.”

Gunaratne’s team plans to develop an end-user friendly package to bring this assay into the clinic, as well as explore the possibility of expanding this application to other diseases.

Proteomics and systems biology pioneer Ruedi Aebersold at ETH Zurich, Switzerland, who was not involved in the research, noted that “the present study is a powerful and well-constructed example of the application of advanced MS technology to an important clinical question. It promises a clear path for translation and broad utility. Further, the approach is readily extendible to new viruses or virus subtypes without the need for expensive and slow antibody development.”

Aebersold's full commentary can be found here.

The A*STAR-affiliated researchers contributing to this research are from the Institute of Molecular and Cell Biology (IMCB).

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Wee, S., Alli-Shaik, A., Kek, R., Swa H. L. F., Tien, W. P., Lim, V. W. et al. Multiplex targeted mass spectrometry assay for one-shot flavivirus diagnosis. PNAS 116(14), 6754-6759 (2019) | article

About the Researcher

Jayantha Gunaratne

Principal Investigator

Institute of Molecular and Cell Biology
Jayantha Gunaratne obtained his PhD degree from Tokyo Institute of Technology, Japan, in 2003 where he received training on gas and liquid chromatography, mass spectrometry and nuclear magnetic resonance technologies. In 2004, he joined the Scripps Institution, University of California San Diego, US, as a postgraduate research scholar. He moved to Singapore in 2007 and was a founder member of the Mass Spectrometry and Systems Biology Laboratory at A*STAR’s Institute of Molecular and Cell Biology (IMCB), pioneering the establishment of high-resolution advanced quantitative proteomics technology in Singapore. He is currently a Principal Investigator heading translational biomedical proteomics research at IMCB and an adjunct Associate Professor at Yong Loo Lin School of Medicine, National University of Singapore.

This article was made for A*STAR Research by Wildtype Media Group