UK-Indonesian Consortium to Identify Biomarkers Predictive of Dengue Disease Severity.

Dengue is the most important mosquito-borne disease of humans. Indonesia bears the largest dengue disease burden and economic cost in Southeast Asia. The incidence of dengue haemorrhagic fever has increased dramatically in Indonesia from 0.05/100000 individuals in 1968 to ~35-40/100000 in 2013. Infection with dengue virus (DENV) causes a spectrum of clinical illnesses, ranging from dengue fever, a debilitating but self-limited condition, to the more severe and potentially fatal dengue haemorrhagic fever, but the factors contributing to differential disease severity are not entirely understood. Although most dengue cases could be managed at home, the lack of a diagnostic test to predict those individuals who progress from mild to severe disease forces patients and healthcare providers to seek hospital admissions for "safety purposes", saturating an already overwhelmed healthcare system.

Our OVERALL OBJECTIVE, by adopting an integrated analysis of peripheral blood from patients who have well-defined clinical outcomes, is to correlate disease severity with a) DENV genetic diversity or other co-morbidity factors (e.g. co-infection), b) host transcriptomic and proteomic changes and c) alterations in platelet function and endothelial activation and permeability. In order to achieve our objective we will analyse a very well characterised biobank of patient samples, collected both retrospectively and prospectively from patients with different dengue disease outcomes as the disease progresses. We will use sophisticated methods to look at the strains of DENV and other arthropod-borne viruses (such as chikungunya and Zika virus) that are circulating in Indonesia and also identify any changes in patient RNA or proteins which we can detect in blood that might be linked to serious disease associated with DENV infection. The methods will be both high throughput and very sensitive so that even changes in the abundance of low-level transcripts or proteins will be detected. We will also be able to detect the frequency of minor changes in the virus and investigate whether these may be linked to disease. Using complementary approaches we will also examine a) platelet number and function in patient blood samples and b) the ability of patient serum from different disease outcomes to mediate changes in endothelial permeability using in vitro assays. The results will produce large amounts of data that will be brought together using computational methods, either available or to be developed by the UK partners. The UK partners have particular experience with the bioinformatics tools needed and will provide these to their Indonesian counterparts through short-term scientific exchange visits throughout the project.

Computational analysis of the datasets will allow us to identify biomarkers in patients, which will be verified using prospectively collected samples. After verification, any biomarkers can then be used to develop diagnostic tests to predict those individuals at risk of progressing to severe disease and to monitor DENV variability. In addition, our metagenomic analysis will also assess whether other arboviruses such as chikungunya and Zika virus, whose status in Indonesia is unknown, may have been misdiagnosed as dengue infection. Overall, the project will significantly support the building of research capacity in Indonesia. Critically, the training and technology transfer provided by the UK partners will help to establish an adaptable technological framework that is also relevant to many other infectious disease areas in Indonesia.

We will adopt an integrated 'omics approach, to correlate dengue disease severity with viral sequence variation and changes in the host transcriptome and proteome, using whole blood and serum samples from dengue patients with well defined clinical outcomes. The 'omics analysis will be supported by biochemical and immunological analysis of platelets in clinical samples and an analysis of the effects of serum on endothelial permeability using an in vitro model. Existing serum samples (from the UI and UP biobanks) from patients sampled at different times after hospital admission (during the febrile, critical and recovery phases) with different clinical outcomes (febrile illness non-dengue, dengue +/- warning signs and severe dengue disease) will be selected for viral metagenomic and proteomic analysis. Metagenomics will generate viral sequence data for analysis by the UoG's bespoke bioinformatics pipeline and identify other non-dengue viruses causing febrile illness. Serum proteins will be identified and quantified by tandem mass tagging of the peptides prepared from serum proteins by tryptic digest followed by high-throughput LC-MS/MS analysis at the UoB. A second phase of 'omics analysis will utilise prospectively collected samples (UI, UP, UJS). The samples will correspond to the above disease conditions. Along with sample storage, clinical variables for patients and data concerning the viral infection will be collected. These blood and serum samples will be analysed by metagenomics, RNAseq transcriptomic, proteomic studies and biochemical and immunological assays. The clinical and 'omics datasets will be integrated using a bespoke bioinformatics pipeline and analysed using statistical methods. This will identify candidate mRNA and protein biomarkers which will be further verified by qRT-PCR and targeted proteomics, using a larger prospective sample collection. Verified biomarkers will form the basis for novel diagnostic tests to predict severe dengue disease.

Who will benefit from the research?
A key element of our proposal will be the creation of a collaborative network between Indonesian and UK groups that will build mutually beneficial relationships for the future. The UoB and the UoG will bring a range of state-of-the-art expertise to Indonesia, promoting the use of novel technologies and analytical tools in bioinformatics. This will facilitate translatable applications both in the area of predictive diagnosis and viral surveillance. Within such a broad collaborative network, there will be unique opportunities for training young scientists from both countries via exchange visits. This will yield benefits for young Indonesian scientists, who will have access and training in the most recent innovations in 'omics technologies and a suite of bioinformatic tools, and to young UK scientists, who will benefit from the opportunity to address the challenges of applying advanced systems in a developing country.

In the longer term, this collaborative endeavour will stimulate economic growth through science and technology by developing relevant products for health and industry. In a wider context, the collaborative interaction between the UK and Indonesian groups will advance dengue and arbovirus research in both countries and nurture the growing number of young graduates wishing to pursue a scientific career to supply the much needed critical mass of local scientific experts. The identification of biomarkers that can be used to predict dengue disease progression will lead to the development of new diagnostics which can be exploited by technology business incubators in Indonesia and provide opportunities for scale-up manufacturing and the rise of new leaders in biotechnology in the region. An increased understanding of arbovirus circulation in Indonesia will be of immediate benefit to those involved in developing public health policies both in Indonesia and throughout the region.

How will they benefit?
The research plan will be achieved through an integrated strategy involving Indonesian and UK researchers. The expertise of the UoB and UoG researchers in high-throughput 'omics along with any integrated bioinformatic analytical tools will be transferred to Indonesian researchers through short term scientific exchange visits and joint workshops throughout the project. This will significantly support the Indonesian research capacity enhancement program. Critically, the training will help to establish an adaptable bioinformatic framework that is relevant to many other infectious disease areas in Indonesia. In this context, the coupling of two major UK centres with complementary strengths and expertise in virus research brings synergistic benefits to the Indonesian partners and considerably enhances the scope of the project. A major objective of the program is the creation of novel dengue diagnostic platforms that will not only provide the tools for identifying the presence or absence of the disease but more importantly provide clinicians with an objective basis for risk stratifying dengue cases that require hospitalisation. In addition, the project will significantly improve our understanding of the viruses circulating in Indonesia that lead to febrile disease that are not readily distinguishable from dengue. At present this knowledge is lacking but urgently required in the face of emerging viruses and the future deployment of dengue vaccines. The described collaborative research programme between scientists in the Indonesia and the UK will hopefully be the start of a long-term relationship that will create a bridge between clinical and basic research, not only focused on dengue but on other biomedical problems of interest to both parties.