Differential proteomic analysis of virus-enriched fractions obtained from plasma pools of patients with dengue fever or severe dengue

Abstract

Background: Dengue is the most widespread mosquito-borne viral disease of public health concern. In some patients, endothelial cell and platelet dysfunction lead to life-threatening hemorrhagic dengue fever or dengue shock syndrome. Prognostication of disease severity is urgently required to improve patient management. The pathogenesis of severe dengue has not been fully elucidated, and the role of host proteins associated with viral particles has received little exploration.

Methods: The proteomes of virion-enriched fractions purified from plasma pools of patients with dengue fever or severe dengue were compared. Virions were purified by ultracentrifugation combined with a water-insoluble polyelectrolyte-based technique. Following in-gel hydrolysis, peptides were analyzed by nano-liquid chromatography coupled to ion trap mass spectrometry and identified using data libraries.

Results: Both dengue fever and severe dengue viral-enriched fractions contained identifiable viral envelope proteins and host cellular proteins. Canonical pathway analysis revealed the identified host proteins are mainly involved in the coagulation cascade, complement pathway or acute phase response signaling pathway. Some host proteins were over- or under-represented in plasma from patients with severe dengue compared to patients with dengue fever. ELISAs were used to validate differential expression of a selection of identified host proteins in individual plasma samples of patients with dengue fever compared to patients with severe dengue. Among 22 host proteins tested, two could differentiate between dengue fever and severe dengue in two independent cohorts (olfactomedin-4: area under the curve (AUC), 0.958; and platelet factor-4: AUC, 0.836).

Conclusion: A novel technique of virion-enrichment from plasma has allowed to identify two host proteins that have prognostic value for classifying patients with acute dengue who are more likely to develop a severe dengue. The impact of these host proteins on pathogenicity and disease outcome are discussed.

Fig. 1

Characterization of the viral-enriched fraction of DV-infected HepG2 supernatant or plasma obtained from patients with dengue. a After infection with DV at a MOI of 1 or 10, HepG2 cell culture supernatants were purified by ultracentrifugation (Step 1) followed by the use of Viraffinity™ (Step 1 + 2). b Silver-staining assessment of the protein complexity of DV-infected HepG2 cell culture supernatant before and after purification; not diluted (lane “not purified”), 1/10 diluted (lane “not purified 1/10”) DV-infected cell supernatant before purification or the same sample as shown in lane 1 after purification (lane “Step 1 + 2”) were separated by denaturing polyacrylamide gel electrophoresis and silver-stained. c Electron microscopy of DV-infected HepG2 culture supernatant after ultracentrifugation. Grids were negatively stained using uranyl acetate. Bars: 50 nm, 200 nm. d Representative Western blotting analysis of plasma pools obtained from patients with dengue after purification by ultracentrifugation and Viraffinity™ using an anti-E monoclonal antibody. MW: molecular mass

Fig. 2

Ingenuity Pathway Analysis for host proteins identified in the viral-enriched plasma fraction of patients with dengue. Pathway classification according to canonical pathways was performed using IPA software. The x-axis represents the pathways identified. The y-axis (left) shows the − log of the P-value calculated using Fisher’s exact test. The ratio (y-axis, right) represented by the line is calculated as follows: number of proteins in a given pathway that meet the cutoff criteria divided by total number of proteins that make up that pathway

Fig. 3

Assessment of the protein concentrations of C1r, CP, OLFM4 and PF4 in the individual plasma samples of Colombian patients using specific quantitative ELISAs. Each value corresponds to the mean of two independent experiments, each performed in duplicate. *: 0.01 < p < 0.05; **: 0.005 < p < 0.01; ***: p < 0.005

Fig. 4

Assessment of the protein concentrations of OLFM4 (a) and PF4 (b) in the individual plasma samples of Cambodian patients using specific quantitative ELISAs. Patients were taken during both the acute phase (DF acute and SD acute) and at time of discharge from the hospital (DF discharge and SD discharge). T: healthy patient samples. Each value corresponds to the mean of two independent experiments, each performed in duplicate. **: 0.005 < p < 0.01; ***: p < 0.005