Pathophysiology of chikungunya virus infection associated with fatal outcomes

Summary Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that causes acute, subacute, and chronic human arthritogenic diseases and, in rare instances, can lead to neurological complications and death. Here, we combined epidemiological, virological, histopathological, cytokine, molecular dynamics, metabolomic, proteomic, and genomic analyses to investigate viral and host factors that contribute to chikungunya-associated (CHIK) death. Our results indicate that CHIK deaths are associated with multi-organ infection, central nervous system damage, and elevated serum levels of pro-inflammatory cytokines and chemokines compared with survivors. The histopathologic, metabolite, and proteomic signatures of CHIK deaths reveal hemodynamic disorders and dysregulated immune responses. The CHIKV East-Central-South-African lineage infecting our study population causes both fatal and survival cases. Additionally, CHIKV infection impairs the integrity of the blood-brain barrier, as evidenced by an increase in permeability and altered tight junction protein expression. Overall, our findings improve the understanding of CHIK pathophysiology and the causes of fatal infections.


Supplemental Figures
. The serum proteomic profiles of chikungunya patients (deaths, n=13, and survivors, n=11) were compared with healthy controls (n=15).A) Heatmap analysis of selected proteomic markers for chikungunya survivors compared with blood donors (healthy) shows the relativity intensities.The relative intensities (Z-score) are scaled from blue (low intensity) to red (high intensity).Samples were clustered according to Pearson correlation coefficient distance.B) Volcano plot displaying the log2-fold change (xaxis) against the t-test-derived −log10 statistical P value (y-axis) for all proteins differentially expressed between chikungunya survivors and blood donors (healthy).Proteins with significantly decreased levels (p < 0.05) are shown in blue, and proteins with significantly increased levels are noted in red.The gray circles indicate proteins non-regulated.All proteins are shown.C) Heatmap analysis of selected proteomic markers for chikungunya deaths compared with blood donors (healthy) shows the Log2 FC.The relative intensities (Z-score) are scaled from blue (low intensity) to red (high intensity).Samples were clustered according to Pearson correlation coefficient distance.D) Volcano plot displaying the log2-fold change (x-axis) against the t-test-derived −log10 statistical P-value (y-axis) for all proteins differentially expressed between chikungunya deaths and blood donors (healthy).Proteins with significantly decreased levels (p < 0.05) are shown in blue, and proteins with significantly increased levels are noted in red.The gray circles indicate proteins non-regulated.All of the proteins are shown.Gene symbols (abbreviation names) for proteincoding genes followed the nomenclature available on HUGO Gene Nomenclature Committee.Legend: F, female.M, male.CSF, cerebrospinal fluid.

Figure S1 .
Figure S1.Clinical features of chikungunya patients, related to Figure 2. A) Symptoms and signs of fatal chikungunya cases.B) Symptoms and signs of chikungunya survivor cases.C) Percentage of females and males per group (CHIK-deaths, CHIK-survivors, and healthy blood donors.D) Comorbidities among fatal chikungunya cases.E) Percentage of RT-qPCR positive per biological sample in fatal chikungunya cases.CHIK, chikungunya.PFU, plaque-forming units.CSF, cerebrospinal fluid.RT-qPCR, Reverse transcription-quantitative polymerase chain reaction.COPD, Chronic obstructive pulmonary disease.

Figure S3 .
Figure S3.Principal component analysis of the metabolomics data from the serum samples of chikungunya patients (i.e., deaths and survivors) compared with healthy blood donors, related to Figure 3.Each dot represents one patient sample, color-coded for the different groups as described in the figure.

Figure S4 .
Figure S4.Heatmap analysis of selected metabolomic markers for chikungunya fever patients (i.e., fatal and survivors) compared with blood donors (healthy), related to Figure 3. Normalized logtransformed ion intensities (Z-score) are scaled from blue (low intensity) to red (high intensity).Samples

Figure S5 .
Figure S5.Proteomics signatures of chikungunya patients compared to healthy controls, related to Figure 4.The serum proteomic profiles of chikungunya patients (deaths, n=13, and survivors, n=11) were compared with healthy controls (n=15).A) Heatmap analysis of selected proteomic markers for chikungunya survivors compared with blood donors (healthy) shows the relativity intensities.The relative intensities (Z-score) are scaled from blue (low intensity) to red (high intensity).Samples were clustered according to Pearson correlation coefficient distance.B) Volcano plot displaying the log2-fold change (xaxis) against the t-test-derived −log10 statistical P value (y-axis) for all proteins differentially expressed

Figure S6 .
Figure S6.Protein-protein interaction analysis from chikungunya survivors compared to healthy blood donors, related to Figure4.Gene symbols (abbreviation names) for protein-coding genes followed the nomenclature approved by the HUGO Gene Nomenclature Committee.

Figure S7 .
Figure S7.Protein-protein interaction analysis from chikungunya deaths compared to healthy blood donors, related to Figure4.Gene symbols (abbreviation names) for protein-coding genes followed the nomenclature approved by the HUGO Gene Nomenclature Committee.

Figure S8 .
Figure S8.Molecular dynamic stimulations of R242H substitutions in envelope 2 protein of chikungunya virus with the Matrix Remodeling Associated 8 (MXRA8) receptor, related to Figure 5.

Figure S9 .
Figure S9.Folding energy differences after alanine-scanning mutagenesis of key residues in E2-H242 and E2-R242 substitutions of chikungunya virus, related to Figure 5 and STAR Methods.A) Chikungunya virus with envelope 2 with arginine (R) in position 242.B) Chikungunya virus with envelope 2 with histidine (H) in position 242.Residues were selected based on an energy threshold of >5.0 kcal/mol or <2.5 kcal/mol.Chains were named according to the Protein Data Bank (https://www.rcsb.org/).