DIFFERENTIAL EXPRESSION OF INTERFERON INDUCIBLE PROTEIN: GUANYLATE BINDING PROTEIN (GBP1 & GBP2) IN SEVERE DENGUE Sanchita Chakraborty a , Vignesh Mariappan a , Shalinda Adikari b , Lokesh Shanmugam c,d , Joshy M. Easow d , Agieshkumar Balakrishna Pillai a ,* a Mahatma Gandhi Medical Advanced Research Institute (MGMARI) (Formerly Central Inter-Disciplinary Research Facility-CIDRF), Sri Balaji Vidyapeeth (Deemed to be University), Puducherry, 607402, India; b Department of Information System and Analytics, National University of Singapore (NUS), Singapore, 117417, Republic of Singapore; c ICMR-National Institute of Epidemiology (ICMR-NIE), Ayapakkam, Chennai, 600070, India; d Mahatma Gandhi Medical College and Research Institute (MGMCRI), Sri Balaji Vidyapeeth (Deemed to be University), Puducherry, 607 402, India AIM & OBJECTIVES To see the role of GBPs on dengue disease progression by: a) quantifying the mRNA and protein levels of GBP1 and GBP2 in severe and non-severe forms of DENV infected patients during the course of infection b) Assessing GBPs predictive capacity as effective prognostic markers using machine-based mathematical models c) Finding the association between GBPs ,oxidative stress process and severity markers during the course of DENV infection METHODOLOGY Sample Size Used in this Study Severe Dengue Dengue With Warning sign Dengue Without Warning Sign Other Febrile Illness Healthy Control 10 25 15 10 8 SUMMARY AND CONCLUSION Expression pattern of GBP 1 and 2 were found to be negatively correlated with plasma leakage and elevated levels of oxidative stress is associated with the decreased expression of GBPs during the course of Dengue infection. Thus, antioxidant supplement as adjuvant therapy may regulate the expression of GBPs and disease virulence. Nevertheless, machine models found that the plasma levels of GBP1 and 2 along with routine clinical symptoms could predict the dengue disease severity with higher accuracy. A large prospective cohort study may be required to ascertain the role of GBP1 and GBP2 as effective prognostic markers of dengue severity. The findings has been published in Free Radical Biology and Medicine, 194: 131-46, 2023. Acknowledgment - We greatly appreciate the support of Sri Balaji Vidyapeeth for providing facilities for conducting this research. REFERENCES 1. Mariappan V, Adikari S, Shanmugam L, Easow JM, Pillai AB. Differential expression of interferon inducible protein: Guanylate binding protein (GBP1 & GBP2) in severe dengue. Free Radical Biology and Medicine. 2023 Jan 1;194:131-46. 2. Kutsch M, Coers J. Human guanylate binding proteins: nanomachines orchestrating host defense. The FEBS Journal. 2021 Oct;288(20):5826-49. 3. Merabet O, Pietrosemoli N, Perthame E, Armengaud J, Gaillard JC, Borges-Cardoso V, Daniau M, Legras-Lachuer C, Carnec X, Baize S. Infection of Human Endothelial Cells with Lassa Virus Induces Early but Transient Activation and Low Type I IFN Response Compared to the Closely-Related Nonpathogenic Mopeia Virus. Viruses. 2022 Mar 21;14(3):652. ABSTRACT Dengue virus is reported to activate endothelial cells (EC), but the precise cause for severe dengue (SD) is not known. Guanylate binding proteins (GBPs) are IFN-inducible proteins secreted by ECs and are involved in the anti-oxidant and anti-viral response. The involvement of GBPs in the pathogenesis of dengue remains under explored. We have quantified the mRNA and protein levels of GBP1 and 2 during different phases of infection and have corelated with the oxidative stress in plasma samples of different study groups. The efficacy of the proteins in predicting disease severity was done by Support Vector Machine (SVM) model The study recorded a decreased expression of GBPs during critical phase among severe dengue. The GBP levels were found to be negatively correlated with plasma leakage and emerged as a strong predictor of disease outcome based on machine models. 1 • 3 mL of venous blood was collected under aseptic condition at the Day of admission (1 st day, DOA) , Day of defervescence (4rth day, DOD) and Day of convalescence (11 th day, DOC) from patients. 2 • The PBMCs were isolated from the whole blood samples, RNA extracted using trizol method Finally, 1 μg of RNA was converted into complementary DNA (cDNA) and the cDNA was used for qPCR analysis. 3 • To assess the plasma (Protein) concentration of GBP1 and 2, a quantitative human GBP1 and GBP2 sandwich ELISA kit was used. 4 • Plasma MDA( Lipid peroxidation) was estimated by the Kei Satoh method. 5 • The status of protein and DNA oxidation in the plasma samples were estimated using an advanced oxidation protein production (AOPP) and 8-hydroxy-2-deoxyguanosine (8-OHdG) ELISA kit. 6 • Data was collected and Statistical Analysis of Result were performed with 5% level of significance (P≤0.05). RESULTS AD DWOW DWW SD OFI 0.01 0.1 1 10 Study Group GBP1 m RNA Levels DOA DOD DOC *** *** ** *** *** ** ** AD DWOW DWW SD OFI 0.1 1 10 Study Group GBP2 m RNA levels DOA DOD DOC *** *** ** mRNA levels GBP1 and GBP2 GBP1 and GBP2 expression was measured by qRT-PCR and normalized by β-actin. DOA of all the groups is taken as a baseline P-value≤0.05 is considered significant. ELISA (PLASMA LEVEL) GBP1 and GBP2 Plasma levels of GBP1 and GBP2 in all the study groups. Wilcoxon signed-rank test was used to compare within the groups. P-value ≤0.05 is considered significant. AD DWOW DWW SD OFI 0 100 200 300 400 500 Study Group GBP1 (ng/L) DOA DOD DOC *** ** * * ** * AD DWOW DWW SD OFI 0 2 4 6 8 Study Group GBP2 Conc (ng/m l) DOA DOD DOC *** *** * ** ** ** ** LEVEL OF MDA (lipid peroxidation) Plasma levels of MDA in all the study groups. Wilcoxon signed-rank test was used to compare within the groups. P-value ≤0.05 is considered significant. AD SD DWW DWOW OFI 0 5 10 15 20 Study Group MDA (M/ml) DOA DOD DOC *** ** ** *** ** ** * LEVEL OF DNA OXIDATION Plasma levels of DNA damage in all the study groups. Wilcoxon signed- rank test was used to compare within the groups. P-value ≤0.05 is considered significant. AD DWOW DWW SD OFI 0 100 200 300 Study Groups 8-OHdG (ng/L) DOA DOD DOC ** * * * * *** *** LEVEL OF PROTEIN OXIDATION Plasma levels of AOPP in all the study groups. Wilcoxon signed-rank test was used to compare within the groups. P-value ≤0.05 is considered significant. AD DWOW DWW SD OFI 0 20 40 60 80 Study Group AOPP (mol/L) DOA DOD DOC *** *** * * * ** ** DISCUSSION • . MACHINE LEARNING BY SVM MODEL We observed that the protein levels & mRNA levels of GBP1 and GBP2 along with other clinical symptoms could predict the disease severity with an accuracy of 98% and 100% respectively. Interestingly, the SVM model showed that plasma levels of GBP-2 with clinical symptoms could predict dengue severity with 100% accuracy. Differential Expression of GBP1 and GBP2 in response to Oxidative Stress • During the febrile phase (DOA), GBP1 showed an elevated mRNA and protein level compared to Other Febrile illness, and towards the critical phase (DOD), a significant decrease in the mRNA and protein level of both GBP1, as well as GBP2, was observed in severe Dengue (SD) cases, indicating the importance of these two molecules in disease manifestation. • Lipid Oxidation (MDA ) showed a negative correlation with GBP1 as well as GBP2 in Severe Dengue groups, particularly at the critical phase of infection. • GBP1 and GBP2 showed a negative association with protein and DNA oxidation respectively. • Increased oxidative stress may downregulate both GBP1 and 2 thereby enhancing disease manifestation by creating a suitable micro-environment for virus propagation accompanied by endothelial dysfunction. • Thus, the study found that the expression pattern of GBP 1 and 2 were found to be negatively correlated with plasma leakage, and elevated levels of oxidative stress are associated with the decreased expression of GBPs during Dengue infection.