4. Discussion
In this study we demonstrated our results of the first non-targeted CCHF metabolomics study to improve the current understanding of the pathogenesis of CCHF for diagnostic purposes and aid studies on development of potential therapies against CCHF. Our main findings revealed considerable increases in the levels of SAH, GTP, Carnosine, Maleate, 2-Deoxyuridine, IMP, AMP and NADP+ in the blood serum of CCHF patients, suggesting these metabolites could play crucial roles in the pathogenesis of CCHF and therefore, potentially serve as important biomarkers for early detection and monitoring of the disease progression.
Metabolomics has emerged as a pivotal approach for inspecting the modifications in host metabolism elicited by viral infections. Up until the present, only a single omics study has been conducted by MS that explores the interaction between the host and CCHFV and the ensuing pathogenesis (12). NMR and MS are the two commonly utilized methods used for metabolomics studies with each having certain strengths and weaknesses depending on their purpose of use. NMR spectroscopy offers better reproducibility, minimal sample preparation, and the ability to analyze complex biofluids, whereas MS has relatively higher sensitivity and better for targeted metabolomics studies. Although reproducibility and superior comparability of NMR spectra across different instruments and laboratories compared to MS makes it preferable for non targeted metabolome analysis for diagnostic and prognostic analysis of several diseases (18). NMR spectroscopy in metabolomics is used in analysis of infection on host metabolism and its implications for the pathogenesis of viruses such as HIV (20), Dengue virus (DENV) (21), Chikungunya virus (22) Metabolomics analysis has been employed in the study of other viral hemorrhagic fevers, such as Ebola, Marburg, and Dengue, with promising results for early diagnosis and prognosis prediction (23). Our study demonstrates a new path by harnessing the power of NMR metabolomics analysis to further decipher the intricate interplay between the host and CCHFV and to elucidate the mechanisms of CCHFV pathogenesis.
S-adenosyl homocysteine (SAH) is an intermediate metabolite, a precursor to homocysteine and adenosine(24). Moreover, SAH is the substrate of SAH hydrolase enzyme which is a crucial enzyme involved in the S-adenosylmethionine (SAM/AdoMet) regeneration cycle (Figure 4a) (25). This cycle is noteworthy in the context of CCHF patients, as evidenced by increased SAH levels from the beginning of hospitalization. Furthermore, a genomic study investigating the effect of Methylenetetrahydrofolate reductase (MTHFR) polymorphism showed that MTHFR polymorphism creates a predisposition to milder CCHF (26). MTHFR’s primary role as an enzyme in folate metabolism extends to functions related to methylation as well. Thus, we hypothesized that alterations in methylation pathways during the viremic phase of CCHF may serve as valid indicators of prognosis in CCHF patients. Furthermore, SAM is understood to function as a primary methyl donor in various cellular methylation reactions, including those necessary for the 5’ RNA capping process (27). Similar to the methylation process in Flaviviruses (28, 29), the Ebola virus’ L protein has been found to exhibit methyltransferase activity, affecting co-transcriptional modifications at the cap structure and internal adenosine-2’-O-methylation (30). This additional complexity in RNA methylation within some viral families could provide a framework to consider the implications of SAM-related domains in replication complexes as potential drug targets.
GTP, AMP and inosine monophosphate (IMP) are nucleotides involved in purine metabolism, which play a vital role in energy metabolism (Figure 4b), and nucleic acid synthesis (31). Increase in the level of GTP and IMP are known indicators for infection of several viruses belonging to Orthornavirae kingdom (32). In fact, increase in purine compounds can be attributed to infection of CCHFV considering known effective broad spectrum antivirals such as Ribavirin as a nucleotide analog targets the substrate-binding site of the IMPDH enzyme and prevents binding of IMP causing reduction of RNA synthesis in infected cells (33, 34) through down regulating GTP synthesis. Monitoring the levels of these metabolites may provide vital insights into the efficacy of antiviral treatments, especially nucleotide analogs like Ribavirin. Identifying shifts in GTP and IMP concentrations could be a good addition to therapeutic strategies for clinical trials, optimizing the use of antiviral agents that target purine metabolism, and thereby enhancing the response to CCHF infection. (35, 36) Carnosine (beta-alanyl-L-histidine) (Figure 4c) is a dipeptide with antioxidant, free radical scavenging; anti-glycation, and antiinflammatory properties which is predominantly found in skeletal muscle and in the brain (29). Increase in carnosine levels may reflect changes in the antioxidant defense system, reduction of muscle mass due to hospitalization and infection related cell death and as a possible anti-inflammatory response to the CCHFV infection. Carnosine is considered as a potential therapy for Zika and Dengue viruses, (37) and SARS-CoV-2 (38) as it showed significant reduction in viral replication and ease the symptoms of these diseases, however the effectiveness of carnosine as a therapeutic against CCHF requires further investigation. 2’-Deoxyuridine (2’-dU) is an intermediate in the synthesis of thymidylate (Figure 4d), which acts as a precursor for DNA synthesis and Edoxudine as an antiviral therapeutic. (39). Increase in the levels of 2-deoxyuridine can have significant implications for viral pathogenesis as 2’-dU variants such as BVDU is effective against Herpes simplex virus type 1 (HSV-1) and varicella-zoster virus (VZV) (40). Nevertheless 2’-dU is an antiviral of DNA viruses and used as an indicator to determine presence of viruses which have DNA as their genetic material (41). Presumably 2’-dU levels increased in the blood serum may indicate a dysregulation in the pyrimidine and energy metabolism in accordance to our other findings which signal metabolic disruption of these pathways. Nicotinamide Adenine Dinucleotide Phosphate (NADP+), another key metabolite, serves as a crucial cofactor in various enzymatic reactions, primarily within the pentose phosphate pathway, where it facilitates the synthesis of NADPH, essential for fatty acid synthesis and the regeneration of reduced glutathione. Additionally, NADP+ plays a vital role in supporting the body’s antioxidant defense system and is involved in the reduction-oxidation (redox) reactions. It is difficult to definitively pinpoint why NADP+ increases in CCHF patients, as the underlying cause could be multifaceted, encompassing a range of biological, metabolic, and external factors. Furthermore in relation to our other findings it is possible that elevation of NADP+ also overlaps with increase of GTP, IMP, and AMP which take part in energy metabolism and nucleotide synthesis, as a possible indication of a coordinated increase in use of energy and nucleic acid synthesis.. This is further supported by our final analysis between severe and moderate infection CCHF in Fig 3, considering AMP, IMP and NAAD levels increased more significantly in severe cases signaling increased activity in metabolic pathways including these compounds. Maleate (cis-butenedioic acid), a dicarboxylic acid, and trans-isomer of fumaric acid is a metabolite taking role in nicotinate and nicotinamide metabolism. (42) Previous studies on rats have demonstrated that the injection of sodium maleate can induce a generalized renal transport defect resembling Fanconi syndrome. Furthermore, maleate has been shown to reduce cellular levels of coenzyme A, inhibit the tricarboxylic acid cycle, lower ATP concentrations, and affect various enzymatic activities. Its impact even extends to the ultrastructure of kidney cells. The elevated presence of maleate in the blood serum of CCHF patients could signal underlying metabolic and cellular disruptions, warranting further investigation into its potential role and implications in the disease. (43,44)