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)