In a recent study posted to the medRxiv preprint* server, an international team of researchers evaluated metabolic responses to acute coronavirus disease 2019 (COVID-19) and identified biomarkers for acute viral infections.

Study: SARS-CoV-2 Infection Biomarkers Reveal an Extended RSAD2 Dependant Metabolic Pathway. Image Credit: NIAID

*Important notice: medRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.

Background

The COVID-19 pandemic has caused unprecedented morbidity and mortality globally. Effective therapeutics and tools for early detection and prompt treatment must be developed to mitigate disease. Research is required to improve our understanding of the biological characteristics of the causative virus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

COVID-19 is characterized by pulmonary and extrapulmonary symptoms with complex pathophysiological and immunological effects on major organs. The combined metabolic disruption and immunological stimulation in COVID-19 patients can lead to persistent symptoms and the development of post-acute coronavirus disease 2019 syndrome (PACS) or long-COVID.

Serological biomarkers such as ferritin, C-reactive protein (CRP), and lactate dehydrogenase (LDH) have been identified to track the development and recovery from COVID-19. In addition, altered levels of serological and urinary lipids, amino acids, lipoproteins, cytokines, and tryptophan metabolites have been observed among COVID-19 patients.

About the study

In the present study, researchers elucidated candidate COVID-19 biomarkers by performing a spectroscopic analysis.

Two-dimensional (2D) nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry were used to characterize and quantify metabolites with potential diagnostic value for viral diseases in urine. The correlation between the urinary nucleoside analogs and serological cytokines, such as interleukin-10 (IL-10), interferon-alpha2 (IFN-α2), and interferon-gamma (IFN-γ) was investigated to determine the relationship between the virus inhibitory protein, endoplasmic reticulum-associated and interferon-inducible (viperin) enzyme [or radical S-Adenosyl methionine domain-containing 2 (RSAD2)] and the endogenous protective innate immunological responses against viruses such as SARS-COV-2.

In particular, the team investigated whether the antiviral deoxy-didehydronucleosides (ddhN) were measurable in the urinary samples and evaluated their role as a biomarker. A family of endogenous cytosine metabolites with potential antiviral activity was biosynthesized in vivo during SARS-CoV-2 replication. The team modeled urine spectral data from 273 SARS-CoV-2-infected individuals and 77 non-infected individuals (controls) using principal component analysis (PCA) and OPLS-DA. COVID-19 patients were recruited between July 2022 and January 2023 in ambulatory settings in the urban areas of Heidelberg.

To assess COVID-19 severity and its impact on the excretion of ddhN metabolites, the group of COVID-19 patients was subdivided into non-hospitalized and hospitalized individuals. The team investigated the excretion profiles of nucleosides in the initial week of SARS-CoV-2 infection among six COVID-19 patients who were diagnosed using polymerase chain reaction (PCR) or rapid antigen tests (RAT). Flow cytometry was performed to quantify serological cytokines. Linear regression modeling was performed to determine associations between the ddhN metabolites.

Results

The study analyzed urine from acute COVID-19 patients, revealing a spectroscopic signature associated with anomeric CH-1′ ddhN protons. Nuclear magnetic resonance spectroscopy showed a distinct spin-system motif, which indicated that several metabolites were present in urine during the acute SARS-CoV-2 proliferation.

Mass spectrometry revealed the most abundant serological metabolites with diagnostic potential for viral diseases. The findings revealed an extended innate viperin-dependent pathway contributing to an adaptive response to viral agents like SARS-CoV-2. In total, 10 nucleoside analogs (uracil- and cytosine-based) were identified, of which eight have not been reported previously in humans, indicating more evolutionary and metabolic adaptability to viral infections than previously believed.

The analysis also revealed a correlation between the urinary analogs and viperin enzyme-related serological cytokines. The detection of ddhNs in serum was associated with inflammation, with the mean concentration of the three most abundant nucleosides being a minimum of one order of magnitude greater among individuals infected with SARS-CoV-2 compared to controls.

The major urinary ddhN metabolites accurately distinguished SARS-CoV-2-infected individuals from controls and showed strong correlations with serological interferon-alpha2 and interferon-gamma levels, with weaker correlations with IL-10, 17, and 18. Interferon-alpha is raised in COVID-19 and is linked to an increase in the number of T cell subsets implicated in initial responses to viruses. Interferon-alpha and interleukin-10 are linked to lung damage, predict COVID-19 severity, and were found in greater amounts in symptomatic individuals.

Time trajectories indicated rapid ddhN clearance from the body through urine. The structural similarities between the nucleosides and their antiviral equivalents, which have brief half-lives in humans, indicated that a decrease in levels over time for the endogenous antivirals could indicate the recent introduction of short-term enzyme activity induced by immunological stimuli.

Overall, the study findings highlighted the presence of an extended RSAD2-dependent metabolic pathway that may contribute to an endogenous innate immunological defense mechanism against viral infections such as COVID-19. Measurement of the excretion of ddhN metabolites may be used reliably as a direct indication of viral infection and potential severity. The observed biomarkers may prove valuable in developing tools for early detection and monitoring of viral infections and may result in clinical actionability concerning acutely ill patients.