The viral genome

The SARS-CoV-2 genome is a 29.9-kb RNA that consists of 14 open reading frames (ORFs) encoding two large polyproteins (ORF1a and ORF1b) and 13 small ORFs that encode viral structural proteins and other polypeptides. Polyproteins from the large ORF1a/b are further arranged into 16 non‐structural proteins (nsp1 to nsp16)^1,29. The structural proteins comprise of nucleocapsid (N), membrane (M), envelope (E), and spike (S) proteins. The M and E proteins are located among the S-proteins in the viral envelope³⁰. Based on the structural map of SARS-CoV-2, about 6% of the viral proteome mimics human proteins, while nearly 7% has been implicated in cellular hijacking mechanisms, and about 29% of proteome self-assembles into heteromeric components to support viral replication³¹.

Virus–host interactome

Virus-human host protein–protein interactions play a major role in clinical outcomes of acute SARS-CoV-2 infection and its long-term sequelae, the PASC. A ribonucleoprotein (RNP) capture has identified a direct binding of SARS-CoV-2 RNA with 109 human host factors³². A comprehensive virus–host interactome of 29 viral (i.e., non-structural/structural) proteins, and 18 host/human cellular proteins (i.e., CSR, proteases, as well as restriction, replication, and trafficking molecules), showed an extensive involvement of >4780 unique high-confidence interactions of SARS-CoV-2 with human metabolome¹⁴. These diverse virus–host interactions could reprogram/dysregulate host cellular functions such as genomic, mitochondrial, lipidomic, and innate defense activities at various levels in human metabolism.

Viral binding/attachment to human cell surface receptors (CSRs)

The virulent outcome of a SARS-CoV-2 infection depends on (i) binding/interaction of viral S-protein with human cell surface receptors (CSR) and (ii) priming of S-protein by human cellular proteases^54,55. This infectious process is accomplished by viral hijack of cellular metabolic machinery through sequential steps of viral attachment, invasion, RNA replication, and propagation⁵⁶. The viral S-protein serves as an anchor to interact with host tissue, followed by sequential cleavage of S-protein to facilitate viral entry^57,58. The viral hijack of host cellular metabolic machinery during SARS-CoV-2 infection is depicted in Fig. ​Fig.1Fig.1

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Fig. 1

Viral hijack of host cellular metabolic machinery.

SARS-CoV-2 infection of a susceptible host is achieved through viral spike (S)-protein-mediated hijack of human cell surface receptors (ACE2 and/or NRP1) and cell membrane proteases. The S1-region on viral S-protein contains a receptor-binding domain (RBD) that specifically recognizes host cell surface receptor(s) and exposes the S2 site⁵⁵. For fusion with host cell membrane, the viral S-protein hijacks specific cellular proteases for activation (‘priming’) of viral S-protein at the S1/S2 region. Subsequent conformational changes to viral S-protein lead to S1 shedding by cleavage of S1/S2 fragments. This process facilitates insertion of fusion peptide (FP) into host membrane. Accordingly, proteolytic cleavage by cellular enzymes TMPRSS2 and/or furin accomplish the task of viral FP insertion into host cell membrane. Alternatively, SARS-CoV-2 could also hijack lysosomal protease cathepsin L (CTSL) for direct viral endocytosis, where the viral membrane fuses with luminal face of the endosomal membrane facilitating viral RNA transfer into the cytosol. Thus, SARS-CoV-2 could infect the human by hijacking these 5 major host cellular factors via different routes of entry and elicit a wide range of clinical outcomes. The angiotensin-converting enzyme 2 (ACE2)/TMPRSS2-mediated viral infection and/or the ACE2/CTSL-mediated endosomal route may result in full-spectrum symptomatic COVID-19. The alternative neuropilin 1 (NRP1)/furin-mediated route^62,63 may down-regulate human pain receptors and manifest as asymptomatic to mild disease outcomes.

The SARS-COV-2 S-protein hijacks human angiotensin-converting enzyme 2 (hACE2) to anchor on the host cell surface. The SARS-CoV-2 S/ACE2 complex undergoes conformational change for proteolytic priming/activation. The N-terminal S1 subunit contains receptor-binding domain (RBD) region, which avidly binds to the carboxypeptidase (CPD) domain on the hACE2 receptor and exposes the S2 site⁵⁵. Co-expression of ACE2 with membrane serine proteases is high on ileal absorptive enterocytes in the GI tract, nasal goblet secretory cells and type II pneumocytes in the respiratory tract, as well as on the urogenital epithelia^59,60.

SARS-CoV-2 may also infect host cells independent of the ACE2 receptor binding. The carbohydrate moieties on viral S-protein surface could facilitate viral internalization via innate immune factors, such as neuropilin (NRP)-1, C-lectin type receptors (CLR), and toll-like receptors (TLR), as well as the non-immune receptor glucose-regulated protein 78 (GRP78) for systemic spread of infection⁶¹. NRP1, a transmembrane glycoprotein involved in cardiovascular (CV), neuronal, and immune regulation, is also hijacked by SARS-CoV-2 for host cell surface binding⁶². NRP1, widely expressed in olfactory and respiratory epithelia, is shown to enhance TMPRSS2-mediated viral cell entry⁶³. NRP1 binds to S1 through a multi-basic furin-cleavage site (FCS) and promotes S1 shedding to expose the S2′ site for TMPRSS2 priming⁶⁴.

The S-protein of SARS-CoV-2 has a polybasic insertion (PRRAR) region at the S1/S2 site, which is readily cleaved by furin enzyme⁶⁵. Furin cleavage site (FCS) is an important determinant of SARS-CoV-2 transmission in the human population. After binding to ACE2 and/or NRP1 receptors, the S-protein is proteolytically pre-activated by human proprotein convertase furin⁶⁶. High-affinity interaction of ACE2 and/or NRP1 with the RBD of viral S-protein, followed by cell-mediated furin pre-activation could effectively facilitate host cellular entry of SARS-CoV-2 while evading host immune surveillance⁶⁷.

COVID-19/phase-I: hypoxia/hypoxemia

SARS-CoV-2 binding to host CSRs (i.e., ACE2, NRP1) is an initial step in the pathogenesis of COVID-19. Viral binding to ACE2 receptors on alveolar epithelia affects renin-angiotensin-aldosterone system (RAAS), subsequently lowers the blood pressure and lung function of an infected host⁸⁵. The reduced O₂ transport (hypoxia) triggers a mitochondrial metabolic reprogramming/dysregulation via alteration of OXPHOS/TCA cycle and activation of anaerobic glycolysis, known as the ‘Warburg Effect’^86,87. This shift in mitochondrial energy metabolism (or ATP synthesis) is regulated by different cellular systems, of which the hypoxia-inducible factor (HIF)-1α plays a critical role⁸⁸. HIF-1α induced HMRD affects the available host energy reserves for immune function⁸⁹. Ultimately, HIF-1α could impair host immune response, exacerbate inflammation, and inflict tissue damage. SARS-CoV-2 could evade host innate immunity and sustain intracellular viral replication cycle by altering the mitochondrial dynamics through targeting the mitochondria-associated antiviral signaling (MAVS) pathways⁹⁰ HIF-1α could up-regulate vascular endothelial growth factor (VEGF) to cause vascular leakage, damage epithelial barriers of alveoli and vascular endothelia^91,92. Therefore, phase-I of COVID-19 is considered a hypoxia-induced blood disorder, associated with FeRD and HMRD^27,93.

COVID-19/phase-II: hyperferritinemia

Severe COVID-19 is characterized by hyper-inflammation with elevated proinflammatory cytokines that stimulate the synthesis of both ferritin and hepcidin (which ultimately mediate FeRD)⁹⁴. The iron homeostatic imbalance is reflected by high iron content in reticuloendothelial cells and elevated serum ferritin levels. When the iron-binding capacity of transferrin (TF) in the blood exceeds, free iron is released into plasma in a redox-active state known as the labile plasma iron (LPI), which forms tissue-damaging free radicals and cause fibrosis⁹⁵. A ferritin/TF ratio >10 predicts a five-fold higher risk of ICU admission and an eight-fold higher risk for need of mechanical ventilation in COVID-19 patients⁹⁶. A dysfunctional hyperimmune response in tandem with macrophage activation could trigger hyperferritinemia, and ‘cytokine storm’ or cytokine release syndrome (CRS). CRS is characterized by fulminant activation of a large number of lymphocytes that release inflammatory cytokines and result in severe tissue damage with multi-organ dysfunction syndrome (MODS)⁹⁷. Hyperferritinemia, and FeRD collectively play a detrimental role in disease progression of COVID-19^27,98. Phase II of COVID-19 is considered a wide-spectrum hyperinflammatory disease, amplified by CRS from HMRD²⁷.

COVID-19/phase-III: thrombocytopenia

Acute COVID-19 due to severe iron toxicity from oxidized iron could modulate several systemic pathways of coagulation cascade and cause thromboembolism⁹⁹. SARS-CoV-2 could invade blood vessels, induce vascular damage, and activate systemic thrombotic events with severe to fatal coagulopathies in COVID-19 patients¹⁰⁰. Such coagulopathies (or blood clots) are characterized by elevated procoagulant factors such as fibrinogen, along with high levels of D-dimers linked to increased CFR^101,102. Hematological parameters such as anemia of inflammation (AI), reduced numbers of peripheral blood lymphocytes and eosinophils with increased neutrophil-to-lymphocyte ratios are recognized as major risk factors^103,104. This clinical phase along with hypoxia, could exhibit signs of hemolysis with the release of heme proteins and accumulation of free heme. The hemolysis-derived heme could initiate inflammatory OxS that may cause microvascular thrombosis, organ ischemia and MODS in severe COVID-19^102,105.

Virus-induced hypoxia/’Warburg’ effect

SARS-CoV-2 hijacks host cellular metabolic machinery to extract adequate energy and carbon skeletons to facilitate viral entry and facilitate molecular constructions for viral progeny inside a host cell for replication and propagation. The SARS-CoV-2 infection initiates complex human host-pathogen interactions and alters mitochondrial function with significant disruption of glycolysis/TCA cycle (Warburg effect), affecting several metabolic pathways of amino acid (AA), fatty acid (FA), nucleotide, and antioxidant synthesis^139,140. The virus-induced hypoxia/Warburg effect could potentially compromise endocrinal, cardiovascular, neurocognitive, gastrointestinal, pulmonary, and reproductive functions that demand high levels of mitochondrial O₂ consumption, OXPHOS, and ATP reserve. Failure to reset hypoxia/Warburg effect after viral clearance in COVID-19 survivors, could eventually evoke PASC with metabolic impairments including new onset T2DM, myocardial infarction, chronic fatigue syndrome (CFS), brain fog, and blood clotting issues¹⁴¹. Accordingly, PASC could be described as a SARS-CoV-2-induced chronic and self-perpetuating comprised state of m-Dys, where OxS potentially drives inflammation and shifts energy metabolism towards glycolysis while down-regulating OXPHOS^27,142,143. Long-term consequences of virus-induced hypoxia/Warburg effect could amplify potential risks of HMRD with chronic multi-organ impairments in PASC (Fig. ​(Fig.44).

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Fig. 4

Virus-induced HMRD: hypoxia and ‘Warburg’ effect.

Dysregulation of glycolysis/TCA cycle is a key feature of HMRD. COVID-19 patients exhibit elevated serum glucose levels with an upregulation of glycolytic intermediates. Glutamine deficiency and hyaluronan over synthesis are HMRD-induced metabolic events in SARS-CoV-2 infection⁷⁸⁵. M1 macrophages express nitric oxide synthase (NOS), which oxidizes arginine to nitric oxide (NO•) and citrulline. NO• modulates vascular tone, blood pressure and hemodynamics. Disrupted arginine metabolism further down-regulates NO• synthesis, aggravates endothelial dysfunction and triggers severe coagulopathies in COVID-19¹⁸⁴. Downstream generation of amino acids ornithine, citrulline, arginine in the circulation also indicates a severe renal dysfunction⁵¹. Degradation of sphingomyelin by acid sphingomyelinase (ASM) generates stimulatory ceramides, the docking molecules for phospholipase A2 (PLA₂). The hydrolysis of phospholipids (i.e., phosphatidyl choline) by PLA₂ elevates arachidonic acid levels, a precursor for broad spectrum eicosanoids produced by cyclooxygenase (COX) and lipoxygenase (LOX) enzymes. These enzymes further convert arachidonic acid to prostaglandins (PGs), thromboxanes (TXs), and leukotrienes (LTs), which collectively contribute to the development of vascular inflammation and disease severity in COVID-19⁷⁸⁶. Virus-induced HMRD alters host lipid metabolism with major impact on sphingolipid and arachidonic acid pathways⁷⁸⁷. A decline in fat-soluble antioxidants’ vitamin E and carotenoids could compromises ROS quenching capacity in the plasma membrane, causes lipid peroxidation and OxS. Elevated serum lipase levels indicate damaging clinical outcomes in COVID-19 patients⁷⁸⁸. The virus-induced HMRD alternations to glucose, amino acid, and lipid metabolism could aggravate the severity of COVID-19 and may extend to PASC pathology.

Iron (Fe)-redox dysregulation (FeRD)

During SARS-CoV-2 infection, free iron released into the circulation induces inflammation of alveolar macrophages and causes oxidative damage to the lungs¹⁴⁴. Increased iron load increases blood viscosity with recurrent diffused micro/macro circulatory thrombosis leading to high levels of D-dimers in COVID-19 patients. Altered iron metabolism, iron-restricted erythropoiesis from hyperinflammation causes FeRD^27,145. In COVID-19 patients, FeRD could trigger several clinical manifestations including (i) decrease functional hemoglobin (Hb), (ii) increase cellular iron overload, (iii) release free toxic heme into the circulation, (iv) manifest hypoxemia and systemic hypoxia, (v) reduce nitric oxide (NO•) synthesis, (vi) activate coagulation pathway(s), (vii) trigger ferroptosis with OxS and lipid peroxidation, and (viii) induce mitochondrial degeneration^27,146.

On the other hand, viral protein sequences could form complexes with porphyrin, affect heme on the 1-β chain of Hb, and release free iron¹⁴⁷. SARS-CoV-2 envelope (E) protein directly binds to heme (from Hb) released from damaged erythrocytes and lysed phagocytes¹⁴⁸. The viral genomic ORF8 protein could interact with the 1β-chain of Hb, capture the porphyrin and inhibit heme metabolism in the body¹⁴⁹. Such an array of SARS-CoV-2 interactions with Hb could induce hemolysis and/or form complexes with released heme, generate dysfunctional Hb (hemoglobinopathy) with reduced ability to transport O₂/CO₂ and lead to O₂ deprived multi-faceted syndromes, including coagulation disorders^146,150. In severe stages of COVID-19, other Hb-associated markers such as bilirubin and ferritin progressively increase and worsen the clinical outcomes.

The FeRD-induced hyperferritinemia strongly correlates with different inflammatory phases of SARS-CoV-2 infection^98,101,151. In SARS-CoV-2 infected patients, the plasma levels of ferritin and IL-6 steadily decrease with gradual recovery from COVID-19^152,153. FeRD is highly prevalent among hospitalized COVID-19 patients and this clinical condition may continue for weeks or even months in PASC patients. Biomarkers of iron metabolism (i.e., ferritin, transferrin (TF), lactoferrin (LF), etc.) and Hb could provide risk stratification strategies for COVID-19 management. FeRD determinations are specific and sensitive to predict disease severity in COVID-19 and PASC patients^27,154.

Mitochondrial dysfunction (m-Dys)/altered bioenergetics

The mitochondrion is the cellular powerhouse involved in oxidative phosphorylation (OXPHOS), ATP synthesis, and regulation of calcium (Ca²⁺) signaling, redox homeostasis, lipid metabolism, cell differentiation, immune system, apoptosis, and cellular senescence (aging)^155,156. These vital processes are perturbed when the host cellular machinery is hijacked by SARS-CoV-2, which ultimately manifests as mitochondrial dysfunction (m-Dys). SARS-CoV-2 infection leads to m-Dys including mitochondrial membrane depolarization, mitochondrial permeability transition pore opening, increased release of reactive oxygen species (ROS), and disrupted. mitochondrial redox homeostasis^157,158. SARS-CoV-2 infection also affects fusion/fission kinetics, size, structure, and distribution of mitochondria in the infected host cells. COVID-19 patients with underlying primary mitochondrial disease and secondary m-Dys are prone to increased disease severity and CFR compared to patients with healthy mitochondrial functions¹⁵⁹. Thus, m-Dys could heavily compromise host bioenergetics with detrimental consequences on COVID-19 and long-term PASC patients^160,161.

After host cell entry, the ORF9b of SARS-CoV-2 RNA could directly manipulate mitochondrial function to evade host cell immunity, facilitate viral replication and trigger the onset of COVID-19. The ORF9b could further manipulate host mitochondria by releasing mitochondrial DNA (mt-DNA) into the cytoplasm to activate mt-DNA-induced inflammasome and suppress innate as well as adaptive immunity¹⁶². SARS-CoV-2 may also manipulate mitochondrial function via ACE2 regulation. A decline in ACE2 function in aged individuals, coupled with the age-associated deterioration in mitochondrial functions results in chronic metabolic disorders like diabetes or cancer, and predisposes the host for increased susceptibility to infection, vulnerability to health complications, and intensifies the risk of mortality¹⁶³.

SARS-CoV-2 invades mitochondria and evades host defense by the formation of double-membrane vesicles. These virus-induced vesicles could damage mitochondrial membrane integrity, release mt-DNA into circulation, compromise innate immunity, and trigger an exacerbated pro-inflammatory response in COVID-19 patients¹⁶⁴. SARS-CoV-2 infection could alter mitochondrial function(s), activate TLR9 signaling, induce hyper-inflammation and disrupt endothelial activity¹⁶⁵. The viral infection could also cause rapid T lymphocytopenia with functional impairment of T cells, which may onset OxS, pro-inflammatory state, cytokine production, and apoptosis^166,167. Hyper-inflammation (with CRS or cytokine storm) due to massive outburst of ROS, is a prominent clinical feature of COVID-19¹⁴⁵. The mitochondrion is a significant source of ROS in human cellular metabolism that could trigger the onset and development of cytokine storm¹⁶⁸.

SAR-COV2 could induce m-Dys, activate mitochondrial-dependent intrinsic apoptotic pathways, and cause microglial and neuronal apoptosis leading to neuropathological symptoms in COVID-19 and PASC patients^169,170. In the current pandemic, about 40% of COVID-19 patients demonstrated neurological symptoms, lingering neuro-inflammation, where neuronal damage in PASC patients has emerged as a novel syndrome, the ‘Neuro-COVID’^169,171. Peripheral blood monocytes of such patients demonstrate altered bioenergetics and reduced basal respiration, reduced spare respiratory capacity, and decreased proton leak¹⁷². The m-Dys-induced exercise intolerance with elevated arterial blood lactate levels and reduced fatty acid β-oxidation rates is a major health issue in PASC¹⁷³. These patients complain about chronic fatigue during exercise, despite no obvious heart or lung abnormalities¹⁷⁴.

During the aging process, progressive m-Dys occurs due to the loss of thioretinaco-ozonide-oxygen-ATP complex from mitochondrial membranes through the opening of mitochondrial permeability transition pore¹⁷⁵. Disruption in mitochondrial OXPHOS could elevate OxS and activate sepsis cascade through HIF-α/Sirtuin pathway. Due to m-Dys, senescent cells fail to meet the hyper-metabolic demands of sepsis in COVID-19 patients. A decline in mitochondrial function in the aging population could be a possible risk factor for increased mortality in COVID-19 and PASC¹⁷⁶. Furthermore, as a hallmark of the aging population, m-Dys could onset chronic inflammation with massive cytokine release and cause multi-organ failure with fatal outcomes in elderly COVID-19 patients¹⁷⁷. Age-related comorbidities (metabolic syndromes) such as, obesity, T2DM, asthma, and CVD, could also increase severity and mortality in elderly COVID-19 patients. Preventive therapies to improve mitochondrial turnover, dynamics and activity could prove beneficial in protection against COVID-19 severity¹⁷⁸. Therefore, nutritional targeting of mitochondrial metabolism could showcase as an effective treatment regimen for PASC management.

Oxidative stress (OxS)/cellular damage

Oxidative stress (OxS) is a nonspecific pathophysiological condition that reflects a redox imbalance between increased production of ROS (free radicals) and the inability of antioxidant defenses to neutralize the reactive intermediates or to repair the ensuing damage^179,180. ROS disrupts cellular metabolism by inflicting DNA strand breaks, protein degradation, lipid peroxidation, and cellular damage¹⁸¹. Combined with inflammation, OxS contributes to cardinal patho-mechanisms of both COVID-19 and PASC¹⁸².

After SARS-CoV-2 infection, the viremia stage could increase OxS, elevated levels of ROS/inflammation markers (i.e., peroxide, NO•, carbonylated proteins, and IL-6) and inflict severe cellular/tissue damage. This clinical condition may compromise mitochondrial functions and trigger apoptosis of leukocytes¹⁸³. Hyper-inflammation, pro-oxidant cytotoxic milieu, and early apoptosis of leukocytes from SARS-CoV-2 infection, could cause severe endothelial-alveolar injury and MODS¹⁸⁴. PASC patients exhibit a wide range of tissue/organ damage involving pulmonary, cardiovascular, neuro-cognitive, GI, reproductive, and dermatological systems^5,127,185.

Immuno-pathogenesis/hyper-inflammation

SARS-CoV-2 infection could disrupt host immune homeostasis, inflict tissue injury, and may persist during the post-recovery phase of COVID-19 survivors and manifest as PASC²¹⁰. Cell-mediated immune responses with antigen-specific T cells decrease in COVID-19 patients and affect viral clearance from infected host cells²¹¹. Cytotoxic T cells elevate in peripheral blood and bronchoalveolar lavage of PASC patients with severe airway dysfunction with persistent respiratory symptoms that last for 3 to 6 months²¹². SARS-CoV-2 induced T-cell imbalance resolve over time; however, the markers upregulated from T-cell exhaustion may remain up to 1 year in PASC patients^213,214.

Elevated levels of neutrophils with neutrophil extracellular traps (NETs and NETosis) and associated immune-thrombosis are prominent features of COVID-19 pathology^215,216. Monocytes and macrophages mediate lung fibrotic tissue injury, a drastic consequence in the immunopathogenesis of SARS-CoV-2 infection²¹⁷. Myeloid cells may also incite local fibrosis-mediated tissue injury and sustain proinflammatory cytokine levels contributing to the clinical development of PASC. COVID-19 patients with severe disease exhibit increased monocyte counts with higher frequencies of classical monocytes, lower frequencies of intermediate/non-classical monocytes and elevated plasma levels of C-reactive protein (CRP) and serum TF in comparison to mild disease. This abnormal immune response may persist for >6 months after COVID-19 recovery²¹⁸. Monocyte alterations in acute COVID-19 patients include aberrant expression of leukocyte migration molecules that extend to convalescence and correspond to specific symptoms of PASC. Monocytes from PASC patients with ongoing fatigue show a sustained reduction of prostaglandin-generating enzyme, the cyclooxygenase 2 (COX-2)²¹⁹. Circulating monocytes may remain dysregulated, especially in convalescent subjects for 1 to 3 months of post-COVID.

‘Cytokine storm or CRS’, a clinical state of hyper-inflammation, is a prominent feature of COVID-19 severity, linked to respiratory dysfunction, ARDS with adverse disease outcomes^220,221. About 10% of patients recovered from COVID-19 show persistent symptoms up to 6 months after initial SARS-CoV-2 infection. Cytokine storm, in tandem with lymphopenia, lymphocyte dysfunction, and granulocyte/ monocyte abnormalities, could increase the disease severity of COVID-19²²². Immune cytokine signatures of PASC patients reflect an ongoing chronic inflammation and angiogenesis with elevated plasma levels of IL-17a, stem cell factor, IL-12p70, IL-1β, macrophage inflammatory protein-1β (MIP-1β), brain-derived neurotrophic factor (BDNF), and VEGF²²³ Also, among other immune mediators, reduced levels of cortisol strongly correlate with pulmonary-PASC symptoms²²⁴. Hyperimmune activation and autoimmunity are considered as potential causative factors in the onset of PASC²²⁵.

Autoimmunity

In COVID-19 patients, autoantibodies against nuclear bodies (auto-nuclear antibodies—ANA), phospholipids, type I interferon (IFN), melanoma differentiation-associated protein 5 (MDA5), and ACE2 have been reported²²⁶. These autoantibodies against immunomodulatory proteins (including cytokines, chemokines, complement components and cell-surface proteins) could attack tissues of patients, thereby impair host cell signals, perturb immune function, damage organ systems, and increase COVID-19 severity³⁶. Human leukocyte antigen (HLA) genetic polymorphism has also been observed in COVID-19 patients²²⁷. HLA polymorphism plays a key role in the onset of several autoimmune diseases²²⁸. Evidently, SARS-CoV-2 infection could elicit auto-inflammatory and autoimmune disorders such as Guillain-Barré syndrome (GBS), autoimmune hemolytic anemia, immune thrombocytopenic purpura, and Kawasaki disease (KD)^12,229,230. Mechanisms of COVID-19-derived autoimmune disorders include (i) viral-mediated host hyper-immune response, (ii) virus-induced excessive NETs formation with neutrophil-associated cytokine responses, and (iii) the molecular mimicry between viral antigenic components and host molecules²³¹.

Latent autoimmunity correlates with humoral responses against SARS-CoV-2 infection and autoimmunity has emerged as a prominent feature of PASC pathology²³². Long-term persistence of immune activation and proinflammation with latent and overt autoimmunity are etiological factors in clinical manifestation(s) of PASC²³³. About 20 distinct autoantibodies that target G-protein-coupled receptors (GPCR) of the CNS and ACE2/RAAS-related molecules were linked to the clinical severity of PASC²³⁴.

Dysbiosis

Gut dysbiosis is defined as the reduction in diversity of GI microflora or depletion of autochthonous or host commensal beneficial bacteria with an enrichment of microbial pathogens that may alter host susceptibility to SARS-CoV-2 infection^235,236. Impairment of short-chain fatty acid (SCFA) and l-isoleucine biosynthesis in gut microbiome persists beyond 30 days after recovery from COVID-19, which could contribute to persistent leaky gut and dysbiosis in PASC patients^237,238. Notably, even after viral clearance, more than half of patients suffer from PASC with persistent dysbiosis, deregulated GI metabolism and compromised host immune response^14,127. Gut dysbiosis and disrupted intestinal barrier function could worsen pulmonary symptoms, augment neurological or hepatic inflammation through translocation of endotoxins and bacteria via portal veins^239–241. Dysbiosis of gut microbiome with ensuing gut barrier dysfunction could severely impact patho-physiologies of both COVID-19 and PASC²⁴².

The GI tract is the largest immunological organ in the body and any aberrant immune response to SARS-CoV-2 infection induced by resident microflora could affect recovery from COVID-19. Dysbiosis may lead to GI impairment with persistent symptoms of diarrhea and abdominal pain²⁴³. Gut dysbiosis could also increase susceptibility to respiratory infections, alter immune responses and affect lung homeostasis (the ‘Gut-Lung Axis’). Persistent gut dysbiosis after resolution of COVID-19 may be linked to PASC, particularly to neurological manifestations²⁴⁴. In a prospective follow-up study from Wuhan, China, patients (n=187) recovered from COVID-19 demonstrated a strong correlation to gut microbiota dysbiosis and PASC symptoms, 1-year after hospital discharge²⁴⁵. In a subset of patients recovered from COVID-19, long-term dysbiosis correlated with PASC symptoms, especially fatigue, joint pain, diarrhea, headache, depression, and anxiety²⁴⁶. Dysbiosis in tandem with excess antibiotic use during the pandemic possibly have contributed to an array of PASC manifestations²⁴⁷. Since the diversity of gut microbiota erodes during aging, dysbiosis could also be a reason for high susceptibility of older adults to severe COVID-19²⁴⁸.

SARS‐CoV‐2 infection inflicts sustained metabolic damage to gut microbiome and GI function; therefore, opportunistic pathogens could selectively enrich in fecal microflora of COVID-19 patients^249,250. Fecal enrichment of bacterial pathogens, such as Coprobacillus spp., Clostridium ramosum, and Clostridium hathewayi, directly correlates with the severity of SARS‐CoV‐2 infection. Conversely, symbiotic gut microflora (Bifidobacteria, Roseburia and Faecalibacteria) with prominent immunomodulatory functions, are extinguished from the gut of PASC patients²⁵¹. Accordingly, SARS-CoV-2 infection could inflict direct cytopathic injury to gut epithelia and elicit indirect immune-mediated damage to endothelial cells²⁵². SARS-CoV-2 could also induce GI inflammation, dysregulate intestinal ACE2 activity, and/or infect gut microflora (similar to bacteriophage-type transduction), as three potential inter-connected mechanisms in gut dysbiosis in PASC²⁵³. Effective clinical management of PASC is contingent upon the following critical evaluation of dysbiosis: (i) the duration of gut dysbiosis after COVID recovery, (ii) the link between gut dysbiosis and long-term persistent symptoms, and (iii) the possible adverse health effects from enriched or depleted specific gut microflora on COVID-19 recovered individuals²⁵⁴. Therefore, reversal of dysbiosis and restoration of normal GI function could alleviate PASC symptoms and support patient recovery.

Reactivation of latent viral pathogens

Since the onset of COVID-19 pandemic, a strong correlation between SARS-CoV-2 infection or COVID-19 vaccination and herpesvirus co-infection/reactivation has been reported²⁵⁵. To date the reactivation of eight human herpesviruses (HHVs) have been identified, including Herpes Simplex Virus types 1 (HSV‐1) and 2 (HSV‐2), Varicella‐Zoster Virus (VZV or HHV‐3), Epstein-Barr Virus (EBV or HHV‐4), Cytomegalovirus (HCMV or HHV‐5), HHV‐6, HHV‐7, and Kaposi’s Sarcoma‐associated Herpesvirus (KSHV or HHV‐8). Almost 100% of the adult population in the world is infected with at least one HHV during their life²⁵⁶. A meta-analysis (n=32 studies) has estimated the prevalence of HHV reactivation in hospital-ICU-admitted COVID-19 cases at 38% for HSV, 19% for CMV, 45% for EBV, 18% for HHV-6, 44% for HHV-7, and 19% for HHV-8, respectively²⁵⁷.

The incidence of HHV reactivation was found high among patients admitted to the ICU for severe COVID-19 and among individuals administered with COVID-19 vaccine²⁵⁸. Simultaneous occurrence of cytokine storm and immune suppression during SARS-CoV-2 infection may lead to reactivation of latent HHV in the body. Lymphopenia with reduced CD8+ levels and elevated CD4+/CD8+ ratio indicate the severity of COVID-19^215,259. This clinical condition leads to an immune-suppressed state, which could ultimately trigger the reactivation of latent HHV and aggravate SARS-CoV-2 infection²⁶⁰. In addition to viral co-infection, anti‐COVID‐19 therapies (i.e., azithromycin, nafamostat mesylate, and remdesivir) could activate various cell signaling pathways and trigger viral lytic reactivation²⁶¹. Remdesivir, a widely administered anti‐COVID‐19 drug, is shown to induce lytic reactivation of KSHV and EBV, from virus‐associated lymphoma cells²⁶².

SARS-CoV-2 infected patients demonstrate a wide spectrum of cutaneous manifestations, including maculopapular or perifollicular rash, urticaria, vesicles, petechiae, purpura, livedo racemosa, and pseudo-chilblains, often referred to as the ‘COVID toes’^263,264. These cutaneous manifestations of COVID-19 are reportedly associated with reactivation of latent HHVs^265,266. Reactivation of HSV-1 coincides with decreased expression of IFN-stimulated genes and concurrent increase in highly activated T-lymphocytes during acute stages of SARS-CoV-2 infection²⁶⁷. Reactivation of EBV could enhance the severity of SARS-CoV-2 infection. SARS-CoV-2 infected patients with EBV co-infection are prone to high fever with elevated levels of CRP, and aspartate aminotransferase²⁶⁸. A study from China reported higher mortality rates in COVID‐19 cases with EBV reactivation (29.4%) compared to EBV-negative patients (8.1%)²⁶⁹.

An exhausted dysfunctional antiviral immune response from SARS-CoV-2 infection could trigger reactivation of human adenovirus with a sequelae effect of ME/CFS in PASC patients²⁷⁰. Immuno-compromised individuals susceptible to HHV infection are at higher risk for SARS-CoV-2 infection, PASC, are vulnerable to develop virus‐associated cancers. Several HHVs, such as KSHV, and EBV are oncogenic viruses; therefore, a follow‐up surveillance of COVID‐19 survivors, PASC patients, and vaccinated individuals for possible risk(s) of latent viral reactivation is an important preventive public health strategy²⁵⁸.

Persistent viral reservoirs

Infectious viral particle clears out and remain undetectable in the body for most COVID-19 cases; however, among certain patients, SARS-CoV-2 could persist for months after post-recovery²⁷¹. Accordingly, total clearance of SARS-CoV-2 RNA or its protein antigens from host infected tissue may take a longer time, while the virus and its antigenic fragments continue to remain dormant for extended periods of time in the body. The persistence of SARS-CoV-2 or its viral components in the body could trigger a dysregulated immune response and proinflammatory cytokine release, which may cause chronic low-grade inflammation and MODS. These acute sequelae also have a genetic basis that may predispose COVID-19 survivors to a compromised immune status consequently affecting viral clearance²⁷².

Multi-organ viral tropism predominantly in cells expressing ACE2, TMPRSS2, or both has been reported^82,83. Viral shedding (as detected by RT-PCR) may be prolonged in certain tissues of post-COVID patients for an extended duration in the lower respiratory tract (59 days), serum (60 days), upper respiratory tract (83 days), and feces (126 days)²⁷³. Such viral persistence could serve as a chronic trigger for inflammation and cellular activation that may further inflict tissue damage and elicit PASC-related symptoms²⁷⁴. In the long-term persistence of COVID-19-associated anosmia (loss of smell), viral transcripts are detected in the inflamed olfactory mucosa. Viral persistence and associated inflammation in olfactory neuro-epithelium may account for prolonged or relapsing symptoms in PASC, such as anosmia²⁷⁵. Delayed immune clearance of SARS-CoV-2 antigen(s) or duration of viral antigen burden in the upper respiratory tract and other anatomical sites during acute COVID-19 could be linked to the development of PASC²⁷⁶.

Long-term shedding of SARS-CoV-2 is widely reported, even after resolution of symptomatic COVID-19. The continuous replication of live SARS-CoV-2, its viral RNA, or viral protein fragments could play a major role in the clinical onset of PASC. SARS-CoV-2 RNA could persist for several weeks in the respiratory tract of COVID-19 survivors²⁷⁴. Viral replication has been reported in multiple respiratory and non-respiratory tissues, including the brain. Persistent shedding of SARS-CoV-2 was detected for months in the feces of patients recovered from COVID-19, regardless of GI symptoms²⁷⁷. In gut mucosa of mild to acute cases of COVID-19 (with IBD as comorbidity), the persistence of SARS-CoV-2 RNA was detected in ~70% PASC patients, whereas the viral nucleocapsid (N) protein was found in ~50% of PASC patients after seven months of post-recovery²⁷⁸. Such SARS-CoV-2 antigen persistence in infected tissues could possibly trigger immune perturbations that may contribute to the development of PASC.

Also, persistent viral RNA has been detected in multiple tissues of recovered patients even months after the onset of COVID-19. In a cohort of COVID-19 patients with persistent symptoms, about 45% showed detectable plasma SARS-CoV-2 RNA. Viral RNA was also found in blood, stool, and urine of PASC patients²⁷⁹. Spike and/or viral RNA fragments could persist in COVID-19 recoverees up to 12 months or longer^280,281. The S1 antigen in peripheral blood monocytes could remain up to 15 months after SARS-CoV-2 infection²⁸². The S-protein of SARS-CoV-2 contains structural motifs that affect T-cell receptors and trigger hyperinflammatory responses observed in severe COVID-19 and multi-system inflammatory syndrome in children (MIS-C)²⁸³. Spike may not activate cytokine storm in PASC patients; however, it could impair endothelial function via down-regulation of ACE2 and disrupt the integrity of BBB^284,285. In summary, viral persistence could play a major role in PASC, considering the ability of the SARS-CoV-2 pathogen to infect and reinfect individuals over a lifetime²⁷⁴.

Viral-hijacked human angiotensin-converting enzyme-2 (hACE2)

SARS-CoV-2 binds to the human angiotensin-converting enzyme-2 (hACE2) as a potential CSR for anchoring to specific cellular tissue sites in the body^286–288. Human cells that express ACE2 are potential targets for SARS-CoV-2 infection; however, other cellular factors such as human proteases that prime the viral S-protein are also critical for the next sequential steps of the viral infection process^55,289. The hACE2 levels are highest in the small intestine, testis, kidneys, heart, thyroid, and adipose tissue; moderately present in the lungs, colon, liver, bladder, and adrenal glands; lowest in the blood, spleen, bone marrow, brain, blood vessels and muscle²⁹⁰. ACE2 exists either in free soluble form (sACE2) or in bound form immobilized on cell membranes (mACE2) of intestinal, renal, testicular, gall bladder, pulmonary, and cardiovascular (CV) epithelia²⁹¹. Both soluble and membrane-bound ACE2 proteins are critical for the regulation of blood pressure in the body.

Viral-hijacked human neuropilin (hNRP)-1

SARS-CoV-2 anchors to human ACE2 as its primary host CSR; however, the broad-spectrum tissue tropism of COVID-19 raises the possible involvement of other host receptors in the binding of SARS-CoV-2 to other target tissue sites. Neuropilin-1 (NRP1) is another prominent CSR that facilitates entry of SARS-CoV-2 into the CNS through olfactory epithelium in the nasal cavity⁶³. Also, NRP1 (but not ACE2) serves as the principal CSR to mediate SARS-CoV-2 infection of astrocytes in the brain tissue. Viral infection of astrocytes resembles reactive astrogliosis with elevated type-I IFN production, increased inflammation, and down-regulation of transporters for water, ions, choline, and neurotransmitters³¹². These events lead to dysfunction and death of uninfected bystander neurons that could inflict severe symptoms of neuro-COVID including anosmia, ageusia, headache, delirium, acute psychosis, seizures, and stroke^171,197.

NRP1 avidly binds to furin-cleaved S1 fragment of viral S-protein and potentiates cellular entry of SARS-CoV-2⁶³. NRP1 is abundantly expressed in the respiratory and olfactory epithelia, with the highest localization in endothelial and epithelial cells. Blocking of NRP1/S-protein interaction with small-molecule inhibitors or monoclonal antibodies (mAbs) may reduce SARS-CoV-2 infectivity and provide potential intervention strategies for COVID-19 management⁶².

Viral-Hijacked Human Furin

SARS-CoV-2, the etiological agent of COVID-19 pandemic, contains a unique insertion of amino acids (AAs), exclusively proline-arginine-arginine-alanine-arginine (PRRAR₆₈₅↓) at the S1/S2 boundary of its S-protein, which is clearly absent in SARS-CoV and other related hCoVs⁷². Interestingly, this PRRAR insertion generates a ‘furin cleavage site’ on S-protein at the S1/S2 multi-basic region, considered as a potential ‘gain of function’ for the viral pathogen. Furin-mediated priming of viral S protein at S1/S2 (PRRAR₆₈₅↓) [the underlined AAs refer to critical residues needed for the furin recognition] is a key determinant in the pathogenesis of COVID-19^55,73,329.

Furin is a member of the proprotein convertase (PC) family of enzymes, known to process latent precursor proteins into their biologically active state³³⁰. Intracellular proteolytic processing of precursor polypeptides is an essential step in the maturation of many proteins such as plasma proteins, hormones, neuropeptides, and growth factors³³¹. Due to their homology with bacterial subtilisin and yeast kexin proteases, PCs are also known as the PC subtilisin/kexin type (PCSK) enzymes³³⁰. Humans encode nine members of the PCSK family (from 1–9), where the PCSK3 represents ‘furin’³³². Most viral envelope glycoproteins, like bacterial exotoxins, also require proteolytic cleavage to mediate entry into host cells. Accordingly, viral pathogens hijack cellular endo-proteases, such as furin/PCSK3 that prime polybasic cleavage sites and provide critical access for tissue tropism and viral spread in an infected host³³³. Accordingly, the canonical polybasic ‘furin cleavage site’ has been reported in several enveloped viruses, including Herpes-, Corona-, Flavi-, Toga-, Borna-, Bunya-, Filo-, Orthomyxo-, Paramyxo-, Pneumo, and Retroviridae³³².

Viral-hijacked human transmembrane protease, serine 2 (TMPRSS2)

Human transmembrane protease, serine 2 (TMPRSS2) has been identified as a key host cell factor that determines the route of viral entry for SARS-CoV-2 infection and the pathogenic spectrum of COVID-19³⁵⁵. Specifically, TMPRSS2 processes the SARS-CoV-2 S-protein and enables the viral entry into host cells within <10min in a pH-independent manner. In TMPRSS2-defecient cellular tissue sites, SARS-CoV-2 is endocytosed into lysosomes, and an alternative route of viral entry into the cytosol is achieved in about 40–60min of post-infection via acid-activated CTSL protease³⁵⁶. TMPRSS2 cleaves the viral S protein at multiple sites, including the canonical S1/S2 cleavage site³⁵⁵. TMPRSS2 expression is high in the human prostate gland under androgenic hormone regulation. As an apical surface serine protease, TMPRSS2 regulates epithelial sodium homeostasis in the prostate gland and plays a vital role in male reproduction³⁵⁷. In normal prostate, TMPRSS2 is involved with proteolytic cascades to activate prostate-specific antigen (PSA) in the seminal fluid (like fibrinolytic blood coagulation)³⁵⁸. TMPRSS2 expression is high in ciliated cells and type I alveolar epithelia (AT1), which is known to upregulate with ageing³⁵⁹. This may explain the relative protection of infants and children from severe respiratory illnesses. Recently, both TMPRSS2 and ACE2 were detected in human corneal epithelia, which suggests that the ocular surface is a potential route of cellular entry for SARS-CoV-2³⁶⁰.

Nutritional reset of hypoxia/’Warburg’ effect

Dysregulation of glucose metabolism with elevated serum glucose levels and upregulation of glycolytic intermediates is a prominent feature of COVID‐19. Glucose metabolism supports OXPHOS/TCA cycle (ATP synthesis) in mitochondria and generates malate, an indicator of mitochondrial activity. Interestingly, plasma malate levels dwindle after SARS‐CoV‐2 infection, which suggests m-Dys due to hypoxia/Warburg effect in the pneumonia state^27,51. The SARS-CoV-2-induced hypoxia activates gluconeogenesis and porphyrin (or iron) metabolism, thereby steers the clinical onset/progression of COVID-19. Furthermore, the hypoxia-mediated ‘Warburg’ effect also alters both TCA cycle as well as lipid metabolism through perturbation of tryptophan biosynthesis, aminoacyl-tRNA degradation, phenylalanine, and arachidonic metabolism⁴⁰⁶. Dysregulated arachidonic acid metabolism and fatty acid β‐oxidation in tandem with platelet aggregation and bile acid synthesis help identify SARS-CoV-2 infected asymptomatic individuals, the hidden drivers of COVID-19 pandemic as well as massive victims of PASC^21,407.

l-Arginine metabolism is vital for immune and vascular functions in the body⁴⁰⁸. Its metabolic functions include conversion to nitric oxide (NO•) by NO synthase (NOS) or arginine catabolism to ornithine by arginase⁴⁰⁹. NO• is master regulator of cardiovascular function, metabolism, neurotransmission, and immunity⁴¹⁰. Upregulation of arginase depletes serum levels of arginine, subsequently inhibits NO• production. Accordingly, low availability of plasma arginine has been implicated in endothelial dysfunction, T cell dysregulation, and thrombocytopenia (coagulopathy) in ARDS patients, the most severe form of COVID-19^408,411. Thus, restoring optimum levels of arginine through oral supplementation could maintain immune homeostasis, particularly to reverse altered T cell activity and reset T cell/macrophage functions in PASC patients^412,413.

Perturbations in l-arginine metabolism is prevalent among COVID-19 and PASC patients, across all disease stages^414,415. Oral supplementation of l-arginine (1.66g twice a day) for 10 days in COVID-19 patients (n=101) show significant reduction both in respiratory/ventilation support (71.1%) and in-hospital stay⁴¹⁶. Oral supplementation with l-arginine (1.66g/d), in a human RCT (n=110) during COVID-19 pandemic showed patient improvement in cardiac rehabilitation after myocardial infarction and cardiac revascularization⁴¹⁷. Interestingly, arginine depletion with arginine-metabolizing enzymes has also been suggested as a therapeutic approach to block viral proliferation during acute COVID-19⁴¹⁸. However, the persistent hyper-inflammatory state and immune dysregulation which is common among virus-free PASC patients, arginine supplementation may prove beneficial to this group. In PASC pathology, the markers of NO• bioavailability are low; accordingly, oral supplementation of these patients with l-arginine + vitamin-C for 4 weeks could significantly elevate serum l-arginine levels and thereby increase NO• bioavailability⁴¹⁹. In another human RCT (n=50), oral supplementation of l-arginine (1.66g/d) + vit-C (500mg/d) for 4 weeks restored physical performance, endothelial function, fatigue, and relieved persistent symptoms in PASC patients⁴¹⁵. Oral supplementation of l-arginine could be a promising nutritional remedial to reset HMRD-associated cardiovascular disorders and immune dysfunction in PASC patients.

l-Tryptophan metabolism is the most prominent pathway affected by HMRD during SARS-CoV-2 infection. The virus-induced re-wiring of l-tryptophan metabolism alters kynurenine pathway, dysregulates host inflammatory and immune responses⁴²⁰. The l-tryptophan-kynurenine pathway is a regulatory ‘hub’ for canonical and non-canonical transcription, macrophage release of cytokines, which could trigger hyper-inflammation and cause poor clinical outcomes in COVID-19 and PASC⁴²¹. Major clinical manifestations in PASC such as depression, fatigue, sleep disturbances, attention disorders, anxiety, muscle weakness, and dyspnea are directly linked to ‘kynurenine shunt’, which is known to increase l-tryptophan degradation towards kynurenine and away from serotonin synthesis^422–424.

l-tryptophan is an essential AA, vital for biosynthesis of neurotransmitter serotonin (5-hydroxytryptamine, 5-HT), sleep hormone melatonin and co-factor NAD⁺ through its downstream metabolic pathways⁴²⁵. l-tryptophan catabolism by indoleamine-dioxygenase (IDO) through the kynurenine pathway generates several bioactive metabolites collectively referred to as kynurenines⁴²⁶. SARS-CoV-2 infection could deplete plasma l-tryptophan levels and increase IDO-stimulated generation of neuroactive tryptophan catabolites, including kynurenine⁴²⁷. Kynurenine is precursor for the vital cellular effector NAD⁺ and for several other intermediate metabolites that modulate immune and neuronal functions. SARS-CoV-2 infection deprives the host for NAD⁺ by inhibiting the biosynthetic pathway from quinolinic acid, and simultaneously acquiring NAD⁺ from nicotinamide in a salvage pathway⁴²⁸. Thus, l-tryptophan metabolism via kynurenine pathway produces niacin (vit-B3), a building block for NAD⁺ synthesis. In oxidized form, NAD⁺ is a potent inhibitor of pro-inflammatory cytokines and ventilator-induced acute lung injury (ALI) in COVID-19⁴²⁹. Accordingly, reduced serum levels of niacin or NAD⁺ reflects l-tryptophan deficiency and increased severity of COVID-19⁴³⁰. Activation of kynurenine pathway in elderly COVID-19 and PASC patients is a major cause for cerebrovascular damage⁴³¹. Virus-induced HMRD of tryptophan metabolism and its clinical implication on neuro-cognitive function(s) is shown in Fig. ​Fig.66.

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Fig. 6

Virus-induced HMRD of tryptophan metabolism/neuro-cognitive implications.

The SARS-CoV-2-induced HMR affects tryptophan metabolism by lowering the levels of tryptophan, serotonin, and indole-pyruvate, while elevating the levels of kynurenine, kynurenic acid, picolinic acid, and nicotinic acid^51,424. After conversion to kynurenine, the tryptophan catabolism divides into different branches, leading to the formation of 3-OH-kynurenine, anthranilic acid or kynurenic acid. The 3-OH-kynurenine catabolism further leads to the generation of picolinic acid, quinolinic acid, and nicotinamide. The neuroprotective kynurenic acid is present mainly in astrocytes, neurotoxic 3-OH-kynurenine and excitotoxic quinolinic acid are found in microglial cells. Besides directly targeting neurotransmitter receptors, the tryptophan metabolites, in particular 3-OH-kynurenine and 3-OH-anthranilic acid, are redox active that impact brain physiology⁷⁸⁹. The modulation of the tryptophan-kynurenine pathway is an indicator for a coherent metabolic shift⁷⁹⁰. The tryptophan-nicotinamide pathway is associated with inflammatory signals and coordinator of cell metabolism in SARS-CoV-2 infection⁷⁹¹. The broader virulence spectrum of SARS-CoV-2 with ability to cross the BBB and inflict a plethora of neuropathological manifestations by HMRD in host brain metabolism has been elucidated as ‘Neuro-COVID-19’¹⁷¹.

Serotonin (5-HT) is a precursor for melatonin, a chrono-biotic pineal hormone, which may elicit potential adjuvant effects to combat COVID-19 and PASC⁴³². Melatonin could also reverse aerobic glycolysis in immune cells and inhibit SARS-CoV-2-induced hyper-inflammatory response in COVID-19 patients⁴³³. l-tryptophan deficiency could deplete melatonin levels and aggravate pathophysiological risks of COVID-19 and PASC. l-tryptophan deficiency could also lower serum levels of 5-HT and augment disease manifestations such as anosmia, ageusia, and dysfunctional chemesthesis in COVID-19 and PASC⁴³⁴. Furthermore, 5-HT deficiency could worsen silent hypoxemia, weaken hypoxic pulmonary vasoconstriction, and compromise the recovery of COVID-19 and PASC patients.

The gut-brain’ axis is a bi-directional system that links emotional and cognitive centers of the brain with the peripheral functioning of the GI tract. The serotonergic system forms a diffuse network within the CNS and plays a neuroprotective role while regulating mood and cognition⁴³⁵. Based on 11 human RCTs, oral supplementation of l-tryptophan (0.14 to 3g/d) with regular meal seem to improve the mood of individuals⁴³⁶. l-Tryptophan supplementation, especially at ≥1g is shown to improve sleep quality and resolve insomnia⁴³⁷. Nutritional reset of l-tryptophan levels, and optimization of peripheral and central 5-HT levels could help alleviate neuro-cognitive dysfunction in PASC patients.

Nutritional reset of Iron (Fe)-Redox dysregulation (FeRD)

SARS-CoV-2 mediated acute lung injury (ALI) induces death of inflammatory cells with sloughing of alveolar epithelia and damage of pulmonary vasculature with hemolytic consequences⁴³⁸. Free heme released during hemolysis could induce pro-inflammatory, pro-oxidative, and pro-thrombotic effects. FeRD is highly prevalent among hospitalized COVID-19 patients. Serum levels of iron and hepcidin are low in COVID-19 patients, whereas erythropoietin (EPO) and haptoglobin levels significantly decline in critical and deceased patients⁴³⁹. Other biomarkers of iron metabolism (i.e., ferritin, TF, LF, etc.) and Hb could provide risk stratification strategies for COVID-19 management, as initial anemia is strongly linked to increased CFR. Altered iron-redox homeostasis (Fe-RH) with elevated ferritin/TF ratio predicts subsequent insufficient pulmonary oxygenation (with the need for ICU admission) and mechanical ventilation⁹⁶. Serum TF levels decrease within the 1^st week of hospitalization in many COVID-19 patients; however, a continuous decline is prominent among subjects with fatal outcomes⁴⁴⁰. Therefore, nutritional strategies to reset FeRD with Fe-redox regulators and ferroptosis inhibitors could be an effective strategy for optimal recovery of COVID-19 patients and in post-recovery management of PASC^94,441.

Fe-Redox regulators

In the human body, the total iron content is ~3-g for women and ~4-g for men, distributed in two main forms as heme-iron, mostly found in the Hb, myoglobin, and cytochromes (2 to 2.7-g); and as non-heme-iron, a cofactor for several enzymes⁴⁴². Free iron levels in human body fluids are regulated at <10^–18 M to avert microbial infections as well as to prevent the precipitation of insoluble ferric hydroxides and the formation of toxic free radicals. Innate Fe-Redox regulators such as lactoferrin (LF), heme oxygenase-1 (HO-1), erythropoietin (EPO), and hepcidin (HEP) serve as first innate barriers against free radical damage and hyper-immune responses during COVID-19 and PASC²⁷. In COVID-19 recovered virus-free PASC patients, the host Fe-RH is disrupted for an extended period; while the Hb, ferritin, and TF levels slowly restore back to normal after onset of initial FeRD, around a median of 122 days after discharge from the hospital⁴⁴⁰.

Lactoferrin (LF), an iron-binding glycoprotein present in milk as well as several body fluids including saliva, tears, nasal secretions, gastric/cerebrospinal/synovial fluids, sperm, vaginal secretions, and neutrophils, is a key component of innate host defense⁴⁴³. Due to iron-chelation and iron-transport properties, LF is considered an innate iron regulator with a multifunctional role in scavenging iron-catalyzed free radicals (i.e., ROS, RNS) and maintain Fe-RH in the body¹⁷¹. Free radical scavenging mechanisms of LF involve stimulated glycolysis, increased ATP generation to sustain ion gradient with membrane potential and morphology of the cell⁴⁴⁴. Oral supplementation of LF could prevent oxidative damage by heme iron and reverse ferritin-bound iron overload during chronic inflammation and aging⁴⁴⁵. Notably, both OxS and related metabolic syndromes are considered as potential risk factors in COVID-19 pathology⁴⁴⁶. As an innate regulator of Fe-RH, LF could combat OxS at the cellular level, modulate inflammatory responses at the tissue level and play a therapeutic role in clinical management of COVID-19 and PASC^27,447.

Several studies have elucidated a broad-spectrum antiviral activity for milk LF against SARS-CoV-2^{171,448–450}. Breast milk from several positive COVID-19 mothers were found negative for SARS-CoV-2 pathogen^451,452. The antiviral spectrum of milk LF include (i) direct interaction with the viral protein target(s) and blockade of viral attachment to host target cells⁴⁵³; (ii) binding to heparan sulfate proteoglycans (HSPGs) on the host cell surface with subsequent inhibition of viral attachment and cell entry⁴⁵⁴; and iii) interference with intracellular trafficking of the virus⁴⁵⁵. Also, LF is a potent anti-inflammatory agent that modulates hepcidin and ferroportin (FPN) synthesis through down-regulation of IL-6^456,457; thereby inhibits intracellular iron overload⁴⁴². Oral administration of 20–30% iron-saturated milk LF (corresponding to 70–84μg of elemental iron) twice a day could down-regulate IL-6 and restore FPN-mediated iron export from cells to blood in both hepcidin-dependent or independent pathways⁴⁵⁶. LF regulates both pro-inflammatory and anti-inflammatory responses⁴⁴⁷; thereby could prevent viral insult-induced cytokine storm⁴⁵⁸. LF also activates plasminogen that regulates coagulation cascade and antithrombotic activity, a promising clinical intervention for COVID-19 and PASC^22,459,460.

Apo-LF is a normoxic mimetic of hypoxia that effectively stabilizes redox-sensitive transcription factors HIF-1α and HIF-2α⁴⁶¹. In hypoxia, such as during the early stages of SARS-CoV-2 infection, these transcription factors could provide synergistic protection through activation of the Keap1/Nrf2 signaling pathway⁴⁶². Also, LF could block HIF-1α activity and provide therapeutic benefits to retinal neuronal cells during neuro-COVID^171,463. Taken together, milk LF could reverse iron overload and reduce inflammation, both considered as critical factors in the pathogenesis of COVID-19 and PASC; accordingly, LF could serve as a promising all-natural intervention to resolve FeRD and reset virus-induced HMRD in the ongoing new onset global metabolic syndrome.

Heme Oxygenase-1 (HO-1) also known as the ‘heat shock protein-32’ (hsp32), is an inducible intracellular enzyme upregulated by >100-fold during infections and clinical conditions such as sepsis, renal ischemia-reperfusion injury and acute lung injury (ALI)⁴⁶⁴. At the cellular level, HO-1 exists in the endoplasmic reticulum, mitochondria, nucleus, and plasma membrane⁴⁵. HO-1 mediates catalytic breakdown of heme, a potent pro-oxidant and pro-inflammatory molecule⁴⁶⁵. HO-1 regulates Fe-RH and provides cyto-protection via endogenous mechanisms to sustain body’s antioxidant response against OxS⁴⁶⁶. Human HO-1 binds to the SARS-CoV-2 ORF3a protein and inhibits virus-induced inflammation and tissue damage via the NLRP3 pathway^23,467. The ability of HO-1 to protect against SARS-CoV-2 infection is probably an emergency inducible defense mechanism to ameliorate OxS from heme-released oxidants²⁷. Thus, the cyto-protective function of HO-1 is a promising intervention strategy to control SARS-CoV-2 infection and alleviate virus-induced cytokine storm as well as the subsequent lung dysfunction during COVID-19 and PASC^468,469.

Several natural compounds could modulate HO-1 expression. Nimbolide, a limonoid tetranortriterpenoid isolated from neem plant (Azadirachta indica) could upregulate the HO-1 enzyme⁴⁷⁰. Phytochemicals such as, the Resveratrol (3,4’,5-trihydroxy stilbene) and the Curcuminoids (with α, β-unsaturated carbonyl groups) are potential inducers of HO-1 expression through Nrf2/antioxidant-responsive element (ARE) pathway^471,472. Quercetin, a polyphenolic flavonoid found in a variety of fruits and vegetables could induce HO-1 expression via mitogen-activated protein kinase (MAPK)/Nrf2 pathway⁴⁷³. In response to inflammation and OxS, the activated HO-1 is a powerful down-regulator of pro-coagulant factors to prevent thrombotic events, endothelial injury from vascular inflammation, resolve FeRD and reset HMRD in COVID-19 and PASC patients^27,46.

Erythropoietin (EPO) is a hypoxia-inducible growth factor expressed in various organs and tissues of the body⁴⁷⁴. Erythropoiesis is the single largest consumer of iron, quintessential for hemoglobin (Hb) synthesis in the body. Critical as well as deceased COVID-19 patients demonstrate significantly lower serum levels of EPO, haptoglobin, and hepcidin compared to survivors or mild cases⁴³⁹. EPO treatment could restore Hb levels, increase red blood cell (RBC) count, and improve O₂ delivery to the tissues, thereby help in the recovery of both COVID-19 and PASC patients⁴⁷⁵.

Overexpression of proinflammatory cytokines during COVID-19 results in cytokine storm, which ultimately leads to the clinical development of ALI and ARDS. Such hyper-inflammatory conditions could trigger NO• release and inhibit EPO synthesis causing anemia of inflammation (AI)⁴⁷⁶. Notably, fatal cases of COVID-19 demonstrate 2.5 times lower serum EPO (2.8 vs. 7.1 mU/mL), and 1.24 times lower Hb levels (14.0 vs 17.4g/dL) compared to survivors⁴⁷⁷. COVID-19 patients also show ground-glass opacities localized to alveoli indicating the presence of ALI^478,479. In a clinical study, recombinant human (rh)-EPO showed effective attenuation of ARDS symptoms and facilitated recovery from COVID-19 via multiple mechanisms including cytokine modulation, anti-apoptotic effects and leukocyte release from the bone marrow⁴⁷⁵.

EPO could potentially benefit neuro-COVID-19 patients with acute and chronic-progressive downstream sequelae of the CNS and peripheral nervous system (PNS)^171,480. Therapeutic benefits of EPO on COVID-19 patients may include (i) respiratory improvement at several levels including lung, brainstem, spinal cord, and respiratory muscles⁴⁸¹; (ii) counteract hyperinflammation caused by cytokine storm/inflammasome^482,483; (iii) neuro-protection and neuro-regeneration in brain and peripheral nervous system⁴⁸⁴. Thus, EPO could be a potential bio-replenishment to reverse FeRD in COVID-19 and help reset HMRD in PASC patients^27,484,485.

Ferroptosis inhibitors

Ferroptosis is an iron-catalyzed, non-apoptotic form of regulated necrosis that causes oxidative lipid damage in cell membranes leading to m-Dys⁴⁹⁸. Ferroptosis with characteristic accumulation of oxidized phospholipids (or their breakdown products) in myocardial and renal tissue is responsible for ischemia-reperfusion injury, which is a detrimental factor for cardiac damage and MODS in COVID-19 patients⁴⁹⁹. Ferroptosis is more immunogenic than apoptosis and plays a detrimental role in hyper-inflammation such as the CRS⁵⁰⁰. Accordingly, ferroptosis might serve as a potential treatment target for COVID-19 and PASC management^27,112. A hallmark of ferroptosis is iron-dependent lipid peroxidation, which could be inhibited by the key ferroptosis regulator Gpx4, free radical trapping antioxidants and ferroptosis-specific inhibitors⁵⁰¹. Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) could suppress ferroptosis and resolve cellular Fe-RH in COVID-19 and PASC⁵⁰².

Vitamin-E (Vit-E) or α-tocopherol is a fat-soluble antioxidant that prevents ROS formation during lipid oxidation. Vit-E could effectively prevent ferroptosis by reducing the Fe³⁺ center to inactive Fe²⁺ in lipoxygenase (LOX), thereby inhibit enzyme activity of LOX. Notably, LOX oxidizes cell membranes via peroxyl (ROO•) radicals and forms lipid hydroperoxides. Vit-E could neutralize ROO• radicals and prevent formation of lipid hydroperoxides. Afterward, GSH, GPX4, and other antioxidant agents may detoxify oxidized lipids and inhibit ferroptosis⁵⁰³. A meta-analysis (n=19 studies) of Vit-E supplementation (mean dosage: 384mg/d) has shown to reduce OxS in hemodialysis patients⁵⁰⁴.

Ferrostatin-1 (FER-1) (Ethyl 3-amino-4-cyclohexylamino-benzoate) is a potent lipophilic free radical scavenger that could reduce cellular accumulation of lipid peroxides and peroxyl radicals⁵⁰⁵. The anti-ferroptotic activity of FER-1 is associated with its ability to scavenge alkoxyl radicals and remain unconsumed while inhibiting iron-dependent lipid peroxidation⁵⁰⁶. During SARS-CoV-2 infection of bronchial epithelia, the inflammatory IL-6 could induce ferroptosis as well as ROS-dependent lipid peroxidation, leading to severe FeRD⁵⁰⁷. A combination of FER-1 and N-acetylcysteine (NAC) could reverse ferroptotic effects of IL-6 and help resolve FeRD in COVID-19 and PASC patients^27,508. Also, chrono-biotic hormone melatonin could effectively reduce ferroptosis through activation of Nrf2 and HO-1 signaling pathways⁵⁰⁹. The strong ability to inhibit ferroptosis and platelet activation, makes melatonin a potential nutritional intervention to treat hemolytic, thrombotic, and thrombocytopenic conditions⁵¹⁰, which are widespread among COVID-19 and PASC patients.

Dietary phytochemicals could serve as natural ferroptosis inhibitors to resolve FeRD-related manifestations in COVID-19 and PASC²⁷. Glycyrrhizin, the main extract from licorice (Glycyrrhiza glabra), is a natural antioxidant, anti-inflammatory, antifibrotic and antiviral agent widely used in the treatment of chronic hepatitis⁵¹¹. Glycyrrhizin shows significant reduction in the degree of ferroptosis and inhibits OxS and provides anti-ferroptotic liver protection through up-regulation of Nrf2, HO-1 and Gpx4; and down-regulation of lactate dehydrogenase (LDH), Fe²⁺, malondialdehyde (MDA), and ROS⁵¹². Quercetin (pentahydroxy flavone), a natural flavonoid, could up-regulate the GSH levels and inhibit ferroptosis by reducing MDA and lipid ROS levels in the renal proximal tubular epithelia⁵¹³. Two tannin hydrolysates, chebulagic acid and chebulinic acid, were identified as potent ferroptosis inhibitors. Their ferroptosis inhibition is mediated by regular antioxidant pathways (ROS scavenging and iron chelation), rather than the redox-based catalytic recycling pathway by FER-1⁵¹⁴. Curcumin could inhibit myoglobin (Mb)-induced ferroptosis in renal tubular cells. Curcumin could reduce Mb-mediated inflammation and OxS by inhibiting the TLR4/NF-κB axis and activating the cytoprotective enzyme HO-1⁵¹⁵. Dietary polyphenols such as piceatannol and astringin could strongly inhibit ferroptosis via preferential transfer of hydrogen atoms at the 4’-OH position as conventional antioxidants^154,516.

Nutritional reset of mitochondrial dysfunction (m-Dys)

Mitochondrial dysfunction (m-Dys) is characterized by a loss of efficiency in the OXPHOS and reductions in the ATP synthesis, a causative mechanism for several metabolic syndromes including PASC. m-Dys leads to fatigue (with reduced tolerance to exercise), a common persistent symptomatic feature amongst COVID-19 survivors. Nutritional strategies to resolve m-Dys should comprise specific bioactives and cofactors essential for mitochondrial bioenergetic pathways, as well as provide effective free radical (ROS) scavengers to prevent OxS, maintain Fe-RH, and reset virus-induced HMRD.

Nicotinamide adenine dinucleotide (NAD⁺) is a vital cofactor in mitochondrial bioenergetic pathways, including glycolysis, fatty acid β-oxidation, and the TCA cycle⁵¹⁷. It exists in both oxidized (NAD⁺) and reduced (NADH) forms, the latter is generated by NAD⁺ accepting high-energy electrons from glycolytic and TCA intermediates and acts as a primary electron donor in ATP synthesis to drive mitochondrial OXPHOS⁵¹⁸. NAD⁺ also regulates non-redox NAD⁺-dependent enzymes such as poly-ADP-ribose-polymerases (PARPs) and sirtuins⁵¹⁹. The macrodomain-containing protein, nsp3 of SARS-CoV-2 could counteract the host antiviral defenses from PARPs, SARM1, sirtuins and CD38¹⁵⁷. Therefore, NAD⁺ and NAD⁺-consuming enzymes are critical for immune responses, cellular bioenergetics, and to design antiviral strategies for COVID-19 and PASC.

Furthermore, NAD⁺ plays a key role in several essential cellular processes including DNA repair, immune cell function, senescence, and chromatin remodeling⁵²⁰. Cardiac tissue is dense with mitochondria; as one of the most metabolically demanding organs, the heart has the highest NAD⁺ levels. A decline in NAD⁺ metabolism and low tissue levels of NAD⁺ is a common trait among the elderly. Alterations to NAD⁺ metabolism and/or decline in tissue NAD⁺ levels are directly related to m-Dys and pathological conditions such as CVD⁵²¹. SARS-CoV-2 infection dysregulates NAD⁺ metabolism, which could manifest m-Dys and lead to chronic fatigue syndrome (CFS) in PASC patients⁵²². Therefore, nutritional reset of m-Dys with NAD⁺ or its natural dietary precursors could be effective in improving myocardial bioenergetics and function.

NAD is synthesized de novo from tryptophan or bio-replenished through NAD⁺ precursors such as nicotinic acid, nicotinamide, or nicotinamide riboside, collectively referred to as niacin/B₃ vitamins. Dietary NAD⁺ could partially resolve SARS-CoV-2-induced dysregulated gene expression and mitochondrial metabolism⁵²³. NAD supplement could also alleviate intestinal barrier injury by protecting mitochondrial function in gut epithelia⁵²⁴. Also, NAD⁺ supplement could directly inhibit PARP-1 and prevent pro-inflammatory cytokines and resolve hyper-activated immune system. Oral administration of NAD⁺ precursors, such as tryptophan, nicotinic acid (niacin), nicotinamide, and nicotinamide riboside (NR), could increase tissue NAD⁺ levels^525,526. Food supplement cocoa flavanol is shown to boost the NAD⁺ and NADH content, stimulate sirtuin metabolism, reduce cellular H₂O₂ production as well as OxS, and improve mitochondrial function in PASC patients⁵²⁷. Increasing NAD⁺ levels could also stabilize telomeres and help recovery of elderly PASC patients⁵²⁸.

Alpha-lipoic acid (ALA) is an intracellular redox regulator that reduces OxS, blocks activation of NF-kB and lowers both ADAM17 activity and ACE2 upregulation⁵²⁹. ALA upregulates ATP-dependent K⁺ channels in the cell, subsequently raises intracellular pH and thereby inhibits viral entry into host target cells⁵³⁰. Furthermore, ALA could increase intracellular GSH levels and reinforce human host antiviral defense⁵³¹. Therefore, ALA could be considered an effective intervention against SARS-CoV-2 as well as a potent redoxeutical to resolve m-Dys-associated clinical manifestations in COVID-19 and PASC^532,533. A combination of ALA (50µM) and palmitoyl-ethanolamide (5µM) could reduce OxS (overproduction of ROS and NO•) and modulate the major inflammatory cytokines (IL-β, IL-6,TNFα, and IL-10) involved in COVID-19 infection⁵³⁴. A human RCT from Wuhan, China, showed that ALA therapy (1200mg/d) for 7 days, could reduce a 30-day all-cause mortality rate (37.5%) in critical COVID-19 patients⁵³⁵.

Coenzyme Q10 (CoQ10) or ubiquinone is a lipophilic cofactor in the mitochondrial ETC of the OXPHOS system that exerts powerful antioxidant, anti-apoptotic, immuno-modulatory and anti-inflammatory effects in cellular metabolism⁵³⁶. CoQ10 is also a potent anti-inflammatory agent that effectively down-regulates cytokines (i.e., TNF-α, IL- 6, CRP) and could optimize viral-disrupted ACE2/RAAS system, by exerting anti-angiotensin II effects and decreasing OxS in COVID-19 patients^537,538. Excess release of cytotoxic ROS during m-Dys leads to OxS, which may hyper-activate platelet function and pose risk of thrombosis in COVID-19 patients. As a potent mitochondrial redox regulator, CoQ10 could prevent thrombotic events in COVID-19 and PASC patients by resolving ROS-induced platelet aggregation⁵³⁹. CoQ10 is expressed in all tissues; however, its biosynthesis drops down with ageing and sharply declines during OxS in COVID-19⁵⁴⁰. Thus, CoQ10 as an adjuvant combined with other mitochondrial nutrients could provide potential therapeutic options to resolve hyper-inflammation and reset HMRD in COVID-19 and PASC^541,542.

Creatine could replenish mitochondrial viability and restore cognitive function(s) by down-regulating toll-like receptors (TLRs) involved in neuroinflammation and neurodegeneration. The potent antioxidant activity of creatine could also protect mitochondrial DNA from ROS-mediated oxidative damage^543,544. Orally administered guanidino-acetic acid (GAA) could positively affect creatine metabolism, alleviate several aspects of fatigue and improve both physical as well as work capacity in patients with CFS⁵⁴⁵. Human RCTs have suggested that creatine (monohydrate form) could revitalize cellular bioenergetics, neuro-metabolism, and immune function, thereby may provide a multifunctional benefit in recovery of PASC with MS/CFS complications^546,547.

Vitamin-B12 (Vit-B12) also known as cobalamin, is vital for cardiovascular function, as well as for immune regulation and antiviral defense^548,549. Vit-B12 is also an essential nutrient for ‘skeletomuscular–gut–brain’ axis to maintain skeletal muscle, neuro-cognitive functions, and modulate gut microbiota^550,551. Vit-B₁₂ has ranked among the top four bioactive nutrients for potential management of COVID-19 and PASC⁵⁵². Thus, vit-B₁₂ combined with clinical nutrition is a potential adjuvant to reset HMRD in COVID-19 and PASC patients.

Nutritional reset of oxidative stress (OxS)

Virus-induced OxS with excess levels of ROS i.e., superoxide anion (O₂^•-), hydroxyl radical (^•OH), singlet oxygen (¹O₂), and hydrogen peroxide (H₂O₂), could trigger severe clinical manifestations including hyper-inflammation, tissue damage, thrombosis, and MODS in COVID-19, which may continue in PASC^553,554. In the body, O₂^•- anions are intended products of redox signaling enzyme cascade and byproducts of several metabolic processes including mitochondrial respiration⁵⁵⁵. Superoxide (O₂^•-) anions are scavenged by redox enzyme superoxide dismutase (SOD), whereas H₂O₂ by catalase (CAT), glutathione (GSH), GSH-peroxidase (GPx), thioredoxin peroxidase (Trx), and peroxiredoxins (Prdx)⁵⁵⁶. Any decline in redox enzymes may increase free radical generation with subsequent induction of lipid peroxidation, protein oxidation, and DNA/RNA degradation^557–559.

Serum levels of SOD, CAT, GSH, and GPx are significantly altered in COVID-19 patients^560,561. The depleted total antioxidant capacity (in blood) of SARS-CoV-2 infected individuals serves as a predictive marker for COVID-19 severity⁵⁶². Both OxS and hyper-inflammatory state during the acute phase of COVID-19, could also predict severity of chronic fatigue, depression, and anxiety symptoms even after 3 to 4 months in the virus-free PASC patients. Based on cluster analysis, a majority of PASC patients show severe abnormalities in SpO₂, increased OxS and reduced antioxidant indices⁵⁶³. Therefore, antioxidant enzymes could be considered an effective nutritional strategy to resolve OxS and reset HMRD in COVID-19 and PASC.

Superoxide dismutases (SODs) are metalloenzymes that trigger endogenous antioxidant machinery, the first-line defense against ROS in the body⁵⁶⁴. SOD catalyzes the conversion of superoxide (O₂^•-) into O₂ and H₂O₂⁵⁵⁷. The H₂O₂ is further hydrolyzed to water via CAT and GPX enzymes⁵⁶⁵. Three isoforms of SOD exist in human body: the cytosolic Cu-, Zn-SOD (SOD1), the mitochondrial Mn-SOD (SOD2) and the extracellular Cu-, Zn-SOD (SOD3)⁵⁶⁶. PC-SOD (recombinant human SOD1 covalently coupled to four molecules of lecithin) is a potent superoxide-radical scavenger with a 100-fold increase in protective effects against endothelial cell injuries, compared to unmodified SOD^567,568. OxS plays a critical role in COVID-19 and PASC; therefore, the therapeutic use of SOD and SOD-mimetics (e.g., Mangafodipir) may prove beneficial in PASC recovery^565,569.

Catalase (CAT), a heme enzyme that catalyzes the decomposition of H₂O₂ to water + molecular O₂, provides a vital cellular antioxidant defense⁵⁷⁰. Excessive production of H₂O₂ in mitochondria could damage lipids, proteins, mDNA, resulting in necrosis or apoptosis; where then CAT could protect such cells from H₂O₂-induced oxidative injury⁵⁷¹. Apart from its main substrate H₂O₂, the CAT enzyme could also process other oxidative species such as O₂^•-, ^•OH, ¹O₂, hypochlorous acid (HOCl), NO•, and peroxynitrite (ONOO^-). A number of these free radicals are formed under oxidative ‘eustress’ (good stress) and ‘distress’ (bad stress), where CAT could help regulate the cellular redox-oxidative status⁵⁷². CAT-mediated decomposition of H₂O₂ to water minimizes the downstream flow of excessive ROS, which otherwise could trigger OxS and m-Dys in COVID-19 and PASC patients. CAT plays a crucial intermediary role in S-protein binding to hACE2 receptors, thereby affects the host susceptibility to SARS-CoV-2 infection⁵⁷³. CAT could also regulate cytokine production in leukocytes, protect alveolar cells from oxidative injury, and block SARS-CoV-2 replication⁵⁷⁴.

Solid lipid nanoparticles based on phosphatidylcholine stabilizers, is a functional CAT supplement designed to resist enteric digestion and deliver potent antioxidant activity⁵⁷⁵. Supplemental CAT is shown to alleviate OxS in the GI tract and improve gut microbiota⁵⁷⁶. Lactic acid bacteria, Lactobacillus casei BL23, L. delbrueckii subsp. bulgaricus CRL 864, and Streptococcus thermophilus CRL 807, produce both antioxidant enzymes CAT as well as SOD, and provide intrinsic immunomodulatory benefits in the GI tract^577,578. Programmable probiotics are promising dietary adjuvants to prevent OxS in the gut, curb intestinal inflammation, and resolve certain persistent GI pathologies (e.g., colitis, inflammatory bowel disease, and dysbiosis) in PASC patients⁵⁷⁹.

Glutathione (GSH) (γ-l-glutamyl-l-cysteinyl-glycine) is a tripeptide synthesized in the cytosol by two ATP-consuming enzymatic reactions⁵⁸⁰. GSH reaches millimolar levels (1–10mM) within cells, micromolar levels (10–30μM) in plasma, and its low redox potential (E^′₀=−240mV) makes GSH an ideal cellular redox buffer^581,582. GSH is commonly found in reduced GSSG form in cytosol, nucleus, mitochondria, and endoplasmic reticulum⁵⁸³. The GSSG/GSH redox couple interacts with other antioxidant enzymes to maintain mitochondrial function and cellular redox homeostasis⁵⁸⁴. The GSSG/GSH redox couple plays a vital role in several enzymatic reactions, including the elimination of peroxides by GSH peroxidases (GPx), in covalent addition of cysteines to proteins by glutaredoxin, and in detoxification of electrophiles by GSH-S-transferase (GST)^585–587. GSH plays the role of ‘master antioxidant’ in tissues; where the high millimolar levels of GSSG in reduced form emphasizes its regulatory role in processes such as detoxification, protein folding, antiviral defense and immune response⁵⁸⁸. Mitochondria are the main source of ROS, generated from the ETC/OXPHOS and any excess release of toxic free radicals could trigger OxS and m-Dys⁵⁸⁹. GSH is the main cellular antioxidant to reduce H₂O₂ and lipid hydroperoxides (LOOH) catalyzed by GPXs^589,590. A key enzymatic step in antioxidant clearance with GSH redox cycle is converting H₂O₂ to water, which is further catalyzed by GPx. This reduction step occurs via oxidation of GSH to GSSG, and the GSSG is subsequently reduced back to GSH in the body via the enzyme glutathione reductase and NADPH⁵⁹¹.

The SARS-CoV-2-induced FeRD, its ensuing OxS could deplete cellular antioxidant reserves and increase severity of COVID-19 and PASC²⁷. Decreased expression of GSH synthesis leads to low free GSH levels, resulting in elevated ROS, immune dysfunction, and increased disease severity in COVID-19 patients⁵⁹². SARS-CoV-2 infection causes GSH deficiency in the body through inhibition of nuclear factor erythroid 2–related factor 2 (Nrf2) pathway, the up-regulator of GSH synthesis⁵⁹³. During OxS condition, Nrf2 is transported from cytoplasm into the nucleus by karyopherins, where SARS-CoV-2 interferes with transfer process and reduces GSH synthesis⁵⁹⁴. GSH precursors, particularly NAC, are widely used to revert OxS and replenish low GSH levels in pulmonary episodes such as ARDS, bronchitis, or emphysema in COVID-19 and PASC⁵⁹⁵. Furthermore, comorbidities such as hypertension (56.6%), obesity (41.7%), and diabetes (33.8%) are frequently linked to OxS and chronic inflammation in hospitalized COVID-19 patients⁵⁹⁶. In obese patients, OxS is associated with diminished GSH levels and decreased GSH/GSSG ratio⁵⁹⁷. Low GSH levels could also increase viral replication, pro-inflammatory cytokine release, endothelial damage, and immune-thrombosis, which is a hyper-coagulative clinical condition that could exacerbate morbidity and mortality in COVID-19 and PASC⁵⁹⁸. Since m-Dys and OxS jointly contribute to both COVID-19 and PASC pathology, nutritional to replenish optimal GSH levels could be a promising strategy to reset HMRD and support patient recovery⁵⁸².

Ferroptosis, the programmed cell death due to iron and lipid dependent peroxidation, is associated with ageusia and anosmia, the early clinical manifestations of COVID-19^197,599,600. Ferroptosis is regulated by lipid repair enzymes, which also include GSH and GPx4 reducing lipid hydroperoxides (L-OOH) to lipid alcohols (L-OH)⁶⁰¹. Ferroptosis could cause severe tissue damage and MODS in COVID-19 patients. Administration of liposomal GSH could boost intrinsic GSH levels, enhance GPx4 function and reduce tissue damage from ferroptosis in COVID-19 and PASC⁶⁰².

N-Acetyl-L-Cysteine (NAC) is a sulfur-containing AA that breaks disulfide bonds, increases viscosity of mucoproteins and serves as an antioxidant in pulmonary mucous secretions of the respiratory tract⁶⁰³. NAC is widely used as a mucolytic agent to improve airway clearance in chronic respiratory diseases. Glucose 6-phosphate dehydrogenase (G6PD) deficiency predisposes GSH depletion and increases susceptibility to SARS-CoV-2 infection, and such GSH depletion could be reversed with NAC administration⁶⁰⁴. As a precursor for GSH synthesis, adjuvant therapy with NAC could resolve SARS-CoV-2-induced OxS via GSH release and help restore cellular redox homeostasis during COVID-19 and PASC⁶⁰⁵.

SARS-CoV-2 infects type II pneumocytes and disrupts the cellular Fe-RH with increased ROS release causing severe OxS. Treatment of COVID-19 with NAC (dosage: 600mg) could provide a prophylactic benefit, and at higher dosage (1200mg) could serve a therapeutic regimen when administered at the first onset of symptoms⁶⁰⁶. Oral administration of NAC (600mg every 8h) to COVID-19 patients (n=19,208) with high-risk comorbidities (i.e., hypertension, dyslipidemia, diabetes, and COPD) is shown to significantly lower mortality rate⁶⁰⁷. NAC intervention (1200–1800 mg/d) markedly improve oxygenation (SpO₂/FiO₂) parameters in 10 days, reduce inflammatory markers, and shorten the length of hospitalization in COVID-19 patients⁶⁰⁸. In a retrospective study, COVID-19 patients (n=1083) receiving NAC (1200mg/d) had a shorter length of hospital stay⁶⁰⁹. In a cross-sectional study, COVID-19 patients (n=164) receiving NAC with standard therapy had an average hospital stay duration of 12 days, a 97% rate of discharge, an average duration of O₂ therapy for 8 days with limited transfer to ICU, and only one case of fatality⁶¹⁰. NAC, as a precursor for reduced GSH, demonstrates antioxidant, anti-inflammatory and immunomodulatory effects, which may prove beneficial in modulating any excess inflammatory activation during COVID-19^611,612. Therefore, nutritional supplementation with NAC could effectively resolve OxS and target pathophysiological pathways involved in SARS-CoV-2 infection and persistent pulmonary fibrotic sequelae in PASC⁶¹³.

Glutamine is a precursor for several bioactive molecules in plasma and skeletal muscle, largely utilized for gluconeogenesis in the liver. Glutamine increases cellular GSH levels, improves antioxidant capacity, reduces OxS and inflammation in the body⁶¹⁴. Severe OxS with elevated blood levels of high sensitivity-C-reactive protein (hs-CRP) is a hallmark of hyper-inflammatory state in COVID-19 and PASC^290,553,615; therefore, proper nutrition rich in antioxidants is critical for recovery of these patients⁶¹⁶. Glutamine is also a widely used nutritional antioxidant in several hospital ICU-admitted patients with respiratory infections⁶¹⁷. Glutamine supplementation (10g/3x daily) for 5 days could reduce serum levels of IL-1β, hs-CRP, TNFα and increase appetite in COVID-19 patients with pulmonary complications⁶¹⁴.

Nutritional reset of virus-hijacked ACE2/RAAS

ACE2 is a key component of the renin-angiotensin-aldosterone system (RAAS) that plays a vital role in regulating blood pressure, vasoconstriction, sodium retention, tissue remodeling, pro-inflammatory and pro-fibrotic functions^633,634. The viral hijack of human ACE2 receptor disrupts RAAS activation, upregulates NF-κB pathway, triggers cytokine storm, hypertension, cell proliferation, inflammation, and fibrosis, where all elicit detrimental effects on every bodily organ during SARS-CoV-2 infection⁶³⁵. Nutritional supplementation with l-carnitine could mitigate these pathobiological processes by inhibiting NF-κB and down-regulating NOX1/NOX2, thereby enhance the antioxidant effects of angiotensin II⁶³⁶. As an immunomodulator, l-carnitine could decrease proinflammatory cytokines (i.e., TNF-α, IL-6, and IL-1) and help reduce cytokine storm. l-carnitine could also protect against SARS-CoV-2-induced cardiotoxicity resulting from dysregulated ACE2 signaling pathway^637,638.

l-Carnitine, a micronutrient composed of essential AAs (i.e., lysine and methionine), is a cofactor that converts long-chain free fatty acids to acyl-carnitine and transfers these metabolites into the mitochondrial matrix⁶³⁹. This hydrophilic AA is widely distributed in CNS, PNS, heart and skeletal muscle, holding >95% of body’s total carnitine^638,640. l-Carnitine plays a vital role in lipid metabolism and its deficiency could induce feeling of tiredness or general fatigue. Therefore, fatigue could possibly be relieved by restoring serum carnitine levels through supplementation⁶⁴¹. l-carnitine levels in patients with chronic fatigue syndrome (CFS) is 30 to 40% lower than healthy subjects. Oral administration of l-carnitine (3g/d) with omega-3 fatty acids could increase carnitine palmitoyl-transferase-I activity and relieve clinical symptoms of CFS⁶⁴². SARS-CoV-2 infection requires a high basal energy expenditure for immune activation, hyper-inflammation (‘cytokine storm’), anorexia followed by muscle loss, weakness, and fatigue⁶⁴³. Circulating autoantibodies may also have a major role in the manifestation of long-term fatigue in PASC patients⁶⁴⁴. Acetyl- l-carnitine supplementation could generate energy from mitochondrial oxidation of fatty acids and help mitigate fatigue in PASC patients⁶³⁷.

Nutritional reset of virus-hijacked NRP1/neuro-cognitive impairment

NRP1 is expressed in olfactory epithelium, astrocytes, and neuronal cells (which lack ACE2 expression) and serve as major CSR for SARS‐CoV‐2 infection of the central nervous system (CNS)^645,646. Viral hijack of NRP1 facilitates CNS invasion of SARS-CoV-2 through the blood brain barrier (BBB), and consequential neuro-cognitive symptoms such as anosmia, ageusia, headaches, confusion, delirium, and strokes in early COVID-19⁶⁴⁷. The ensuing pathophysiology involves virus-induced neuronal damage, neuroinflammation, rupture of the BBB, microvasculitis and hypoxia⁶⁴⁸. Neuroinflammation with hypometabolic lesions cause chronic cognitive impairment in COVID patients⁶⁴⁹. Neurological manifestations of COVID-19 and PASC include damage to CNS and PNS, encephalitis, myelitis, myositis, Guillain Barré syndromes, and cognitive impairments^171,650. These neuro-complications are prevalent among one third of COVID-19 cases, and this clinical condition may persist as chronic symptoms in PASC patients as frequent complaints of brain fog (81%) and fatigue (58%)^651,652. Nutritional reset of neuro-cognitive dysfunction from viral hijack of NRP1 indeed is of high priority in clinical management of PASC.

Melatonin (N-acetyl-5-methoxytryptamine) is a derivative of tryptophan, synthesized/secreted by the pineal gland and reaches peak levels in plasma during the night hours^653,654. This chrono-biotic hormone serves as a photo-periodic switch, influencing the activity of suprachiasmatic nucleus and facilitates human sleep-wake. Melatonin plays multi-functional roles including the regulation of circadian rhythms, immune modulation, oxidative processes, apoptosis, and mitochondrial homeostasis⁶⁵⁵. Melatonin deficiency may lead to CVD with manifestations of hypertension and myocardial ischemia/reperfusion injury, which are prevalent in COVID-1; as well as neuro-cognitive complications such as brain fog, sleep disorders and myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS), which are persistent among PASC^656,657.

Melatonin with its antioxidant, anti-inflammatory, immune-modulatory, and anti-apoptotic effects, is considered a potential therapeutic against COVID-19 and PASC. As a powerful antioxidant, melatonin scavenges toxic free radicals (ROS/RNS) and prevents oxidative damage of DNA through activation of DNA repairing pathways⁶⁵⁸. This neuro-hormone could also elevate levels of antioxidant enzymes (i.e., SOD, CAT, GSH and GPx), and inhibit detrimental effects of NLRP3 inflammasome⁶⁵⁹. Melatonin could resolve hyper-inflammatory conditions in COVID-19 and PASC by down-regulating proinflammatory cytokines (i.e., TNF-α, IL-1β, IL-6, and IL-8) and increasing anti-inflammatory cytokines such as IL-10^660,661. The anti-inflammatory activity of melatonin may also involve Sirtuin-1 induction, suppression of NF-κB activation, and stimulation of Nrf2⁶⁶². Furthermore, the anti-apoptotic and cyto-protective effects of melatonin could stabilize mitochondrial membrane and help reset m-Dys⁶⁶³.

Based on several human RCTs, melatonin is considered an effective intervention to resolve delirium, ameliorate respiratory stress (i.e., ARDS), and restore circadian balance in COVID and PASC patients^664–666. Furthermore, as a cyto-protectant, melatonin helps alleviate several COVID-19 comorbidities, including T2DM, metabolic syndromes, and both ischemic as well as non-ischemic CVD⁶⁵⁷. In an open-label RCT (n=96), oral administration of melatonin tablets (3mg/d, 1h before sleep, for 7 days) with standard treatment, substantially improved sleep quality and blood O₂ saturation parameters in hospitalized COVID-19 patients⁶⁶⁷. In another RCT (n=80), prolonged-release melatonin (PRM 2-mg) therapy showed a significant improvement in sleep hours and reduction in delirium episodes among hospitalized insomniac COVID-19 patients⁶⁶⁸. Melatonin activates two of the G-protein-coupled receptors: MT1 that regulates vigilance states of rapid eye movement (REMS), and MT2 that controls non-REMS. In accordance with the circadian rhythm, melatonin release into blood at night could improve sleep quality of insomnia patients through activation of MT1 and MT2 receptors⁶⁶⁹. Finally, melatonin could help alleviate neurological complications such as brain fog, ME/CFS, anxiety, and sleep disorders; resolve ALI/ARDS with related vessel permeability issues. Based on its multi-functionality, high safety profile, melatonin could be considered a promising adjuvant support for nutritional reset of neuro-COVID in PASC patients^171,665,670.

Nutritional reset of virus-hijacked serine proteases

In healthy lungs, type II transmembrane serine proteases (i.e., TTSPs: TMPRSS2, CTSL, HAT) play a major role in cellular regeneration, repair, and homeostasis^671,672. Furthermore, anti-proteases (i.e., secretory leukocyte protease inhibitor, SLPI) are important for proteolytic inhibition and host defense⁶⁷³. A functional balance between proteases and anti-proteases is vital to ensure respiratory homeostasis. Any imbalance towards increased protease expression and activity may lead to overt inflammation and trigger chronic lung disorders such as COPD and emphysema^674,675. Furthermore, respiratory serine proteases that belong to the TTSP family, increase host susceptibility to SARS-CoV infection-2⁵⁵. Anti-proteases, such as SLPI, inhibit the activity of serine proteases and block viral entry into host target cells⁷⁰. Therefore, the protease/antiprotease balance not only is critical for respiratory homeostasis but also serves as a powerful determinant of SARS-CoV-2 pathogenesis. Nutritional antioxidants could stimulate anti-protease secretion, decrease protease activity and protect epithelial cells against viral infection⁶⁷⁶. Thus, antioxidants serve as regulators of the protease/antiprotease balance that could effectively combat viral infection(s) including COVID-19.

Respiratory epithelium is constantly prone to inhalation insults from excess release of toxic free radicals, ROS/RNS, and peroxides, all cumulatively exert OxS⁶⁷⁷. Nrf2, the innate transcription factor, regulates synthesis and activity of several antioxidant enzymes to help resolve OxS, and prevent tissue damage in lungs⁶⁷⁸. Nrf2 also regulates TMPRSS2, protease/antiprotease balance and protects the respiratory milieu against viral infections^676,679. Thus, bioactive nutrients that activate Nrf2 could reduce persistent OxS as well as help in functional optimization of viral-hijacked serine proteases and reset HMRD in PASC patients⁷⁰.

Flavan-3-ols found in green tea, such as epigallcatechin-3-gallate (EGCG), is shown to induce SLPI secretion, reduce TMPRSS2 secretion, and decrease viral replication⁶⁸⁰. Antioxidant supplementation with flavan-3-ols may serve as a possible nutraceutical therapy to protect against lung disease in the context of a viral infection.

Sulforaphane (SFN), a sulfur-containing isothiocyanate compound naturally found in cruciferous vegetables (i.e., cauliflower, broccoli, brussels sprouts, and cabbage) could enhance Nrf2 activity. SFN could induce cellular antioxidants such as heme oxygenase (HO)-1 and NADPH quinone oxidoreductase 1 (NQO1) activities, thereby inhibit proinflammatory cytokine release⁶⁸¹. SFN supplementation could also increase SLPI secretion, regulate TMPRSS2 expression and plays an important role in modulation of protease/antiprotease balance^676,679.

Nutritional reset of immune impairment

SARS-CoV-2 infection triggers immune response, a regular host defense strategy to restrain viral entry and constrain disease progression. However, when immune system exhausts and/or compromised, the viral infection become aggressive in vulnerable hosts and evolves into a severe pathophysiological state with hyper-inflammation, extensive tissue damage, and MODS⁶⁸². Such hyper-inflammatory state could lead to loss of appetite, altered intestinal absorption, impaired gut permeability, and malnutrition^683,684. Malnutrition in turn could aggravate inflammatory pathways, compromise the immune system, onset dysbiosis, increase risks of new microbial infection(s) as well as reactivate latent pathogens⁶⁸⁵. Therefore, nutritional resolution of immune dysfunction is an important aspect of recovery from severe inflammation, malnutrition, and sarcopenia during clinical rehabilitation of COVID-19 and PASC.

Vitamin D3 (Vit-D) is a lipid-soluble seco-steroid that exists in two forms as D₂ (ergocalciferol) derived mainly from plant sources and D₃ (cholecalciferol), which is present in higher animals⁶⁸⁶. Both endogenous and exogenous forms of vit-D are inactive and require two successive hydroxylation steps by cytochrome P450 (CYP) enzymes to form fully active vit-D. In humans, vit-D3 is produced by the skin with conversion of 7-dehydro-cholesterol to cholecalciferol via exposure to sunlight⁶⁸⁷. Circulatory vit-D is initially transported to liver by vit-D binding protein⁶⁸⁸. Vit-D is mainly known to regulate calcium/phosphate homeostasis and bone metabolism. However, vit-D is also vital for several biological pathways including modulation of innate and adaptive immune responses⁶⁸⁹. Immuno-modulatory role of vit-D could be categorized into three essential functions: (i) physical barrier, (ii) natural cellular immunity, and (iii) adaptive immunity⁶⁹⁰. Vit-D could activate the release of cathelicidin and defensins to inhibit viral replication; furthermore, could down-regulate release of proinflammatory Th1 cytokines (i.e., TNF-α and IFN-γ) and stimulate macrophages to generate anti-inflammatory cytokines to minimize the risk of ARDS in COVID-19⁶⁹¹. Vit-D could also inhibit NF-κB activation to down-regulate proinflammatory cytokine synthesis and other key activators of cell-mediated immunity⁶⁹². Vit-D receptor (VDR) is expressed on antigen-presenting cells, T and B lymphocytes that also synthesize active vit-D metabolite. Vit-D can modulate both innate and adaptive immune responses, and vit-D deficiency is linked to increased autoimmunity as well as increased susceptibility to microbial infections. Vit-D deficiency is prevalent among patients with autoimmune disorders⁶⁸⁹. Vit-D is a negative regulator for expression of renin and interacts with the RAAS/ ACE/ACE-2 signaling axis, therefore could affect SARS-CoV-2 infection process as well as host CV/circulatory function⁶⁹³.

In COVID-19 patients, vit-D deficiency was reported in 41.7% cases, vit-D insufficiency in 46.0%, and the remaining 12.3% of cases with normal vit-D levels. The odds of severe COVID-19 outcomes increase by 38.1 and 5.6 times for vit-D-deficiency and -insufficiency patients, respectively, for each standard deviation decrease in serum 25(OH)D⁶⁹⁴. Low vit-D levels are associated with elevated inflammatory cytokines with increased risk of pneumonia and viral upper respiratory tract infections. Vit-D deficiency is also associated with an increase in thrombotic episodes, frequently reported in COVID-19⁶⁹⁵. Severe cases of COVID-19 demonstrate 64% more vit-D deficiency than mild cases, while vit-D insufficiency could significantly increase hospitalization and CFR⁶⁹⁶. Vit-D deficiency is a risk factor for unregulated cytokine storm and hyper-inflammation in COVID-19 patients⁶⁹⁷. Also, COVID-19 survivors and PASC patients show lower 25(OH)D levels compared to matched-patients without PASC⁶⁹⁸. Vit-D deficiency refers to serum levels of 25-hydroxyvitamin D, 25(OH)D, <20ng/mL (50 nmol/L)⁶⁹⁹.

SARS-CoV-2 pneumonitis could rapidly incapacitate the lung and lead to severe ALI/ARDS, including death in some patients. Vit-D deficiency and/or failure to activate the vit-D receptor could trigger a cytotoxic response in stellate cells of the lung and aggravate respiratory complications in COVID-19 and PASC³⁹⁶. Vit-D could ameliorate pulmonary inflammation and facilitate repair of epithelial layers, damaged organs with inherent anti-fibrotic properties and help resolve inflammation-induced pathologies, such as fibrosis^690,700,701. CVD sequelae such as cardiomyopathy, arrhythmias, thrombotic complications, and cardiogenic shock are prominent features of COVID-19 and PASC pathologies⁷⁰². Vit-D could activate VDR function, regulate calcium flux, reset optimal myocardial contractility, and help reduce risks of myocardial infarction in COVID-19 and PASC patients⁷⁰³.

In Spain, a population-based cohort study of 4.6 million people supplemented with cholecalciferol or calcifediol, when achieved serum 25(OH)D levels >30ng/mL, showed a reduction of about half the risk of SARS-CoV-2 infection, severe COVID-19, or COVID-19 mortality than those not treated⁷⁰⁴. An observational study at the U.S. Veterans Affairs healthcare facilities, patients (n=4599) when adjusted to 25(OH)D levels from 15 to 60ng/mL, showed a decrease in probability of COVID-19-related hospitalizations from 24.1 to 18.7%, and mortality rates from 10.4 to 5.7%⁷⁰⁴. There is mounting evidence that optimal 25(OH)D levels are associated with reduced risk of COVID-19.

Reactivation of latent Epstein–Barr virus (EBV) is an emerging risk factor with adverse outcomes for both COVID-19 and PASC^269,705. Vit-D supplementation (20,000 IU/week over 96 weeks) could significantly reduce humoral immune responses against latent EBV antigen in relapsing-remitting multiple sclerosis⁷⁰⁶. Although more evidence is needed on therapeutic benefits of vit-D in COVID-19 and PASC, the multifunctional role of this vitamin on immune system is evident. Vit-D deficiency is cost-effective, safe, and readily available supplement strategy for COVID-19 and PASC management⁷⁰⁷. Therefore, individuals at higher risk of vit-D deficiency during COVID-19 pandemic should consider taking vit-D supplements to reset the circulating 25(OH)D levels to optimum (75–125nmol/L) and avoid and/or recover from COVID-19 and PASC.

Omega-3 Polyunsaturated Fatty Acids (Omega-3 or n-3 PUFAs), eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA), and docosahexaenoic acid (DHA), are broad-spectrum anti-inflammatory compounds that modulate several pathways of inflammation including leukocyte chemotaxis, adhesion molecule expression, and leukocyte-endothelial adhesive interactions, inflammatory cytokine synthesis and T cell reactivity⁷⁰⁸. As potent antioxidants, omega-3 PUFAs upregulate Nrf2, mitogen-activated protein kinase (MAPK) phosphatases, GSH and HO-1 genes. Metabolites of omega-3 PUFAs are vital for the synthesis of several inflammatory mediators including prostaglandins (PG), leukotrienes (LT), thromboxanes (TX), protectins, and resolvins⁷⁰⁹. Omega-3 PUFAs modulate both innate and acquired immune systems through activation of macrophages, neutrophils, T-cells, B-cells, dendritic cells, NK cells, mast cells, basophils, and eosinophils. As an integral part of the cellular membrane, omega-3 PUFAs regulate membrane fluidity and complex assembly in lipid rafts⁷¹⁰. PUFAs could help alleviate mitochondrial ROS production in T cells to combat SARS-CoV-2 infection²¹. Virus-induced HMRD of host lipid metabolism may persist as chronic inflammatory condition in PASC patients. Palmitoylethanolamide (PEA), a lipid-derived peroxisome proliferator-activated receptor-α (PPAR-α), is shown to dismantle lipid droplets via β-oxidation and restore innate cellular defenses⁷¹¹.

Omega-3 PUFAs may interact at different stages of SARS-CoV-2 infection, particularly during viral entry and replication phases where persistence of viral antigens may lead to sustained inflammatory state in PASC patients^277,712,713. Omega-3 PUFAs, particularly EPA, are potential remedials to reduce pro-inflammatory cytokines, alter the HPA axis, modulate neurotransmission via lipid rafts, and alleviate neuro-cognitive complications in PASC patients⁷¹⁴. Furthermore, omega-3 PUFAs and their metabolites (i.e., specialized pro-resolvin mediators), could effectively ameliorate uncontrolled inflammatory responses, reduce OxS, mitigate coagulopathy, and restore tissue homeostasis⁷¹⁵. Besides antioxidant and anti-inflammatory activities, omega-3 PUFAs could regulate platelet homeostasis and lower the risk of thrombosis, which indicates its potential use in COVID-19 and PASC management.

COVID-19 patients show significantly low Omega-3 Index (O3I=4.15%) compared to healthy subjects (O3I=7.84%). Lower O3I is associated with an increased risk of developing severe COVID-19 with mechanical ventilation and high CFR^716,717. In a retrospective clinical study (n=80), oral supplementation with omega-3 PUFAs has significantly lowered proinflammatory procalcitonin and IL-6 levels and reduced prothrombin time in ICU/hospitalized COVID-19 patients⁷¹⁸. Therefore, the nutritional status of omega-3 PUFAs is particularly important for the overall immune response, tissue inflammation and repair, which may be an effective nutritional strategy for PASC recovery.

Vitamin-C (Vit-C) (ascorbic acid) is a water-soluble, essential nutrient (cannot be synthesized by the human body), important for antioxidant activity and immune-modulation⁷¹⁹. Vit-C regulates NF-kB release and attenuates pro-inflammatory cytokines production⁷²⁰. In a meta-analysis of 7 RCTs and 7 retrospective studies (n=751 patients), vit-C supplementation showed a significant alleviation in inflammatory response by increasing ferritin levels and lymphocyte counts in COVID-19⁷²¹. Another meta-analysis of 19 RCTs showed a reduced in-hospital mortality rate in vit-C supplemented COVID-19 patients (24.1%) compared to non-supplemented group (33.9%)⁷²². In a retrospective study from UAE (n=63), oral supplementation of COVID-19 patients yielded no difference in vit-C levels across BMI categories; however, a significant correlation was noted between vit-C levels and SARS-CoV-2 clearance rate among the obese patient group⁷²³. Extended fecal viral RNA shedding suggests that SARS-CoV-2 infection in the GI tract could be prolonged in a subset of COVID-19 and PASC patients⁷²⁴. The potential viral clearance activity of vit-C supplementation could be a promising adjuvant combo with other ‘reset’ nutritional strategies to treat certain PASC patients.

Nutritional reset of gut dysbiosis

SARS-CoV-2 infection alters gut microflora composition and function, which leads to intestinal barrier dysfunction and immune activation⁷²⁵. Epithelial tight junction is a critical intestinal barrier, and its disruption could leak toxic substances from the gut into blood circulation and cause systemic injury. The maintenance of intestinal epithelial tight junctions is closely related to energy homeostasis and mitochondrial function³⁹⁷. Such virus-induced dysbiosis triggers cytokine release with NF-κB-mediated hyper-inflammatory response, and the ensuing immune dysregulation could worsen the clinical outcomes of COVID-19²⁴⁰. Gut microbiota plays a multi-functional role in the GI tract, including energy extraction from the diet, immune modulation, synthesis of vitamins and short-chain fat acids (SCFAs)⁷²⁶. A complex equilibrium exists among prebiotics (i.e., fructo-oligosaccharides), probiotics (i.e., LAB), and postbiotics (i.e., bacteriocins, SCFAs), with the involvement of several networks between gut microflora and other organ systems through different axes (i.e., Gut-Lung, Gut-Liver, Gut-Brain axes) that affect a plethora of pathways in health and disease^244,727.

Gut dysbiosis could persist for at least 6 months in COVID-19 patients after hospital discharge, and this chronic inflammatory condition may onset a wide range of neurological and neuropsychiatric symptoms in PASC patients²⁴⁴. Accordingly, several gut metabolic dysfunctions could contribute to long-term neuro-cognitive impairments in PASC, including: (i) perturbed ‘gut-brain’ axis due to loss of SCFA producing intestinal flora, which may cause neuropsychiatric disorders⁷²⁸; (ii) cytokine storm-induced immune-metabolic reprogramming, which could elevate ‘kynurenine:tryptophan’ ratio and trigger chronic depression syndrome⁷²⁹; and iii) ACE2 activation in the gut could alter l-DOPA production and neurotransmitter synthesis, thereby inflict neurological complications including CFS⁷³⁰.

Probiotics/lactic acid bacteria (LAB) regulate cytokine secretion and affect both nonspecific as well as specific immune responses. Bacteriocins produced by LAB are antimicrobial compounds known to inhibit adhesion and invasion of microbial pathogens in the GI epithelia⁷³¹. Probiotics could block SARS-CoV-2 proliferation in host cells, via potential immuno-modulation, and inhibit NLRP3 inflammasome activation⁷³².

Oral probiotic therapy of hospitalized COVID-19 patients (n=70) with Streptococcus thermophilus, L. acidophilus, L. helveticus, L. paracasei, L. plantarum, L. brevis, B. lactis, and B. lactis, experienced remission of diarrhea and other symptoms within 72h. These probiotics show a significant ameliorating effect and reduce the risk of developing respiratory failure by almost eight times in SARS-CoV-2 infected individuals⁷³³. Probiotic therapy of COVID-19 patients with a combination of nine different lactobacilli strains (as daily dose), showed significant alterations in gut microbiota, partial restoration of pulmonary dysfunction as well as intestinal dysbiosis. Probiotic therapy also reduced inflammatory markers such as TNF-α, IL-1β, IL-4, and IL-12⁷³⁴. Co-administration of L. rhamnosus EH8 with mycelia could potentially inhibit inflammatory cytokine release induced by SARS-CoV-2 membrane (M) glycoprotein⁷³⁵. A human RCT with COVID-19 patients (n=300) treated with L. plantarum and Pediococcus acidilactici for 30-days, showed total remission of 53.1% in the probiotic-treated compared to 28.1% in the placebo groups, respectively. Probiotic therapy showed significant reduction in pulmonary infiltrates as well as in lowering the nasopharyngeal viral load, shortened the duration of GI and non-GI symptoms, decreased the D-dimer levels, and boosted the synthesis of specific IgM and IgG responses against SARS-CoV-2⁷³⁶.

Co-administration of L. plantarum GUANKE (LPG) strain with COVID-vaccine has been suggested to act as an adjuvant, inducing specific and nonspecific immune response(s), thereby extending protection against SARS-CoV-2⁷³⁷. A 3-month administration of probiotic strain Loigolactobacillus coryniformis K8 on SARS-CoV-2 (mRNA) vaccine-induced immune responses in elderly population (n=200) was evaluated. All participants completed mRNA vaccination, while the intervention started ten days after the first dose. The IgG levels in were significantly higher in the treated group; however, at ages >85, probiotic administration increased IgA antibody levels⁷³⁸. Preclinical studies have shown that probiotic therapy confers specific immune responses in inflammatory cytokine expression, prevent cell apoptosis, and induce immunological memory against COVID-19⁷³⁹. Thus, re-balancing of healthy gut microbiota through probiotics, prebiotics, and immune nutrients, could help reduce inflammation, promote anti-inflammatory mechanisms, and reset a functional ‘gut-brain’ axis for optimal recovery of COVID-19 and PASC patients.

Nutritional reset of virus-induced metabolic disorders

HMRD in tandem with chronic metabolic syndromes could result in more severe outcomes of COVID-19 and subsequently extends into PASC⁷⁴⁰. A meta-analysis of 120 studies (n=125,446 patients) reported that most prevalent comorbidities for SARS-CoV-2 infection include: hypertension (32%), obesity (25%), T2DM (18%), and CVD (16%)⁷⁴¹. COVID-19 in conjunction with diabetes and obesity (both characterized by severe insulin resistance) has severe clinical consequences^742,743. Therefore, restriction of dietary lipid and sugar intake could potentially benefit COVID-19 and PASC patients with T2DM, obesity or other metabolic syndromes.

Epilogue—the final note

Life is a genetically programmed, intricately organized, chemical processing thermo-dynamic system, that self-sustains by breaking chemical bonds (catabolism) to release and capture free energy to form complex macro-molecules (anabolism) for specific structure-function (metabolism) in a lipid-based envelope, known as the cell. Viruses infect almost every species and are probably the most abundant biological entities on the planet Earth. Recently emerged coronavirus, the SARS-CoV-2, is a 29.9-kb RNA virus that possess a unique genomic ability to reprogram a mega-sized 3.1-Mb human DNA and its cellular metabolic machinery to prime, alter, and redirect host macro-molecules for its infection, replication, and propagation. The 14 viral ORFs interact with a few thousands of human metabolites in a specific manner for its intra-cellular invasion, replication, and transmission, which results in HMRD in favor of the viral pathogen. Genomic and meta-genomic data have revealed that co-evolution between viral and cellular genomes involves frequent horizontal gene transfer and the occasional co-option of novel functions over evolutionary time⁷⁸⁴, the virus-induced HMRD with PASC, is one such prime example. Therefore, the nutraceutical/food supplement industry should take a serious note that formulating a RESET composition is not a simple blend of food compounds or nutritional ingredients into a dosage form. A deeper meta-genomic insight to assure 3-D structure/function of nutrient molecules, priming/activation of nutrients with co-factors, compositional stoichiometry, optimal milieu (pH, redox, and ionic strengths), molecular interplay between bioactives (synergism/antagonism), target delivery, ADME/Safety/Toxicity profiles, and most importantly the bio-functional activity, are vital prerequisites in the development of nutrient-based remedials to RESET the virus-induced HMRD in long-COVID, the ‘new onset’ global metabolic syndrome. This precision nutrition-based dietary rehabilitation of PASC patients should be developed as an affordable, interventional regimen for divergent socio-economic populations worldwide for effective public health management of any future emergence of virus-induced ‘new onset’ human metabolic syndromes.

This work was supported by the Global Nutrition Healthcare Council, Mission COVID (www.missioncovid.com). We thank Trevor Amy for digital processing of citations, reference manager, and digital art.