Immunobiochemical aspects in the pathogenesis, diagnosis, and management of novel coronavirus (SARS-CoV-2) infection

Background: A new coronavirus (SARS-CoV-2) that emerged from Wuhan, Hubei Province, China, has spread throughout the world and is declared a pandemic by the World Health Organization (WHO). A lot remains to be understood of SARS-CoV-2 and the disease (COVID19). SARS-CoV-2 has until recently been identified as responsible for both asymptomatic and serious life-threatening infections. The unavailability of specific therapeutic agents is a major hurdle in the treatment and management of COVID-19 patients. The present review attempts to evaluate the immunobiochemical aspects of the pathogenesis, diagnosis, and management of SARS-CoV-2 infection. Main body: This review is a comprehensive evaluation of the data collected through various sources, including Google Scholar, PubMed, and Scopus. The articles were searched and selected using key words such as “Coronavirus disease (COVID-19)”, “Diagnosis of COVID19”, Pathogenesis of Covid-19”, “management of COVID-19”, “Immunology of COVID-19”, and “Complications of COVID-19”. The study noted that the novel Coronavirus infection could result in an exaggerated immune response, causing a cytokine storm and damaging several organs of the body. The infected patients develop several complications, including immunological, hematological, and biochemical alterations. Consequently, COVID-19 patients may develop cardiovascular, liver, renal, and neurological complications, among others. Conclusion: An increased understanding of the immunobiochemical aspects of the disease may contribute to better management of SARS-CoV-2-infected persons, as evidenced from the available literature. A holistic approach to the management of COVID-19 patients taking into Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 24 September 2020

The mortality rates of infection with COVID-19 varied significantly among the affected countries. A recent study from Italy revealed a mortality rate of 7.2% [5,6]. The case fatality rate, as reported from the Chinese mainland, hovered at approximately 4%. Additionally, the Chinese Center for Disease Control and Prevention (China CDC) reported that only 1% of cases were reported among children under 10 years and adolescents [7]. COVID-19 affects more men than women, as evidenced by the results of a previous study [8]. In the most recent development, a multisystem inflammatory syndrome was reported in children infected with SARS-CoV-2.
Children and adults with confirmed COVID-19 were noted to suffer from a clinical condition remarkably similar to Kawasaki disease (a systemic vascular inflammatory condition) and toxic shock syndrome. The patients presented with lymphopenia, hyponatremia, abnormal liver enzymes, thrombocytosis (increased platelets), and elevated activities of inflammatory markers such as erythrocyte sedimentation rate, C-reactive protein, procalcitonin, ferritin, D-dimer, troponin, and interleukin-6 [9]. Additionally, the neurological consequences as a result of SARS-CoV-2 infection are important. As suggested by the WHO, anosmia was recognized as presymptomatic evidence of COVID-19. A recent study evaluated two COVID-19 patients who were diagnosed with inflammatory neuropathy. Histological examination of the olfactory epithelium revealed significant atrophy of the mucosa, leukocytic infiltrates, and axonal damage.
Histopathologic examination of the brain confirmed leukocytic infiltration and thrombosis. The study could not confirm whether the inflammation on the neuronal tissue was a direct effect of the infection or otherwise [10]. In view of the COVID-19 disease complexity and due to the unavailability of specific therapeutic drugs, careful management of COVID-19 patients assumes increased significance. Therefore, this review attempts to delineate the importance of attachment in the case of SARS-CoV [14]. Recent studies have demonstrated that the spike proteins of the novel SARS-CoV-2 also bind to ACE2 receptors, triggering the infection process [15][16][17]. The expression of ACE2 receptors has been found increasingly on type II alveolar epithelial cells of the lungs, heart, ileum, kidney, endothelium, and bladder [18,19,20].
In general, the life cycle of a virus within the host consists of the various phases that include attachment, penetration, biosynthesis, maturation, and release. After the virus binds to the host cell (attachment), it penetrates the cell through endocytosis or membrane fusion. Later, the viral nucleic acid is released inside the host cell. The viral nucleic acid replicates along with the host cell nucleic acid, after which the viral mRNA is used to produce viral proteins (biosynthesis) and develop new viral particles, which undergo maturation and are released. After the virus enters the cell, it becomes exposed to the endosomal proteases present in the host cells [21]. Within the endosome, the fusion peptide is exposed after the S1 subunit is cleaved and inserted into the host cell membrane. The S2 region undergoes a conformational change to bring the HR1 and HR2 regions together. This leads to membrane fusion and release of the viral nucleic acid into the host cytoplasm followed by replication, which results in the formation of daughter viral particles. The viral particles are released when the host cell disintegrates and the virus spreads to the other cells [22]. In addition to the S protein, the plasma membrane-associated type II transmembrane serine protease (TMPRSS2) was found to facilitate the entry of SARS-CoV-2 into the host cell [23].
Previous research has suggested that the RBD of SARS-CoV-2 binds to ACE2 with a higher affinity than SARS-CoV [24].

Immune sensing
Viral antigens are recognized by cells of the innate immune system, such as antigen-presenting cells (APCs). These cells process and present antigens to cells of the cell-mediated immune system, such as natural killer cells (NK) and TCD8+ cytotoxic cells. Hence, both innate and adaptive immune systems are activated. This results in the enormous stimulation and secretion of pro-inflammatory mediators that include cytokines and chemokines. Such an excessive stimulation of inflammatory cytokines creates a storm that results in damage to the tissues, organs, and vascular endothelium, causing multi-organ failure and even death [25,26].
These pro-inflammatory cytokines were noted to be released into the blood of affected patients [27,28].
PRRs such as endosomal Toll-like receptors (TLR-3 and TLR-7) recognize PAMPs in the extracellular milieu, the activation of which leads to the nuclear translocation of transcription factors such as the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) and interferon-regulatory factor family 3 (IRF3). Triggering NF-kB causes an increase in the expression of IL 1, IL 6, and tumor necrosis factor-α (TNF-α). The second set of cytosolic PRRs, such as retinoic acid-inducible gene I (RIG-I) like receptors (RLRs) such as RIG-I, melanoma differentiation--associated protein-5 (MDA-5), and laboratory of genetics and physiology-2 (LGP-2), recognize intracellular PAMPs. RIG-1 and MDA5 activation results in the stimulation of IRF3 through the mitochondrial adaptor antiviral signal (MAVS) protein. In turn, these trigger increases in the expression of type 1 IFN, which is considered most important for antiviral defense. This leads to the activation of the IFN-α receptor complex (IFNAR) and causes phosphorylation/activation of signal transducers and activators of transcription (STAT) family, transcription factors 1 and 2 [29][30][31][32]. Additionally, the binding of DAMPs with RLRs triggers the synthesis of inflammasomes, which trigger the conversion/maturation of procaspase-1 to caspase-1 and pro IL-1β to IL-1β, which are potent pro-inflammatory cytokines [33].
Because of the activation and production of proinflammatory cytokines by the innate immune system, the host defense mechanism then channelizes the adaptive immune system against  and multiorgan failure [35,36].

Cytokine storm
The cytokine storm is caused by an increased secretion of pro-inflammatory cytokines from activated macrophages and monocytes. Some factors that are elucidated for cytokine storm include impaired viral clearance due to immune evasion by the virus, presence of low levels of type 1 INF (T1:INF) due to inhibition of T1:INF signaling as it happens in antibody directed enhancement (ADE), deficiency or inactivation of MDA5, increased neutrophil extracellular traps (NETs), and pyroptosis, which is an inflammatory and caspase 1-dependent programmed cell death. All these factors lead to the release of pro-inflammatory intracellular contents [37].

Immune evasion
Viruses may escape host immune responses by evading PRR sensing. They can do so either by evading or antagonizing PRR action. SARS-CoVs and most likely SARS-CoV-2 could affect the inactivation of cytoplasmic RNA sensors (RIG-I and MDA5) [34]. ORF9b suppresses MAVS signaling, which is required to induce the nuclear translocation of interferon regulatory factor 3 (IRF3) [37]. IFN release is suppressed by counteracting T1IFN signaling and the transcription factor phosphorylation of STAT family proteins [38]. Additionally, TNF receptor-associated factors (TRAFs) 3 and 6 are blocked, which are important for the activation of IRF3 and IRF7 [34]. It was noted that the novel coronaviruses replicate in the host cells by suppressing the host innate immune system and antiviral response mechanisms. Furthermore, apart from stopping IFN signaling, the virus activates alternative inflammatory pathways by secreting accessory viral proteins such as open reading frame 3a (ORF3a), ORF8b, and E proteins, which improve the formation of inflammasomes and the release of IL-1b and IL-18 [39,40]. Viral non-structural proteins (NSP9 and NSP10) have been shown to suppress the NKRF gene, which codes for endogenous NF-kB repressor proteins [41,42]. These factors could contribute to enhanced inflammation and cytokine storm.

Transmission pattern
According to the latest guidelines and from observations made by the Chinese and several other scientists throughout the world, the novel CoV SARS-CoV-2 appears to transmit mainly through droplets, aerosol inhalation, and mucosal contact transmission. Droplets and aerosols are viral particles containing respiratory and oral secretions that are released into the environment when an infected person coughs, sneezes, and talks. Aerosols are also produced during various laboratory (centrifuging) and clinical procedures (tracheal intubation). The distance travelled by the droplets depends on the size of the droplets, wherein droplets >5 μm may not travel more than one meter, but those that measure <5 μm may be floating/suspended in the air for longer periods and may potentially be carried to a longer distance. Transmission by contact (direct or indirect) may occur when a person comes into skin contact with a surface, object, or a fomite contaminated with the virus, which accidentally enters through mucosal contact with mouth, nose, and conjunctiva. [43, 44].

Clinical manifestation and diagnosis
Patients infected with SARS-CoV-2 develop fever, cough, and dyspnea, with pneumonia occurring in complicated cases. It was also observed that several infected patients remain asymptomatic or develop only mild symptoms. The diagnosis of COVID-19 is made based on the travel history of a person (recent travel to places where the disease has shown community spread, contact with an infected or suspected COVID-19 patient), comprehensive clinical, and laboratory examination. The diagnostic methods used include computed tomography (CT) scans of the lungs, antigen/antibody detection by enzyme-linked immunosorbent assay (ELISA), and confirmation by nucleic acid amplification tests such as polymerase chain reaction (PCR) [45].

7.1.Molecular diagnostic methods
Whole genome sequencing (WGS) and real-time reverse transcriptase polymerase chain reaction (rt-RT-PCR) are two frequently applied molecular diagnostic methods for SARS-CoV-2 infection. In view of the high cost associated with WGS, rt-RT-PCR is the most common, effective, and preferred tool for diagnosing COVID-19 from specimens that include nasal and nasopharyngeal swabs, sputum, and other respiratory secretions. Currently, rt-RT-PCR is recommended as the gold standard method to diagnose SARS-CoV-2 infection [45].

Patient Screening
The World Health Organization (WHO) recommended procedure for suspecting and diagnosing the COVID-19 caused by the SARS-CoV-2 is depicted in Figure 2 [44].

COVID-19
All patients who present with acute respiratory illness, along with fever and cough, dyspnea, and a recent history of travel to a place that has reported community transmission of COVID-19 two weeks before the onset of symptoms should be considered suspected patients. Additionally, patients who present with an acute respiratory illness and give a history of coming into close proximity with a COVID-19-positive or suspected COVID-19 case two weeks before the onset

Biochemical Monitoring in COVID-19 Patients
During

8.2.Neutrophil lymphocyte ratio
The ratio of neutrophils to lymphocytes (NLR) is a value calculated by using the absolute neutrophil and lymphocyte counts present in the blood. It is used to predict potential systemic inflammatory conditions. An increase in the counts of neutrophils and a decrease in the lymphocyte counts predict the severity of inflammation and the extent of damage to the immune system, respectively. The NLR can be quickly calculated by routine blood tests, which help in identifying SARS-CoV-2 infection at an early stage. Increased NLR indicates severity and is an independent risk factor for in-hospital mortality. The NLR, platelet-lymphocyte ratio, and monocyte-lymphocyte ratio, when considered in combination, can be used to predict the severity of COVID-19 [48,49].

8.3.Role of ferritin and hyperferritinemia syndrome in COVID -19
Ferritin is a storage protein that participates in iron metabolism. By structure, ferritin contains L and H subunits, which are expressed in the lung and heart, respectively. The H subunit is involved in the inflammatory mechanism by participating in myeloid and lymphoid cell proliferation and stimulating TIM-2, a specific ferritin receptor. H-ferritin plays a key role in immunomodulatory and pro-inflammatory activities by activating several inflammatory mediators, such as IL-1β [48].
Ferritin was found only in the lymph node B area, suggesting its role as an antigen that stimulates macrophage activation related to hyperferritinemia [50].

8.4.Role of cardiovascular markers in patients with COVID-19
The activities of LDH, CK, CK-MB, myoglobin (Mb), cardiac troponin I (cTnI), alphahydroxybutyrate dehydrogenase (α-HBDH), AST, and the N-terminus of the prohormone brain natriuretic peptide (NT-proBNP) were elevated in varied concentrations in COVID-19 patients [52,53]. An elevation in laboratory cardiac markers could be used as a prognostic tool in predicting the necessity of intensive care and critical management of the patients along with the disease outcome [54]. an increase in the prothrombotic milieu, which generates shear stress in the coronary blood vessels, causing increased blood flow. This results in the rupture of plaque, causing vascular blocks and myocardial infarction. Anti-microbial agents used in treating and managing patients infected with COVID-19 may also contribute to cardiovascular events, as evidenced from the available literature. Since SARS-CoV-2 interacts with the renin-angiotensin system, which is a significant factor in balancing body electrolytes, and because of the excessive inflammatory environment, studies have reported hypokalemia and suggested that COVID-19 patients may develop electrolyte imbalance and cardiovascular events such as tachyarrhythmias [55].

8.5.The clinical course of COVID-19 patients as observed in relation to the activities of cardiac troponin
Mild -Patients hospitalized with COVID-19 have been noted to show elevated/fluctuating activities of troponin, typically remaining well below the 99 th percentile upper reference limit.
This appears to be the most common pattern of troponin elevation in patients with COVID-19 and is often associated with no cardiac symptoms. This pattern has been described in patients with COVID-19 who survived after hospitalization.
Moderate time-limited -Elevated activities of troponin, higher than the 99 th percentile of the upper reference limit, which stabilized to normal on subsequent days, were observed in patients who were clinically suspected of developing myocarditis or stress cardiomyopathy.

8.6.Role of CRP and procalcitonin in COVID-19
CRP is an inflammatory substance that is secreted during microbial infections and inflammatory conditions. It is an acute-phase protein secreted by IL-6 in the liver. During injury, infection, and inflammation, there is an increase (>0.6 mg/dL) in the activities of CRP, which returns to normal (<0.6 mg/dL). Therefore, it is considered a potential marker for disease prognosis. As evidenced by the available literature, it was observed that the activities of CRP were significantly increased in COVID-19 patients who required ventilation.

8.8.Role of LDH in COVID-19
LDH is an intracellular enzyme that is essential for the conversion of cell sugars (lactate, pyruvate) into energy. LDH is present in the cells of various organs of the body, including the liver, lung, heart, kidneys, and others. LDH is found in five isoenzyme types, including LDH1-LDH-5. Each LDH isoenzyme is associated with a particular organ of the body. LDH isoenzymes 1 and 2 are found in RBCs and heart tissue. LDH isoenzymes 4 and 5 are present in the liver and skeletal muscles. LDH isoenzyme 3 is found in the lungs, lymphoid tissues, severe infections, tissue injury, and inflammation, causing cell lysis and LDH release. Because LDH isozyme 3 is present in lung cells, infection with SARS-CoV-2 may cause damage to the lung tissue and release of LDH 3, which can be analyzed among COVID-19 patients to assess disease severity [59]. Increased activities of different LDH isoenzymes may predict multiple organ failure and influence the clinical outcome, as evidenced from the correlation of increased LDH with poor clinical outcomes in severe COVID-19 patients [59]. patients [62]. In normal subjects, the serum activities of SAA remain <10 mg/L, and severely ill patients present with SAA >10 mg/L, signifying the importance of SAA as an indicator of disease severity among COVID-19.

Role of renal function test in COVID-19
The kidneys may be affected in COVID-19 patients, who may develop acute kidney injury (AKI) and present to the hospital with proteinuria. Because of the increased expression of ACE-2
The activities of serum urea and creatinine could be used to distinguish severe COVID-19 from mild cases, as they reflect glomerular filtration rates/kidney function. These biochemical parameters may be considered early indicators and used to manage patients with severe COVID-19. The therapeutic efficacy of GS-3754 (remdesivir), an adenosine nucleoside analog, which is currently under trial, has been found to be useful in the treatment of the Ebola virus. Research is currently underway to find its potential usefulness in the treatment of SARS-CoV, MERS-CoV, and other viruses that can pose a serious public health threat against whom there is no specific therapeutic drug [65].

COVID-19 therapeutics
The utility of lopinavir/ritonavir and ribavirin, which have shown promise against SARS-CoV, and MERS-CoV, in treating COVID-19 patients remains to be adequately researched [66]. A combination of lopinavir/ritonavir and interferon-beta-1b, ribavirin, and interferon, which showed promise against MERS-Co-V, also remains to be tested for their efficacy against SARS-CoV-2 infection [67].
HIV protease inhibitors such as lopinavir/ritonavir (LPV/r) in combination and nelfinavir were recommended for the treatment of COVID-19 pneumonia [68,69,70]. Baricitinib is another potential drug for COVID-19 treatment that inhibits viral entry into cells and inflammation [69].
Recently, the anti-rheumatic drug tocilizumab, with its anti-inflammatory properties, was noted to be effective in treating severe cases of COVID-19 [76]. Even teicoplanin, an antibacterial antibiotic that was found to inhibit MERS-CoV, was suggested to treat SARS-CoV-2 infection [77].

Conclusion
The novel Coronavirus disease has now been in existence for more than seven months. The