SARS-CoV-2 (Covid-19): A short update on molecular biochemistry,pathology,diagnosis and therapeutic strategies

Abstract

The ongoing coronavirus (covid-19) pandemic highlights the need for global scientific cooperation to advance our understanding of the immunological, molecular and biochemical mechanisms causing infection by this virus. Better understanding of key processes has allowed the development of vaccines in record time, and of agents with the potential to treat and neutralize current and future coronavirus outbreaks. To date, clinically effective agents for prevention and treatment of covid-19 infections are limited. This review provides a brief synopsis regarding the molecular biology, pathology and laboratory tests commonly used in the diagnosis and prognosis of covid-19, as well as the development of vaccines and therapeutic strategies to manage its current and future mutations.

Introduction

The global pandemic resulting from a new mutation in a coronavirus (CoV) is still in progress. Following the initial genomic characterisation in December 2019, the immediate focus was on preventive measures to minimize the spread of infection. Extensive use of laboratory testing has been and remains a critical part of these measures despite its limitations^1–3 and cost. Simultaneously, there has been an intense research focus on the mechanisms underpinning coronavirus infectivity. Infection begins when the virus recognizes a host cell receptor and binds to it. This is followed by fusion of the viral and host cell membranes, releasing single-stranded viral RNA into the host cell. Replication of viral genomic material then occurs via the virus’s own RNA-dependent-polymerase, replicase, helicase, protease etc. (this has a high error rate; some viral mutations occur at this step) The release of replicated virus material via exocytosis further spreads the infection.

The molecular biology of SARS

The two key steps of coronavirus infection are binding and membrane fusion. These are mediated by the coronavirus spike protein (S). This is found on the viral envelope membrane and that of its host receptor, one of the angiotensin-converting enzyme receptors (ACE2). ⁴ ACE2 is present mainly but not exclusively in the lung alveolar epithelium and intestinal enterocytes. Viral infectivity and transmission is mainly through airways but gut transmission cannot be excluded.

The S protein is present in all coronaviruses that infect humans. It is a 180–200 kDa type 1 transmembrane protein; its N terminus faces the extracellular space and C terminus the intracellular space. Significant progress has been made in characterizing the structure of the covid-19 S protein, including its receptor-binding domain (RBD).^5–7 During the infection process, the S protein is cleaved by furin, a proteolytic enzyme; furin cleavage removes a ‘redundant’ section, leaving two subunits, S1 and S2. S1 contains the RBD that allows coronaviruses to dock and bind to the complementary peptide domain (PD) on ACE2. After the binding, S2 facilitates the fusion of viral and host cell membranes; a host serine protease plays a permissive role. The receptor- binding and membrane fusion domains of the S protein are highly conserved areas and major antigenic determinants. The binding and fusion processes are common to the coronavirus family; interference with these processes would therefore in principle allow cross-functional success, potentially holding the hope of pan-vaccines, drugs and antibodies.

Either the N-terminal or the C-terminal domains of the S1 subunit can serve as the RBD. The part of the RBD that directly binds the receptor is termed the receptor-binding motif (RBM). Generally, but not always, the N-terminal domain mediates viral binding to sugar-based receptors, whereas the C-terminal domain mediates binding to protein-based receptors. Both covid-19 (SARS-CoV-2) and SARS-CoV-1 (which caused an earlier pandemic) utilize the C-terminal domain for binding to ACE2. The greater infectivity of covid-19 (SARS-CoV-2) compared with SARS-CoV-1 has been attributed to its higher (10- to 20-fold) binding affinity to ACE2. ⁸ This may reflect changes in the RBD sequence. However, the ACE2-binding affinities of highly purified SARS-CoV-2 and SARS-CoV-1 are similar. ⁹ Further research is focused on resolving these discrepant findings.

In general, antigen–antibody reactions are associated with conformational changes, the purpose of which is to secure maximum binding affinity. The conformational changes in the S1 subunit expose hidden domains in the native covid-19 virus, e.g. heptad repeat (HR) regions. These highly conserved motifs located in the virus envelope have a six-helix bundle structure and are important in virus fusion. Some of the domains exposed during cell entry, e.g. HR1/HR2 are highly immunogenic, thus providing an additional potential target for viral interruption. So far, however, the structure of HR2 is not yet fully resolved.

Proteolytic cleavage at specific sites of the S2 subunit renders it ‘fusion-competent’. The functional fusogenic element of this subunit is the fusion peptide, a short well-conserved segment of up to 25 amino acids. Fusion occurs at either the plasma membrane or the endosomal membrane; covid-19 utilizes both pathways. As indicated above, a host serine protease (TMPRSS2) is necessary to prime the fusion of viral and host membranes by endocytosis, by causing irreversible conformational change in the S2 subunit. Cryogenic electron microscopy has shown that dimerization of the peptide domain of ACE2 permits binding of two covid-19 simultaneously, thereby enhancing the viral load. Membrane fusion is also heavily influenced by the extracellular environment, with proteases and pH playing direct and indirect roles in enabling fusion. Various ions also influence membrane fusion: calcium promotes it by stabilizing the FP structure, while zinc and magnesium do the opposite. It has also been suggested that cholesterol may directly influence membrane fusion dynamics by facilitating the formation of fusion intermediates; however, formal evidence for this role has not yet been obtained.

Covid-19 pathology

Approximately 80% of adults infected with covid-19 are asymptomatic, or develop mild/transient symptoms,^10,11 being able to mount a timely and coordinated immune response that ultimately leads to covid-19 clearance. It is not yet known whether the immune competence of these patients is intrinsic or a result of prior immune priming, perhaps connected to more rapid resolution of coronavirus infections. A recent study showed that patients with mildest illness have significant immune reactions which help to constrain the viral infection, even at times when humoral responses are not yet high enough to be measured in the blood. ¹⁰ In symptomatic covid-19-infected individuals (∼20%), viral transmission is maximal the day before the development of symptoms.

Laboratory testing

In the current pandemic, reverse transcriptase polymerase chain reaction (RT-PCR)-based tests have been widely used as the ‘gold standard’ in the diagnosis of active covid-19 infection. Amplification of covid-19 viral RNA in a nasopharyngeal swab involves an initial conversion of RNA to complementary cDNA (i.e. reverse transcription) followed by the use of primers which anneal to the specific segments under consideration; this provides a DNA template for amplification by thermostable polymerase. RT-PCR is essentially then an RNA-directed DNA polymerase process of the primed viral RNA within a cDNA. Real-time (one-step) rRT-PCR reduces significantly the detection time of covid-19 in samples. Although less prone to contamination, one-step RT-PCR is prone to degradation of the RNA starting template, resulting in false negatives. The false-negative rate of RT-PCR in new hospital admissions has been reported at ∼15%. ¹ Poor sampling technique, sample degradation, the presence of nucleotides that disrupt the annealing and amplification process, may all explain false-negative results.

Seroconversion occurs 7–14 days post symptoms, so antibody tests carried out thereafter are likely to be positive and may provide evidence of covid-19 infection in those with false-negative RT-PCR. An alternative approach, using exhaled breath condensate followed by RT-PCR, though non-invasive has been tested on a very small number of cases and its accuracy remained to be properly assessed.^12,13

The emphasis on false-negative RT-PCR test results is understandable, given the consequences of undetected infections especially in health and social care settings. The need to investigate false-positive RT-PCR rates has come into sharper focus more recently, with the wider application of RT-PCR as a screening test in asymptomatic and mildly symptomatic individuals. Real-time RT-PCR (rRT-PCR) is binary (positive or negative), producing quick results within 40 min. However, its analytical accuracy is dependent on the choice of primers and the dye used to quantitate dsDNA in all RT-PCR modalities. ¹⁴ Faster tests use lateral flow and are also widely used for mass-screening (results available within 15–20 min). Lateral flow involves a pad with strip coated with antibodies which react with specific covid-19 surface proteins. The antigen–antibody reaction is likewise binary with a positive result appearing as a coloured line. Lateral flow tests are dependent on the actual concentration of covid-19-specific protein(s) in the sample; if present at low concentration, this may produce a negative result; their limitations have discussed elsewhere. ²

RT-PCR is highly sensitive because each sample undergoes 20–40 thermal cycles of exponential amplification. The exquisite sensitivity of the test, especially when higher numbers of thermal cycles are used, renders analysis prone to false-positive results.^1,15,16 This could happen if fragments of old dead virus are detected from previous infection. Other major sources of error are contamination and cross-reaction with other genetic materials and can occur during swab extraction, aerosolisation between samples, amplicon carry-over, etc. A preliminary estimate from 43 EQA of certified laboratories gave a median false-positive rate of 2.3% (interquartile range of 0.8–4%).^1,15 Wider application of RT-PCR in cohorts with low prevalence is likely to produce a higher number of false-positive results. ² Consistent with this, Public Health England recently reported RT-PCR sensitivity and specificity of up to 95%. Both RT-PCR and antibodies tests for covid-19 can produce different false-positive and false-negative rates, the magnitude of which depends in part on disease prevalence in different cohorts.

Saliva is an accessible alternative matrix for detecting covid-19 genetic particles using RT-PCR and/or covid-19-specific IgA antibodies (IgA is the predominant antibody in mucosal surfaces of respiratory and gastrointestinal tracts). In the United States, the Federal Drugs Administration (FDA) has so far issued ‘emergency use authorization’ for five salivary tests, the accuracy of which is probably comparable or lower than tests using nasal swabs. ¹⁷

Among covid-19 hospitalized patients, a minority become progressively ill with some needing mechanical ventilation. Cytokine storm (hypercytokinaemia) is a recognized cause of death in some.^18,19 Both cytokines and interleukins can act as markers of inflammation in patients with CS. A cytokine storm occurs when cytokine release is uncontrolled, leading to hyperinflammation and vascular hyperpermeability. Diagnosis is clinical but laboratory tests help to substantiate the diagnosis and prognosis. Clinically, cytokine storm is marked by the need to increase oxygen supplementation continuously to maintain SpO₂ >93%. Laboratory markers of systemic hyperinflammation include markedly raised CRP, ferritin, D-dimer (a marker of endothelial damage), cardiac troponin, LDH (marker of cell death). Other non-specific abnormalities in routine laboratory investigations include deranged liver function tests and electrolytes, decreased lymphocytes and increased neutrophils and monocytes. Coagulopathy can also occur in cytokine storm^20–23 with serious sequelae. Mechanisms controlling coagulation can be impaired, with defective feedback disrupting the normal procoagulant–anticoagulant balance, predisposing to the development of microthrombosis, disseminated intravascular coagulation and pulmonary emboli.

Generally in primary infections caused by a new pathogen such as covid-19 virus, immunological memory is mediated by specific B or T lymphocytes; they do not participate in the defence during the primary exposure, but can efficiently and quickly differentiate and mount an acquired/adaptive immune response upon subsequent re-infection (T-cell response is generally considered more durable than antibody response). The establishment of such immunity in those who have had covid-19 infection remains has yet to be clearly defined.

Vaccine development

Immunological memory also underpins the principle behind vaccination. The development of safe and effective vaccines has been the focus of intense research activity since the beginning of 2020. This research has benefited from powerful cloud supercomputers ²⁴ and the recently accumulated knowledge of molecular biochemistry and pathobiology of SARS family including covid-19, permitting for example the development and evaluation of 3D model structures of SARS biomolecules, in turn accelerating identification of successful candidate vaccines and/or disruptive drugs. The challenges in developing effective vaccine are discussed and summarized elsewhere. ²⁵

At the time of writing, approximately 150 vaccines have been formulated worldwide, with 11 under phase three clinical trial evaluation. Recent developments exploit innovations, including the use of lipid nanoparticle mRNA, DNA, inactivated virus particles and non-replicating viral vectors such as viral nucleocapsid, spike and the receptor-binding domain of the spike; a comprehensive account is beyond the scope of this article. The monomeric form of the RBD has low immunogenicity, and use of a dimeric form has enhanced this, proving highly effective in provoking an antibody response of neutralising antibody.^26,27 The three most recent vaccines given emergency authorization are well tolerated and generate high titres of neutralizing antibody and cellular immune T-responses against the spike glycoprotein in all age groups including the elderly. This is important because immunosenescence is characterized by progressive decline in T- and B-cells which play a key role in immune response to pathogens and vaccines. Synthetic mRNA-based vaccines provide ‘acquired/adaptive immunity’ essentially by hijacking the replication machinery of the cell; for it to do so, the vaccine genome must be delivered intact to ACE2 target cells by a robust delivery vehicle. The reprogrammed host cells then produce viral antigens which on exocytosis provoke and stimulate an adaptive immune response through the production of antibodies and T-cell response.

The development of vaccines has benefited greatly from the sharing of information/data. For example, the genomic structure of covid-19 was posted on 10 January 2020. It took less than two months to make synthetic modified antigens resembling the key viral structures essential for docking on ACE2 target cells, e.g. an mRNA formulation encoding the viral spike S-2P antigen. Vaccines therefore encode modified viral snippets but do not contain the virus per se. These antigenic structures can in principle be tweaked to neutralize emerging coronavirus mutations, e.g. one originating in Danish mink farms and two other highly transmissible variants which appeared in England (VUI-202012/01) and South Africa. Both contain multiple mutations in the spike (S) protein and the receptor-binding domain. One key mutation (N501Y) is present in both England and South African variants, increasing virus binding to human cells via the ACE2 receptor and contributing to increased transmissibility without necessarily affecting its virulence/pathogenicity. The South African, however, has two additional mutations E484K and K417N in addition to N501Y which appear to reduce the viral visibility by the humoral immune system. Long-term safety and efficacy of recently developed vaccines remain undetermined. Eradicating coronavirus by vaccination is unlikely; reducing viral infection to manageable levels is a more realistic and pragmatic goal.

Therapeutic strategies

As highlighted earlier, exploring the basic mechanisms that underpin viral binding and cell entry is essential for development of structure-based designs of decoy ligands able to ameliorate or even block key processes mediating infectivity, e.g. docking, binding, entry and replication.^28,29 The immediate clinical focus is on (i) agents which reduce or ameliorate covid-19 infectivity and (ii) effective therapeutics to hasten recovery, shorten hospital stay, reduce complications and mortality. The use of powerful supercomputers has been applied to screening the huge library of already licensed drugs which can be therapeutically effective against covid-19, e.g. dexamethasone.

An alternative approach has focused on the prophylactic benefits of natural supplements, e.g. vitamin D^30,31 and zinc.^32,33 A recent meta-analysis^30,31 of 40 studies found that long-term daily doses of vitamin D protected against acute respiratory infections. Meta-analysis of patient data from 11,321 participants in 25 randomized controlled trials showed that vitamin D supplementation protected against acute respiratory tract infections and that patient with very low serum concentrations gain the most.^30,31 Potential mechanisms include enhancement of production of antimicrobial peptides in the respiratory epithelium by 1,25-dihydroxyvitamin D, and regulation of immunopathological inflammatory covid-19 responses.^30,31

The relationship between zinc and covid-19, including how zinc deficiency affects the severity of infection and whether zinc supplements can improve clinical outcomes, is currently under investigation. ³² Increased intracellular zinc concentrations impair replication of some RNA viruses.^32,33 Nevertheless, it is not currently recommended to give elemental zinc supplementation above the recommended dietary allowance for the prevention of covid-19.^32,33 Measurement of serum zinc does not reflect body stores of zinc. Excessive intake for long must be avoided because it could cause copper deficiency with undesirable sequelae. ³²

Treatment of hospitalized patients

Various modalities have been used in the treatment of severely ill covid-19 patients. One therapeutic approach has involved administration of neutralising polyclonal covid-19 antibodies^34,35 harvested from convalescent serum samples of recovered covid-19 patients. This approach has previously been used in previous pandemics, but its efficacy in covid-19 has yet to be established. Alternatively, monoclonal antibodies produced by fusing myeloma cells with selected B-cell clones obtained from recently recovered covid-19 patients are currently under investigation.^36–39 Almost all monoclonal antibodies so far developed target and block the RBD on the viral spike protein. Neutralizing antibodies also have the potential to be used prophylactically for high-risk group given the relatively long half-life of IgG (three weeks).

Remdesivir, a broad spectrum antiviral drug, which inhibits viral replication in host cells by blocking viral RNA-dependent polymerase, is also under evaluation. ⁴⁰ Dexamethasone has been found to reduce mortality in patients with severe illness, ⁴¹ although in that study, it did not benefit older patients and is not recommended in mild cases of covid-19. Dexamethasone acts by dampening an over-reactive immune system reducing the likelihood and complications of cytokine storm; dosage regimen is critical to avoid over-suppression of the immune system. Other ways of dampening an over-reactive immune system are the rheumatoid arthritis (RA) drugs, e.g. otilimab as well as monoclonal antibodies that block cell-signalling protein complements such as C5 and C3.^42,43 An international clinical study on the utility of the monoclonal baricitinib (currently used for rheumatoid arthritis) showed a 71% mortality benefit in elderly patients (median 81 years age) with moderate–severe covid-19. ⁴⁴ Baricitinib reduces viral infectivity by inhibiting kinases associated with viral endocytosis and also has anti-inflammatory activities, thus reducing lung damage and mortality. ⁴⁴

Despite better therapies and reduced mortalities, only 65% of individuals, recovered from acute covid-19 infection regain their previous level of health in two to three weeks after a positive test.^45–49 A sizeable minority remain unwell beyond three weeks, and a smaller proportion (≤1%) for months, enduring long-term health consequences (‘long-covid’). This can affect people of all ages, but older people and those with comorbidities are more prone. Reported symptoms include fatigue, abnormalities of the sleep/wake cycle and cognitive abnormalities. Similar symptoms have been reported with other coronaviruses. The reason why this affects some but not others, irrespective of infection’s severity is not known.

Conclusion

The urgent need to limit and neutralise covid-19 pandemic has galvanized intensive multidisciplinary research and cooperation worldwide. RNA viruses like covid-19 have extremely high mutation rates; mutations can occur due to multiple mechanisms: (i) copying errors during viral replication; (ii) recombination or re-assortment with other viruses that fortuitously happen to be present in the same infected cell and (iii) responding to neutralising agents, e.g. host antibodies. Mutations not only increase the range of virus serotypes in the population but also their potential to cross species barriers, resulting in zoonotic bidirectional transmission as shown in covid-19 presence in minks. The site of this mutation and its contagion are currently being assessed and collated by WHO.

Molecular mutations of covid-19, which reduce the damage to viral capsid (shell) essential for protecting viral genome and replications would enhance its delivery and load to ACE2 target cells, boost infectivity without necessarily altering its intrinsic pathogenicity/virulence per se. Mutations which increase the affinity of spike S-protein to receptors on ACE2 cells could also increase viral load and infectivity.

Vaccination programmes are just beginning to be rolled out across the world. It is too early to assess their effectiveness. ⁵⁰ However, their collective development constitutes the most promising phase yet in the fight to limit the impact of the coronavirus.