Review: Evolving knowledge on Sars-Cov-2 (Covid-19)

In recent months,  the Sars-Cov-2 virus (COVID-19) has become a significant cause for public health vigilance as a result of a pandemic leading 271,410 deaths across six continents (WHO, 2020).  In addition to the death toll, the outbreak has had significant impacts on global economies due to loss of productivity. Due to its infancy, there is much scope for research surrounding the underlying mechanisms of pathology, diagnosis and management of this de-novo virus.  The scope of this review is to summarise and consolidate some of the current understanding to date, and to explore newer findings as more data emerges.

Sars-Cov-2  was first discovered in the city of Wuhan, within the Hubei province of China in December 2019 after reports of large numbers of people mysteriously dying of pneumonia-type illness. As of 7th May 2020, there have been 3.84 million cases identified. In the United Kingdom, cases have risen to an estimated 215,000 confirmed cases and 31, 587 fatalities (14.6%). In stark contrast, the United States has confirmed 1,320, 869, with a 5% fatality rate.  Fatality rates for Spain and Italy are 5.9% and 1.6% respectively (CDC, 2020). The  Commonly identified features of the disease include initial cold-like symptoms that consist of a non-productive cough, loss of olfactory function, pyrexia, fatigue and extreme shortness of breath with severe cases resulting in pneumonia and multi-system organ failure (WHO, 2020). However, these symptoms vary between patients, and the severity of the disease varies – with worse effects on patients with co-morbidities and age-related decline. Based on the phylogeny of the virus, it has been established that Sars-Cov-2 shares close genomic similarities to the βcoronavirus genus, which are groups of positive-sense single-stranded RNA viruses (King, 2011; Sah et al. 2020)

There is well-established evidence that Sars-Cov-2 appears to be of zoonotic origin. The previous sequencing from similar types of infection indicated the implication of bats transferring the virus to intermediate reservoirs. These reservoirs facilitate mutations significant enough to cross over to humans. Examples directly relating to Coronaviridiae family include the Severe Acute Respiratory Syndrome (SARS) outbreak in 2002, followed by  Middle eastern respiratory virus (MERS) in 2010 (Huynh et al. 2012). The association of coronaviruses with bats has led to the speculation that these animals are the progenitor carriers. Reports indicate the presence of both alpha and beta coronavirus in bat faecal matter. Various sequencing studies have shown that human coronavirus can replicate in a plethora of bat species. It has been demonstrated, limitation preventing direct bat-human viral access to cells was via the spike glycoprotein present (RBD). RBD is a 180 amino acid portion of the spike glycoprotein present on the viral surface that shows a high affinity for Angiotensin-converting Enzyme receptor 2 (ACE2) (Huynh et al. 2012).

Concerning SARS-CoV-2 infections, intermediary host such as the Civet, seen in the 2002 SARS outbreak was the likely host to have induced a mutation wide enough to facilitate a change in the tropism of the RDB gene, given the 96% genome match to humans. The latest novel Sars-Cov-2 virus is thought to implicate the zoonotic migration from a bat, to pangolin to human (Ortega et al. 2020). This intermediary reservoir could have provided the opportunity for the RDB mutation to enter human cells (Huynh et al. 2012). The consensus is that the virus utilises ACE-2 receptors present on respiratory ciliated epithelial cells as a portal of entry, where it hijacks the host transcription and translation apparatus to proliferate and migrates to type 2 pneumocytes (Mason, 2020). Pathogenicity studies have demonstrated that the nasal passage offers an optimal route of entry via cough generated aerosols and highlight nasal swabs yield a higher viral load than buccal swabs.

The determinant for the successful entry of the Sars-Cov-2 virus is dependent on RBD-ACE2 interaction followed by subsequent protease activity involving peptidase enzymes TMPRSS2 and Furin (Bestle Et al. 2020). TMPRSS2 plays an essential role in the cleavage of polyproteins in the S2 pockets, and are required for the assembly of viral components, while simultaneously utilising RNA dependent RNA polymerase to yield more copies of its RNA (Cascella et al. 2020). The Newly assembled viruses within the type 2 pneumocyte bud off contributing to pneumocyte damage. These precipitating factors instigate the onset of gaseous exchange complications – presenting a medical crisis caused by a cytokine storm. This cytokine storm is propagated by macrophage hyperactivation and the production of Interleukin 1 (IL-1), IL-6 and Tumour Necrosis factor-alpha (TNF-a).

These cytokines, render the surrounding capillaries of the alveoli more permeable to plasma, which leaks around, and into the alveolar space (Mehta et al. 2020). The outcome of this series of events results in respiratory distress marked by severe hypoxemia and in some cases, death. Post mortem characterisation of lymphatic tissues (nodes and spleen) in Sars-Cov-2 patients were found to have marked changes having undergone necrosis and atrophy. On inspection of the lymph nodes using Immunohistochemistry (IHC), CD68+ and CD169+ Macrophages were shown to have upregulated levels of IL-6 and pro-apoptotic protein FAS (Feng et al. 2020).

It is increasingly evident in worse case scenarios; multiple pathologies arise due to complications secondary to an infection, leading to multiple organ failure. (Feng, et al. .2020). Other immune implications of the virus include Neutrophil migration to the site of infection, which leads to the production of reactive oxygen species, and the formation of NET’s. These NET’s exhibit a damaging effect on both the viral particles and endogenous tissues resulting in alveolar consolidation. The most severe impacts of Sars-Cov-2 have been documented as multi-system organ failure following the development of systemic inflammatory response syndrome (SIRS). Patients who enter this stage of the disease, are frequently places on multi-system support through mechanical ventilators, and dialysis, (Mozzini & Cirelli, 2020).

HAEMATOLOGICAL OBSERVATIONS IN SARS-COV-2

Recent laboratory hospital laboratory findings have implicated sars-Cov-2 infection with dysregulated haematopoiesis and haemostatic mechanisms. Deranged blood cell indices have been highlighted as a standard feature on those with severe disease. Lymphocytopenia has been considered a significant finding with the potential to hold prognostic value. Standard features of Sars-Cov-2 have not been limited to conditions such as increased lactate dehydrogenase (LDH), elevated C-reactive protein (CRP), Increased Interleukin-6 (IL-6), Procalcitonin and Ferritin (Terpos et al. 2020)

In a similar study with a sample that examined the role of lab parameters and diagnostic accuracy in sars-Cov-2 patients, 200 cases were studies. Patients were aged between 19 and 78 years (99.47% female). Of the 200 participants, 35% received a diagnosis for the disease. Patients who tested positive showed a similar increase in biological markers for infection. These tests included elevated neutrophil (P=0.0001), CRP (0.004) and below range WBC (P=0.0001) in addition to other disease markers (Mardani, Et al. 2020).

Lille University Hospital in the North of France reported a case series of Sars-Cov-2 patients in ICU, whereby of 107 patients >20% developed pulmonary embolisms (PE). The virus may be implicated in hypercoagulability disorders similar to those seen in patients with Disseminated Intravascular coagulation (DIC). Standard features to DIC are related to consumptive clotting factors and platelets; however, Increased Fibrin levels seem to differentiate this particular condition from DIC itself. In addition to the aforementioned, elevated D-dimer presence as a result of increased fibrin has also been noted. (Poissy, 2020). In a study containing 449 individuals infected with Sars-Cov-2, 99 patients were prescribed LMW-heparin for one week. There was a positive correlation between the level of D-dimer and mortality rate. Similarly, negative correlation occurred in those with lower platelets. Patients underwent a sepsis-induced coagulopathy (SIC) score. The outcome was that those with a SIC score>4  or markedly elevated D-dimer had more benefit from Low Molecular Weight Heparin (LMWH) administration than those with a lesser score; to which there was little difference between those given heparin and those not given heparin. (Tang et al.  2020). A study conducted at Manchester University Teaching Hospital also identified the benefits of LMWH following successful prophylactic treatment of 99 out of 449 (22%) patients who tested positive for Sars-Cov-2. Their report highlights the rationale for low figures, owing to treatment with LMWH following the discovery of PE in those patients; however, this discovery provided pivotal information for retrospective analysis. Comparatively, this paper also discusses the use of D-dimers as markers for patients with a SIC score >4. Median Tissue factor concentrations were also markedly elevated in Sars-Cov-2 patients compared to non- acute respiratory distress syndrome patients (Thatchil, 2020).

MOLECULAR DIAGNOSTICS

The reverse-transcriptase polymerase chain reaction(Rt-PCR) is the current method employed for the diagnosis of Sars-Cov-2. Due to the fact, the virus itself is an RNA Virus; its genetic material is first transcribed into complementary DNA (cDNA) before amplification. Specially designed forward and reverse primers based on the nucleotide configuration of the 3’ and 5’ end of the Sars-Cov-2 RNA are obtained by correctly sourcing the gene sequence from bioinformatics database, i.e. GenBank.

The gene sequence can be used in conjunction with appropriate software such as Primer3, or Genscript. Primer design is routinely done to the specifications of the practitioner following the basic tenents. Primers should run from 3’ to 5’, be 18-25 nucleotides in length, maintain a 40-60 % GC content and have no long runs. (Warford & Presneau, 2019)

Analysis of PCR product is commonly done on gel electrophoresis using agarose gel as a medium in 10X TBE buffer. TBE is the preferred choice of buffer to use in the analysis of genomic material due to its capacity to give a higher resolution than TAE. RT-PCR may have diagnostic value in the determination of viral load in infected patients. Limitations involved in RT-PCR are primarily related to turnaround time,  labour intensity and cost. Efforts have been concerted into the development if other in-vitro assays such as the recent development of an immunoassay test with reduced turnaround time. The assay is designed explicitly by leading company Abbott, to determine Sars-Cov-19 IgG. One of the many benefits includes 5 minute turnaround time and the capacity to process up to 200 samples at once, relatively high throughput for point of care analysis. This particular assay boasts a specificity and sensitivity>99% for two weeks or more from symptom onset, however, is subject to validation before widespread application in NHS laboratories (Green et al., 2020). At the moment, in the United Kingdom, the Royal College of pathologists in collaborations with Public Health England has issued a testing guidance document outlining the scope and standard operating procedure to the employment of RT-PCR. Other methods, as previously referenced, are currently being validated. In addition, Scientists based as Oxford Biomedical Research Centre have established that Enzyme-Linked immunosorbent assays (ELISA) can be modified for the establishment and quantification of viral antibodies with high sensitivity for Sars-Cov-2 IgG.

TREATMENT AND MANAGEMENT STRATEGIES

Maximal efforts have been made to attain an effective treatment strategy for Sars-Cov-2 patients with severe disease and associated pneumonia. Researchers from the University Quindo, China, began experimental research design to test the efficacy of anti-malarial drugs Chloroquine, and Hydroxychloroquine. At that time, there were 16 trials across ten Chinese hospitals. Studies from this period highlight superior performance compared to placebo controls in >100 patients. The treatment yielded minimal adverse reactions. The mechanism of action is not yet fully understood. Some of the mechanisms proposed relate to endosomal pH reduction and effects on glycosylation (Gao et al. 2020). Glycosylation is a ubiquitous phenomenon achieved by post-translational modification.  It serves diverse functions that help to promote the virulence of the pathogen (Watanabe et al. 2019). Another look into the efficacy of anti-malarial drugs showed success in the treatment of sars-Cov-2; however, this study was in vitro. There can be significant differences in the generation of data between in-vitro and in -vivo studies; therefore, information regarding the use of chloroquine should be viewed with caution. More data should be presented with regards to this form of chemotherapy; much of the literature reviewed appear to present anecdotal evidence accompanied by small sample sizes, not large enough to generate statistical power (Meo et al. 2020). Some of the more creative approaches to developing treatments for the virus lie in understanding the mechanism of entry and synthesising products to inhibit or ACE2 expression, or to formulate ACE2 antagonists that will competitively inhibit interaction with the viral RBD spike; such as the development of anti-RBD antibodies similar those developed after the primary SARS outbreak (Cao et al.  2010)

FINAL WORDS

Given the recently presented evidence, there is much being discovered with regards to the mechanism driving this de-novo illness. With significant efforts being concentrated on impact minimisation, and understanding pathogenicity and drug development. It is beyond any reasonable doubt, that the unprecedented rate at which this outbreak has wreaked havoc on families, public services and global economies causing a worrying time for all. Simultaneously, it has been an exciting time to watch others in the scientific field, sharing knowledge that could be crucial in helping beat this nasty illness and achieve a positive outcome for the health and wellbeing of the globe.

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