The Pathophysiology of Virulence of the COVID-19

Background: On Dec 19, 2019, the public health department of China reported that an outbreak of pneumonia was caused by a novel Coronavirus. The virulence of the new virus COVID-19 was much greater than either the SARs and MERSs viruses and on March 11, 2020, the World Health Department (WHO) declared a worldwide pandemic. Understanding the pathophysiology of virulence of the SARS-COV-2 virus is absolutely necessary for understanding the transmission, virulence factors, reduce risk factors, clinical presentation, predict outcomes of the disease and provide guidance for any current or future treatment protocols. Methodology: A comprehensive PubMed search was performed during December 20, 2019 and April 03, 2020, utilizing the words: Wuhan Virus, COVID-19, SARs coronavirus, ACE2, S-protein, virulence, clinical presentation, epidemiology, genome, treatment, structure, MERs, pathogenesis and/or pathology alone and in combination with other terms. Each paper was evaluated by three content experts for quality, reproducibility, credibility and reputation of the journal. Results: The SARSCOV-2 virus is much more virulent than either the SAR’s or MER’s virus and its ability to cause serious disease inversely corresponds to the person’s ability to produce T-cells which declines linearly with age. The ACE2 receptor binding site does not vary among different ethnic groups but do in ACE-2 expression levels. This variance in expression level may explain for different infectivity rates among men and women and predict and explain different susceptibilities to infection by different ethnic groups. Furthermore, by understanding the underlying pathophysiology one can explain and provide guidance to the clinical effectiveness of any treatment. Conclusions: The underlying pathophysiology of COVID-19 explains not only the virulence, and clinical presentation, but, explains at a molecular level the comorbidity risk factors such as hypertension, sex, and age. Ethnic and anatomic expression patterns of ACE-2 Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 15 April 2020 doi:10.20944/preprints202004.0077.v2

and associated pathophysiology suggests that Native Americans and Asians may be particularly susceptible to this disease.

Introduction
A mysterious illness started from Wuhan, China, has sickened millions throughout the world. It In clinical practice, COVID-19 patients are still mainly affected by the severe respiratory system infection, but evidence of damage to other system organs such as cardiovascular complications has also been reported (Yao et al, 2020;Hu, Wang, & Zhu 2020;Madjid et al., 2020).
The virus is highly contagious, case fatality rate for COVID-19 varies from place to place and country to country with current reported cases growing exponentially. Spreading quickly, affecting mainly elderly and populations with multiple underlying disorders but cases and deaths in young adults have also been reported including one death reported from Illinois of a patient under the age of 1 year. Global trends indicate that SARS-CoV-2 may reach 30-40% of the population, some of the communities may wiped out, and its spread will not slow down without accelerated human involvement in the near future in practices including social distancing, diagnostics, treatment and vaccination (Hagen, 2020). Exacerbating this is the fact the virus can spread from asymptomatic carriers (Rothe et al., 2020) Normally flu, common cold and influenza are the infections that we observe during winter and spring, so is viral pneumonia. Viruses that cause viral pneumonia are commonly influenza/ orthomyxo viruses, others include parainfluenza viruses, adenoviruses, rhinoviruses, cytomegaloviruses, and coronaviruses. These infections might also result in spread or outbreak in small communities or regions, with most common clinical manifestations of fever, soreness, dyspnea, and lung infiltration. Complications are mainly associated with virus load, virulence, route of infection, age and immune status of the host. Higher SARS-CoV-2 viral loads might worsen outcomes, and data from China suggests the viral load is higher in patients with more severe disease. The amount of virus exposure at the start of infectionthe infectious dosemay increase the severity of the illness and is also linked to a higher viral load (Heneghan et al., 2020 Basically, coronaviruses are large, positive-sense enveloped ribo nucleic acid (RNA) viruses in the Nidovirales order. They are divided into four genera i.e. α, β, γ and δ. Three species of βcoronaviruses have caused outbreaks of deadly pneumonia in humans since the beginning of the 21st century. Human coronaviruses, including hCoV-229E, OC43, NL63, and HKU1, cause mild respiratory diseases. These viruses have a broad distribution among humans and other mammals.
Even though most human coronavirus infections are mild, the epidemics of the two betacoronaviruses, severe acute respiratory syndrome coronavirus (SARS-CoV-1), and Middle East respiratory syndrome coronavirus (MERS-CoV), have caused more than 10,000 cumulative cases in the past two decades, with mortality rates of 10% for SARS-CoV and 37% for MERS-CoV. Causative agent of current outbreak is a novel betacoronavirus, the 2019 novel coronavirus (SARS-CoV-2) and there was no evidence or published reports about this species of coronaviruses before December 2019 (WHO 1, 2020).

Survey Methodology
A comprehensive PubMed search was performed during December 20, 2019 and April 03, 2020 utilizing the words: Wuhan Virus, COVID-19, SARs coronavirus, ACE2, S-protein, virulence, clinical presentation, epidemiology, genome, treatment, structure, MERs, pathogenesis and/or pathology alone and in combination with other terms. Initially, 300 research papers were screened and 74 research papers with information related to the chosen topic and scientific merit were selected for further review and evaluation by three content experts considering quality, reproducibility, credibility and reputation of the journal. Expert in medicine/pharmacogenomics, microbiology/infectious disease and molecular pathology separately reviewed each paper.
Mutual agreement on inclusion of information in this review by all three content experts was the threshold of the study.

Clinical Presentation
Most patients who are infected with COVID-19 will have only mild symptoms. Yet due to a high fatality and morbidity rate it is important to identify those at elevated risk. COVID-19 severe illness may be defined as having the endpoints of needing to be ventilated, placed in intensive care units (ICU) or death. Severe disease seems to be correlated linearly with age up to about the age of 65 where the correlation begins to become exponential. (Guan et al., 2020) The incubation period of this disease is about 4-5 days with about 5% of the population not showing clinical symptoms for as long as 14 day after initial exposure. (Guan et al, 2020). Nearly everyone with the disease under the age of 15 will not develop severe disease and most will not show symptoms. Thus, this age group can serve as carriers who are infected unknown to themselves and to many other therafter (Li Q et al., 2020). The disease can pass from those that show no clinical symptoms. (Zou et al, 2020). Studies indicate that the virus sheds at high levels from the nose in both symptomatic and non-symptomatic patients in a way more like the influenza virus than the SARS virus. (Wu, Zhen, & Zheng, 2020) The virus also seems to be able to survive on fomite for 72 hours and in the air as an aerosol for 3-4 hours making transmission possible even if an infected person has not been around for hours from the air or days from inanimate objects. (van Doremalen et al., 2020) The most important symptoms in adults are a fever 88%, cough 67.8%, fatigue 38%, difficulty breathing 18.7%, and myalgia. (Guan et al, 2020). It has been shown however that children may show diarrhea as a sign of infection. (Lu X et al, 2020) For those with clinical symptoms the cough is often followed a day or two later by a slight fever with pneumonia appearing perhaps as early as 2-4 days after the initial cough. The blood labs indicate that about 83.7 percent of those infected have lymphocytopenia and 60.7 percent will have C-reactive protein above 10 mg/liter (Guan et al, 2020). Elevated liver enzymes are also relatively common indicating that the COVID -19 virus has propensity for the liver which may also limit later therapeutic choices.
Lymphocytopenia occurs in 87% of the cases in contrast to SARs where it is at 18%. This may indicate that COVID-19 may be overwhelming the immune system and/or targeting the immune system itself. Chest X-rays are often benign initially or may show signs of interstitial pneumonia with a ground glass appearance. (Holshue et al., 2020). The pneumonia however may progress rapidly into lobar or bronchopneumonia with CT scans similar to what was found with SARS (Franquet, 2009). In terminal cases the chest X-rays may appear completed whited out. There is also evidence that the neuro-invasion by the COVID-19 virus may play a role in respiratory failure. (Li, Bai & Hashikawa, 2020) The severity of pneumonia is often scored with the CURB65 score which uses as criteria 1 point for any of the following conditions: Confusion, Urea > 7 mmoles, Respiratory rate > 30, diastolic BP < 60, and Age > 65. In the absence of other risk factors, any score equals to 2 or more the patient is generally admitted and a score equals to 3 or more the patient may be considered for intensive care. (Barlow, Nathwani, & Davey, 2007). These details were readily available before publication, genome sequence was submitted to NCBI library and provided a great help in diagnosis of early cases. The first PCR tests for COVID-19 were developed very rapidlywithin two weeks of the disease being identifiedand they are now part of the World Health Organization (WHO)'s recommended protocol for dealing with the disease. A positive test for SARS-CoV-2 generally confirms the diagnosis of COVID-19, although false-positive tests are possible. If initial testing is negative but the suspicion for COVID-19 remains, the WHO recommends resampling and testing from multiple respiratory tract sites (WHO 2, 2020).
Negative RT-PCR tests on oropharyngeal swabs despite CT findings suggestive of viral pneumonia, have been reported in some patients who ultimately tested positive for SARS-CoV-2 (Xie et al, 2020) As compared to RT-PCR, serologic tests are considered more rapid and in this kind of outbreak will be able to provide a rapid and in time diagnosis, once largely available, these serological tests (mostly Enzyme Linked Immuno Sorbent Assay or ELISA based) will help the clinicians to identify patients having current or previous infection, asymptomatic patients and patients with negative PCR results. In one study (Guo et al, reported the detection efficiency by IgM -ELISA higher than that of qPCR method after 5.5 days of symptom onset. They mentioned that positive detection rate was significantly increased (98.6%) when they combined IgM ELISA assay with PCR for each patient compared to a single qPCR test (51.9%) (Guo, 2020).
Wenling Wang et. al. (2020) reported presence of Virus in specimens collected from multiple sites. According to them respiratory tract samples most often testing positive for the virus. But the live virus was also detected in feces, implying that SARS-CoV-2 may be transmitted by the glycoprotein to promote host attachment and fusion of the viral and cellular membranes for entry. S is the main antigen present at the viral surface and is the target of neutralizing antibodies during infection. As a result, it is a focus of vaccine design. S is a class I viral fusion protein synthesized as a single polypeptide chain precursor of approximately 1,300 amino acids (Bosch et al, 2003).

COVID-19 cellular pathogenesis
For many coronaviruses, S is processed by host proteases to generate two subunits, designated S1 and S2, which remain non-covalently bound in the pre-fusion conformation. The N-terminal S1 subunit comprises four β-rich domains, designated Cao et al (2020) mentioned that a previous study showed that residues near lysine 31, and tyrosine 41, 82-84, and 353-357 in human ACE2 were important for the binding of S-protein in coronavirus but they were not able to find any mutation among different ethnic groups of these residues. They also reported that there was a lack of natural resistant mutations for coronavirus S-protein binding in populations. Similarly a study by Zhao et al (2020) reported that ACE-2 has been found to be elevated in Asian men, with Caucasian's and Large numbers of SARS-CoV particles and genomic sequences were also detected within circulating lymphocytes, monocytes, and lymphoid tissues, epithelial cells of the respiratory tract, the intestinal mucosa, the epithelium of renal distal tubules, neurons in the brain, and tissue-resident macrophages residing in different organs (Gu et al., 2005) Pathophysiology and virulence mechanisms of CoVs, and therefore also of SARS-CoV-2 have links to the function of the non-structural proteins (nsps), and structural proteins. These nsps are able to block the host innate immune response (Lei, Kusov, & Hilgenfield 2018). With reference to functions of structural proteins, the envelope plays a crucial role as it promotes viral assembly and release. Though, many of these features need more detailed studies.

Pulmonary Damage Cascade
The virus enters the respiratory tract usually through air droplets. There are reports of SARS-COV-2 virus associated cases with mild upper respiratory tract symptoms, suggesting the potential for pre-or oligosymptomatic transmission (Wolfel et al., 2020). Once in the respiratory tract the virus eventually enters the alveoli targeting the ACE2 receptor which is predominant on type II pneumocytes. It is the type II pneumocytes that normally produce surfactant which functions to increase pulmonary compliance, prevent atelectasis at the end of expiration and to help recruit collapsed airways and open these up. The type II pneumocytes are destroyed once infected by COVID-19 during the budding out process from these cells thus, reducing surfactant levels. The destroyed type II pneumocytes then recruit macrophages to destroy the dead debris and tissue. In turn the macrophages secrete interleukin 1, interleukin 6 and tumor necrotic factor TNF α. Interleukin-1 forms an important part of the inflammatory system causing vasodilation, increased body temperature, increased sensitivity to pain and localized fluid build-up.
Interleukin 1 also increases the number of adhesion molecules in endothelium cells enhancing the migration of neutrophils and lymphocytes to the area. Interleukin 6 mediates acute phase proteins to be produced. TNF induces cachexia, fever and cell death of other infected cells. The alveoli begin to become full of fluid and debris which decreases the diffusion of oxygen across the alveoli causing hypoxemia. Additional, bronchial epithelial denudation, loss of cilia, and squamous metaplasia and acute fibrinous with organizing pneumonia in later stages (Bradley & Bryan, 2009) (Gu et al., 2005. This in turn can permanently damage the lung. As the lung is further damaged the pneumonia can turn to acute respiratory disease, septic shock and multi organ failure. Interestingly, the use of ACE inhibitors and ARB's by those with hypertension causes an upregulation of the ACE2 receptor which may partly explain the increase in risks for those with hypertension. Comparatively we saw for MER-CoV infection the target receptor was dipeptidyl peptidase 4 (DPP4; also known as CD26), (Meyerholz, Lambertz, & McCray2016). Targets in the lung included pneumocyte, multinucleated epithelial cells, and bronchial submucosal gland cells.

lopinavir-ritonavir
There are several COVID-19 clinical trials looking at different combinations of Interferons with and without antivirals. A systematic review of the evidence related to lopinavir-ritonavir and SARS and MERS suggests it could be a potential treatment for COVID-19 infections.
Lopinavir/Ritonavir with or without interferons are being used regularly in the treatment of COVID-19 (Yao et al., 2020).
Cultural and lifestyle changes of the last fifty years have created a world in which these newly seen viral infections can spread worldwide in day and weeks. The need to prepare for future pandemics should be obvious.
Rapid identification, basic public health interventions such as social distancing, followed as quickly as possible by development of diagnostic tests to monitor in real time the spread of the disease should become standard reactions to new viral disease. Preparatory measure such as availability of enough personal protective equipments, medication, as well as costly instrumentation (such as ventilators) is essential to overcome the situations like current pandemic. SARS-CoV-2 mutates rapidly, infects with high efficiency, and causes severe illness in a high proportion of infections (Liu Y, et al., 2020). These qualities should be expected in future outbreaks. Entry of the virus via interaction with ACE-2 suggest a variety of potential therapeutic directions to develop and some effort should be made to develop pan-coronavirus interventions. ACE-2 shows patterns of ethnic and anatomic expression consistent with the spread of the disease among ethnic groups and the pulmonary and immune suppression that are major features of the severe acute respiratory syndrome -Coronavirus 2 (SARS-COV-2 syndrome). Public health CANDOR regarding future infections; will prepare the public for realistic scenarios and greater compliance with traumatic interventions ranging from social distancing, stay-at-home orders, job loss, economic costs and end-of-life care that will enhance our ability to conquer future pandemics faster and at less cost than it will require to overcome COVID-19.