The Pathophysiology of Virulence of the COVID-19 Virus

Background: On Dec 19, 2019 it was reported by the public health department of China 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 or MERSs viruses and on March 11, 2020 the World Health Department (WHO) declared world -wide pandemic. Understanding the pathophysiology of virulence of the COVID-19 virus is absolutely necessary in understanding the transmission, virulence factors, reduce risk factors, clinical presentation, predict outcomes of the disease and provide guidance to any current or future treatment protocols. Methodology: A PubMed search was performed 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 COVID-19 virus is much more virulent than either the SARs or MERs 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 do not vary among different ethnic groups but do in 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 Ethnic and anatomic Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 7 April 2020 doi:10.20944/preprints202004.0077.v1

expression patterns of ACE-2 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 was late November 2019 when patients with pneumonia like symptoms started to come and soon on December 19, 2019 first case of novel Coronavirus patient was reported officially by public health department of China. Chinese scientists discovered a new strain of coronavirus that is apparently and genetically different from previously known strains of famous coronaviruses like SARS and MERS (CoV). This is a newly emerged strain that is currently testing the capabilities of this modern world for dealing unfamiliar pathogens like this Novel Coronavirus SARS-2 (SARS-CoV-2) which causes COVID-19. As per briefing of Wuhan's Municipal Health and Health Commission on December 31, 2020, 27 cases were reported with similar clinical manifestations of fever and difficulty in breathing and their chest radiographs showed bilateral lung infiltrative lesions. Overall, the relentlessness of this COVID-19 is unquestionable. Because of the exponential growth of infection, on March 11, 2020; WHO declared this COVID-19 infection a pandemic. As of today March 31 st , 2020-with spread through 177 countries of the world, the COVID-19 remains severe, continuously expanding with worldwide 803,650 cases and biggest number of cases from US (164,785), following Italy, (101,739) Spain (94, 417), China (82,276) and so on. In clinical practice, COVID-19 patients are still mainly affected by the severe respiratory system infection, but evidence of damage to other system organs has also been reported (Yao et al, 2020;Hu, Wang, & Zhu 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 approximately 4.8%, 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 50-60% 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). Exasperating 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, Basically, coronaviruses are large, positive-sense enveloped RNA viruses in the Nidovirales order. They are divided into four genera i.e. α, β, γ and δ. Three species of β-coronaviruses 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  and there was no evidence or published reports about this species of coronaviruses before December 2019 (WHO 1, 2020).

Survey Methodology
A PubMed search was performed 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. Expert in medicine/pharmacogenomics, microbiology/infectious disease and molecular pathology separately reviewed each paper. Agreement on content by all three content experts was the threshold for including the information in this review.

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 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 others. (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 nonsymptomatic 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 as a 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%, 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 lower of 2 or more the patient is generally admitted and a score of 3 or more the patient may be considered for intensive care. (Barlow, Nathwani, & Davey, 2007).
Interestingly the following factors in addition to age may play a role in morbidity: hypertension, diabetes, hepatitis B infection, COPD, immunodeficiency and cancer (Guan et al. 2020; Fang,

Testing
The ideal specimen for the COVID-19 diagnosis as per CDC guidelines is a nasopharyngeal swab. Oropharyngeal, nasal mid-turbinate, or nasal swabs are acceptable alternatives if nasopharyngeal swabs are unavailable. Expectorated sputum should be collected from patients with productive cough; induction of sputum is not recommended. A lower respiratory tract aspirate or bronchoalveolar lavage should be collected from patients who are intubated (CDC, 2020). Molecular techniques have been used successfully to identify infectious agents for many years. Unbiased, high-throughput sequencing is a powerful tool for the discovery of pathogens (Palacios et al, 2008;Armstrong et al., 2019).
Considering ss-RNA nature of viral nucleic acid, and due to the fact that this virus was previously not known, the reliable and suggested method other than culture for SARS-CoV-2 RNA is use of reverse-transcription polymerase chain reaction ( 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 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. 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 fecal route. They also found a small percentage of blood samples positive for COVID-19 PCR test, suggesting systemic infection (Wang et al, 2020).

COVID Virus
Coronaviruses are a group of viruses that cause a significant percentage of all common colds in human adults and children. Four human coronavirus including 229E, OC43, NL63, and HKU1 are prevalent and typically cause common cold symptoms in immunocompetent individuals.  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 It is also reported that the recombinant RBD protein bound strongly to human ACE2 (hACE2) and bat ACE2 (bACE2) receptors. They also found to block the entry of SARS-CoV-2 and SARS-CoV into their respective hACE2-expressing cells, suggesting that it may serve as a viral attachment inhibitor against SARS-CoV-2 and SARS-CoV infection (Tai et al., 2020).

COVID-19 cellular pathogenesis
ACE2 is a type I transmembrane metallo-carboxy-peptidase with homology to ACE, an enzyme long-known to be a key player in the Renin-Angiotensin system (RAS) and a target for the treatment of hypertension (Riordan, 2003). It is mainly expressed in vascular endothelial cells, the renal tubular epithelium, and in Leydig cells in the testes (Ling et al, 2020). That is why this also has been suggested that this novel SARS CoV-2 might affect male fertility. First evidence about such effect reports the alteration of sex-related hormones under COVID-19. They found serum luteinizing hormone (LH) significantly elevated and testosterone (T) to LH ratio decreased in COVID-19 patients, which infer to the potential hypogonadism (Ling et al, 2020).  Additionally, CoVID-19 also needs to be distinguished from other non-viral causative agents that might cause pneumonia-like symptoms and signs for example Mycoplasma, chlamydia, Streptococcus, hemophilus, variety of invasive fungi, vasculitis, and dermatomyositis etc. It is also important to consider that patients with viral pneumonia may also test positive to multiple other viruses, and that is why the severity of a combined virus infection with this COVID-19 or SARS-CoV-2 should not be ignored. Comparing the pathology of SARS-CoV-1, MERS and SARS-CoV-2 it is reported that the predominant visceral macroscopic changes in fatal SARS-CoV-1 cases have been edematous lungs with increased gross weights, multiple areas of congestion, enlargement of lymph nodes in the pulmonary hila and the abdominal cavity, diminished spleen size and reduced spleen weights (Nicholls et al, 2003;Ding et al., 2003).
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. 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 1forms 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.
Reported pathogenesis of MERS-CoV infection include exudative diffuse alveolar damage with hyaline membranes, pulmonary edema, type II pneumocyte hyperplasia, interstitial pneumonia, and multinucleate syncytial cells. Bronchial submucosal gland necrosis had also been observed Interferons are critical for innate and adaptive immunity against viral infections. Additionally, Azithromycin inhibits the bronchial excretion of mucin which when excreted narrows the bronchial passageways inhibiting breathing.Azithromycin may also limit inflammation. (Min & Jang, 2012).

Summary/Conclusion
The COVID-19 pandemic is the third coronavirus-caused disease to arise in the 21 st century.
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 stockpiling common protective measures as well as costly instrumentation (such as ventilators) cannot be left to for-profit health care systems which may find unused stockpiles fiscally unsustainable. 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 Angiotensin Converting Enzyme-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 COVID-19 syndrome. Higher levels of ACE-2 may predict increased susceptibility in men, Asians and Native Americans. 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.