COVID-19

Here is what I have been able to learn about the SARS-CoV-2 coronavirus so far:

The SARS-CoV-2 is a coronavirus which is a type of RNA virus. It is composed of 3 envelope glycoproteins: spike(S), membrane(M) and Envelope(E). These enclose the viral genetic material core, the messenger ribonucleic acid(mRNA). The mRNA is packed tightly within the core by a viral nucleocapsid(N) protein. The S protein is the portion of the virus that is thought to interact with cell membrane surface proteins to allow the virus to enter cells and is the spike depicted on the surface, envelope, of the virus in illustrations. There are six known other coronaviruses which infect humans. Four cause the common cold. Two cause potentially lethal diseases: severe acute respiratory syndrome coronavirus(SARS-CoV) and Middle East respiratory syndrome coronavirus(MERS-CoV). The latter two are believed to have originated in animal populations. 

On day zero of infection with SARS-CoV-2, the virus infects the epithelium of the  nasal sinuses, travels down to the lungs, and by day two the viral load in the lungs is similar to the viral load in the nasal passages. By day four, the infectious process in the lungs is well underway with typical symptoms of viral lower respiratory infection (dry cough, sore throat, shortness of breath, fever). By day seven, the viral activity has mostly been suppressed by people with a normal immune system,  although the viral RNA continues to be detectable as far as 21 days from symptoms onset, suggesting they are still contagious at that point. Although initial viral load likely influences the ability of the body to fight the infection, clinical data indicate that once symptoms develop, serum virus concentration is unrelated to patient outcome. Age correlates positively with poor outcome, likely due to immunosenescence seen normally in aging, as expected.

The virus passes into the bloodstream at the area of the alveoli and capillaries due to direct attack on the vessels by the virus, oxidative damage to endothelial cells caused by exophagocytoized cytotoxic vesicles from white blood cells, and due to the increased permeability of the blood vessels caused by the local inflammatory response. The virus then disseminates hematogenously to the intestinal mucosa, kidneys and spleen with demonstrable presence of virus and damage by day 4 after infection. It is possible that the virus is carried by cilia up the trachea along with sputum to the level of the glottis and reaches the digestive tract through the esophagus. 

The virus is able to enter cells by attaching to the membrane-associated protein type 2 angiotensin converting enzyme(ACE2). ACE2 is present in most cells in the human body, but is highly expressed on cilia-containing cell membranes, including those of lung alveolar type 2 cells, epithelial cells of the gastrointestinal system and renal tubule epithelium in the kidney, explaining why these organs seem to be particularly affected by the virus. ACE2 normally cleaves Angiotensin II(Ang II) creating smaller protein fragments (A1-7) that have anti-inflammatory properties. When abundant virus is present, much of the ACE2 is bound up, so Ang II is not cleaved and builds up. The loss of the anti-inflammatory protein fragment cleavage products is thought to contribute to the aggressive inflammatory response caused by this virus. Angiotensin II is well known to physicians because it causes vasoconstriction, sympathetic nervous stimulation, and aldosterone synthesis in the kidney affecting the body’s water balance. Less well known to physicians is that abundant Angiotensin II also causes induction of growth, cell mitosis of vascular smooth muscle cells, and increased synthesis of collagen type I and III in fibroblasts, leading to fibrosis. This is of particular interest at the moment, in light of the fact that excessive fibrin deposition in the lungs has been one of the puzzling aspects of COVID-19-SARS infection’s consequences in the lungs. 

The virus appears to cause cardiomyopathy without infecting the cells of the heart. This may be due to virus binding to cardiocyte ACE2 membrane-bound protein. This would have two effects. The first is that it would reduce the ability of ACE2 to act on its normal substrate, Angiotensin II(Ang II), causing unusually high amounts of Ang II to accumulate. Ang II is inotropic and is a growth factor for cardiac myocytes, stimulating compensatory hypertrophy. In the long term, excess Ang II has been shown to cause progressive functional loss and cardiac fibrosis, as the synthesis of extracellular matrix is increased by AngII. The second effect could be due to loss of direct signalling to the cardiac myocytes, since soluble ACE2 has been shown to interact with cardiocyte integrins (membrane-bound proteins that induce intracellular responses in response to extracellular stimulus), and soluble ACE2 would also be bound up with virus, unable to perform its usual role. 

COVID-19 has been found to sometimes cause stroke. The mechanism for this is poorly understood. This may be due to the high expression of ACE2 on cerebrovascular endothelial cells. ACE2 is known to indirectly inhibit the inflammatory response in endothelial tissue as well as to cause vasodilation.  In areas affected by the virus, the virus would bind up the available ACE2, there would be less competition for the factors in the blood that cause inflammation and vasoconstriction, so then one would expect increased inflammation and vasoconstriction. Narrowed vessels with excess inflammation can cause clotting due to the slowed flow and the free radicals released in the inflammatory process. The role of free radicals is explained toward the end of this paper. 

The disease process branches into three pathways at day seven. Most people will begin to recover as they would from other viral infections. A subpopulation goes on to develop a cytokine storm. A third population does not recover from the disseminated viral infection, develops diffuse organ failure, and succumbs. It is not clear what percentage of the third population develops a cytokine storm as well. 

In the cytokine storm pathway, cytotoxic immune(CI) cells (cytotoxic T lymphocytes and natural killer cells) attack infected cells that are presenting virus proteins at their surface. Normally, the CI cells will produce a protein called perforin that penetrates the cell membrane of the targeted cell and then the CI cell will release into the targeted cell multiple cytotoxic agents that cause the cell to die. In the meantime, the CI cell is secreting other cytokines (transforming growth factor beta(TGF-ß) and interleukin 6(IL-6)) that summon macrophages to the site in order to clean up the expected debris caused by the destruction of the cell that had been infected with the virus. 

In 10-15% of the population, one of the two copies of the perforin genes are defective making these patients susceptible to perforin malfunction. In the right circumstances, these patients will have an impairment in the ability of the CI cell to utilize perforin to kill the target cell infected with virus. However, the CI cell will continue to secrete cytokines to summon macrophages to the area. The macrophages then summoned to the area, although initially summoned for cleanup, begin to create their own cytokines. In the resulting cytokine storm, the macrophages turn to increasing the deposition of fibrin (rather than debris cleaning). This excessive fibrin deposition leads to the areas of lung restriction in this disease and the ground-glass appearance seen on chest X-ray. The presence of one of the cytokines, (TGF-ß) causes the epithelial cells lining the lungs to convert into connective tissue fibroblasts. The combination of fibrin and fibroblasts appears to increase the chances of inappropriate blood clotting which has been seen frequently in this disease. Specifically, patients with COVID-19 have been noted to have an increased predisposition to develop stroke, and blood clotting in the small vessels of the extremities.

Another mechanism by which COVID-19 causes harm to patients may be by causing a post-infectious immune mediated response directed against their own tissues. A certain portion of patients will develop an autoimmune reaction in response to viral infections or even vaccinations. This is unpredictable and affects an extremely low percentage of people in the population. Autoimmune disorders have been suggested as a cause for the cardiomyopathy, Guillain-Barre syndrome, and vasculitis that have been seen to cause dysfunction in these patients. 

Cytokine storm identification and treatment. The beginning of cytokine storm (day 7-9) is identified by a serum ferritin level in the 10000 range. (This is distinct from the elevated ferritin levels in the 100s often seen in sepsis.) Elevated alanine aminotransferase(ALT), aspartate aminotransferase(AST),  soluble interleukin (IL)-2Rα(CD25) and Fibrin-Split Products(FSP) are also serum indicators of cytokine storm. Elevated levels of lactate dehydrogenase3 isoenzyme(LDH), high-sensitivity C-reactive protein(hs-CRP – a human serum protein originally found to bind to the Capsular polysaccharide found on Streptococcus pneumoniae) combined with low levels of lymphocytes correlate to a 90% degree of accuracy with an elevated mortality rate. LDH is a marker of tissue damage while CRP is a marker of inflammation. LDH >365 U/L, hs-CRP>41.2mg/L & Lymphocyte count <14.7% predicted mortality with 81% accuracy.

Directly inactivating some cytokines has been useful to treat the cytokine storm.  There have also been reports of COVID-19 treatment success with tocilizumab, an IL-6 antagonist. Prior to the COVID-19 crisis, cytokine storm syndrome had been reported  to be successfully managed with high-dose corticosteroids in association with anakinra, an IL-1 antagonist. Anti-INFγ is also being tested.

Plaquenil(generic name hydroxychloroquine), and its more primitive version, quinine, have been utilized since prior to the 1500’s for the treatment of rigors and inflammatory disorders. Plaquenil currently finds significant use in the treatment of a common autoimmune, inflammatory condition of the skin, psoriasis. Prior to the COVID-19 epidemic, Plaquenil had been demonstrated to be useful in the treatment of cytokine storm syndrome in nonviral disorders, possibly due to its ability to directly promote the production of cytokines suppressing the inflammatory immune response. This makes it useful in treating the second phase of the COVID-19 disease process.  Plaquenil may be effective in treating the first phase of the COVID-19 infection by halting virus reproduction and packing by interfering with the way endosomes and lysosomes merge within the infected cell. (The merging requires an acidic pH and Plaquenil makes the pH too basic). Plaquenil is effective against malaria by a completely different mechanism.

Although Chinese controlled trials demonstrated reduction in duration of symptoms in mildly ill patients with COVID19, a large New York study using observational data suggests that Plaquenil may not be effective in reducing rates of death or intubation in symptomatic patients with COVID-19. This study is controversial since the patients who received the drug were sicker than patients who did not. Therefore, the study has also been interpreted to demonstrate that sicker patients treated with the drug had identical outcomes to less sick patients, possibly suggesting the drug has benefit. A prospective study is required. Use of Plaquenil in this patient population appears to be the current standard as of the time of writing of this article.

Zinc is a mineral second only to iron in its importance to the human body. Zinc deficiency compromises the human immune system. It seems that intracellular Zinc is mostly bound up in proteins called metallothionenes which come in 4 classes with multiple subtypes. The first two classes are important, MT1 & MT2. They function primarily as regulators of gene transcription. Several of the MT1 & MT2 gene subtypes are activated by interferons, a type of cytokine particularly important for viral immunity. These MT1 & MT2 genes can cause elevation of intracellular zinc levels. The elevated zinc levels trigger the activation of other MT1 & MT2 genes. It appears that different types of viruses cause different groups of these MT1 & MT2 genes to activate, creating a virus-class specific response. In the case of coronaviruses, through a mechanism not fully elucidated yet,  these genes produce proteins that cause inhibition of the viral protein RNA-dependent RNA polymerase(RdRp), which is a protein used by coronaviruses to copy their RNA genetic code. The zinc-dependent MT1 & MT2 gene products prevent binding and elongation of the code, making the virus unable to replicate. Correcting ZInc deficiency is therefore beneficial.

PPE. Several young, healthy nurses and physicians have developed severe infections requiring intensive care after performing cardiopulmonary resuscitation(CPR) on patients who are COVID positive. It can be assumed that many COVID positive patients requiring CPR have a high viral load which CPR aerosolizes significantly in this setting. Performing CPR is an activity that requires significant exertion, requiring deep breathing by the rescuer. Deep breathing would allow the virus to penetrate deep into the lungs, making the lungs the site of initial exposure rather than the sinuses. This gives the virus access to the alveoli, capillaries and the blood stream before the body’s targeted immune response has had a chance to develop. This may be why individuals with an otherwise healthy immune system succumb to the disease. This emphasizes the need for personal protective equipment (PPE), especially in the CPR setting. 

N-Acetyl-Cysteine(NAC). A trial of IV N-Acetyl-Cysteine in COIVD-19 patients has begun recently. NAC is a non-toxic reducing agent that assists the body’s normal pathways designed to clear free radicals. Free radicals are used by the body to destroy the membranes of bacteria, or cells infected with virus. When free radicals present in excess, they can destroy healthy cells as well as the planned target. These free radicals can induce the clotting cascade by inducing activation of von Willebrand’s factor. Because of the cytokine storm, the macrophages are signalled to keep secreting free radicals.  This overabundance of free radical overwhelms the body’s natural mechanism for clearing them, leads to activation of von Willebrand’s Factor, and contributes to the excessive clotting seen in this disorder. Since NAC helps clean up free radicals it is hoped it will help with the clotting problems seen in COVID-19 patients with a cytokine storm. NAC has been shown to work as an anticoagulant and has been shown to reduce ICU days in patients with severe pulmonary infections from non-COVID causes. For this reason people are studying whether very high doses of NAC can be effective in helping ventilator dependent COVID-19 patients by reducing the tissue damage and the clotting caused by the free radicals.

Azithormycin is an antibiotic drug used to treat bacterial infections. It binds to bacterial ribosomes which prevents the bacteria from making proteins, which causes the bacteria to die. This drug has no direct action against viruses. It is used to treat possible opportunistic bacterial infections that may try to establish themselves in the COVID patient’s injured lungs, in a setting where there is already immune system compromise caused by the virus. It is used to basically help the body keep up defense against bacteria while the immune system is busy fighting the COVID virus. It has not had proven benefit and its continued use in the treatment of COVID-19 positive patients is being debated.

Remdesivir is a ribonucleotide analog, resembling ATP. Viral RNA polymerase will insert Remdesivir into an RNA strand which it is copying . This will then halt further transcription after a few more base pairs are added, preventing the virus from making copies of itself. The drug has had proven efficacy in humans against other RNA viruses including Ebola virus, Lassa virus, respiratory syncytial virus (RSV). In animal models it has been shown to be effective in MERS-CoV, SARS-CoV and SARS-CoV-2. Early anecdotal data in humans describes a modest benefit from the use of this drug so far.

Excessive blood clotting. Above we have described the role that loss of ACE2 activity has in increasing the likelihood of clotting. We also described how the overabundance of free radicals activated von Willebrand’s factor(Factor VIII) to promote clotting. We should also mention that because the cells that line the blood vessels (endothelial cells) express ACE2, they are targets for invasion by the virus. When these cells are killed by the virus, they release tissue factor(TF), which stimulates blood clotting via the extrinsic pathway(by amplifying the effect of factor VIIa). These three mechanisms appear to be the reasons excessive clotting is sometimes seen in patients infected with COVID-19.

Passive immunization is achieved by transfusion of serum from recent COVID19 survivors into patients actively trying to fight the disease. Recent survivors will have large quantities of antibodies to the virus in their serum. There have been anecdotal reports of treatment success by transfusing this serum into patients currently fighting the disease. No published evidence documenting success was found as of the time of this writing. 

Prazosin and Nicotine. The sympathetic and parasympathetic systems can each detect inflammation and modulate it by centrally and locally acting mechanisms. The sympathetic neurotransmitter norepinephrine can act on macrophages by the alpha1 receptor to induce the production of IL-1 and ramp up the inflammatory response. The drug prazosin blocks that receptor and has been shown in a mouse model of the cytokine storm to attenuate the inflammatory response. The parasympathetic neurotransmitter acetylcholine acts on macrophages at the alpha7 subunit of the nicotinic acetylcholine receptor to reduce the production of cytokines, including TNF and IL-6.

Correcting Vitamin D deficiency has been shown to reduce the risk of severe respiratory infections (like influenza) by half. This is based on a 2017 meta-analysis of 25 studies involving 11,000 people.

BCG. Last month an article was published noting the difference in the percentage of the population that was severely affected by coronavirus in Spain and Portugal, with the later being found to be significantly less affected. The author suggested that this might be due to the fact that people in Portugal still receive Bacille-Calmette-Guerin (BCG)  vaccinations for tuberculosis prevention, while the people in Spain do not. Others noticed that there seemed to be a tremendous amount of COVID-19 related death in western countries, whereas in less developed countries the percentage of people who are severely affected is much lower. The use of BCG in the less developed countries was again suggested as a possible reason for the difference. There is a large body of literature describing the protective effects of BCG vaccine on reducing infant mortality from all respiratory causes, including respiratory viruses. This generalized type of immunity might be sufficient to prevent the deaths of people at high risk of exposure, such as health care workers. BCG immunization side effects are well known and the vaccine is already approved by the FDA. Over 4000 healthcare workers in Australia received the BCG vaccine in April in a human clinical trial designed to test the benefits of use of this vaccine to keep our front-line workers healthy. Results of that trial are expected in October of this year. Similar trials have begun in the Netherlands and at Texas A&M.  A recent Israeli study suggested that BCG is not protective against COVID`9, but their vaccinated patient hadn’t had BCG in 50 years and it is not clear if booster BCG had been given, per the normal BCG vaccination protocol.

Survival data from the WHO indicates that about 15% of people over 90 years old who contract the disease succumb to it, and the death rate decreases by about half for every decade younger than 90 the patient is. At 80 there is a death rate of about 8%, at 70 a rate of about 4%, at 60 a rate of about 2%,  at 50 a rate < 1%, and at 40 and younger a rate of about 1 death in 500 patients who test positive who have symptoms. 

There are three unanswered questions physicians have had about this disease:

1)Although elderly and immune-compromised patients are expected to be more severely affected by an infectious disease, why are some otherwise young healthy people more affected than others by this disease? This article suggests that deep inhalation in an area of concentrated virus may be a factor, since this causes initial exposure deep in the lungs prior to exposure in the sinuses, before the immune response has had a chance to start. This article also suggests that some people have a genetic predisposition to developing a cytokine storm due to mutations of their perforin genes.

2) What causes the coagulopathy seen in this disease? This article suggests that the excessive clotting is caused by excessive activation of von Willebrand factor due to overabundance of free radicals, combined with the macrophage-induced increased deposition of fibrin caused by elevated TGFβ expression due to the cytokine storm. 

3) Why do most viruses cause pulmonary failure associated with fluid filled lungs which are heavy at autopsy, whereas this virus seems to cause fibrosis and blood vessel obstruction which causes light lungs at autopsy? This article suggests that the reason may be because this virus reduces functioning ACE2 activity, which leads to a build-up of Ang II, which causes fibroblasts to secrete extracellular matrix. That combines with the cytokine storm, which causes increased production of TGFβ which causes conversion of respiratory epithelium into fibroblasts, increasing the number of producers of extracellular matrix. The increased extracellular matrix, combined with the clotting of the arterioles described above lead to a fibrotic lung picture, rather than a wet lung scenario.

Interesting Questions: 

Autoimmune disorders affect about 3% of Americans. Viral infections have been a suspected cause of autoimmune diseases with reported varying penetrance of ranging from 1% to 3%. Are 1-3% of patients infected with COVID expected to develop autoimmune reactions? Perhaps we can expect up to 30,000 reports of autoimmune disease to be found among the identified COVID-19 positive population, currently about 1MM. This has implications for vaccinations, and the design of vaccines that avoid these autoimmune responses. It also raises questions about the ethics of asking people to put themselves at risk individually for the good of society.

About 20% of people infected with COVID-19 become sick enough to need to go to the hospital. About 5% of those people have died so far. 10-15% of the population has a genetic defect in their perforin genes. Can we prove that the people who develop cytokine storm in relation to COVID-19 have a defect in their perforin genes? [In the 2009 H1N1 flu, several of the fatal cases were found to have perforin gene defects.] If so, can we screen people for perforin defects to identify people who are at higher risk of developing cytokine storm and who therefore need to take more precautions during this crisis? Such screening could be done by either functional assays or by genetic testing. 

Heat. Some strains of coronavirus cause the common cold. In the 1980s it was suggested that the upper respiratory tract (sinus tract) could be cleared of the virus by the administration of moist air >140℉ because the virus capsule becomes increasing less stable as the temperature is increased and completely unstable at 140℉. (Daniel Dimke’s book – where are the peer-reviewed publications?)Would using heated air in ventilators help? Being raised in 120℉ air in Vegas has been safe, so maybe a ventilator running that hot would be safe and might provide some benefit. It seems like a simple study to run. Although this might help with the pulmonary disease, it would not have an effect on virus disseminated to other parts of the body. 

One of my colleagues is assisting with the medical care at a newly constructed prison in California designed to house prisoners who have tested positive for the virus. It is possible that like many non-incarcerated Americans, some prisoners would like to volunteer to be used as test subjects for SARS-CoV-2 vaccine research. Should this be allowed? Should this be viewed as a way to repay a portion of their debt to society? 

Australia. The rate of death in Australia is much lower than in the United States (by a factor of 10 when accounting for population differences). Is this because of the difference in season and temperature? Like the US, they do not vaccinate with BCG, so enhanced immunity should not be the reason for the difference. This suggests that it should be ok to start to normalize things as the weather here gets warmer.

Is the SARS-CoV vaccine effective against the SARS-CoV-2 virus? (This study is underway and we won’t know until June 2021.)

Should Zinc be used prophylactically in healthcare workers? (Likely wouldn’t hurt to bring it up to normal levels.) Should Plaquenil? (The side effects can be pretty significant so this should be reserved for treatment.) Should N-Acetyl-Cysteine? (A trial is underway) Should BCG? (see my recent paper on this)  Should this only be done for people with a proven perforin abnormality?

Should Zinc levels be tested in patients upon hospital admission? Particularly in the elderly, who often have lower than normal zinc levels?

Vaccine design. Vaccine inoculant with Proteins S, N, E would obviously be desirable. Use of agonists for toll-like receptor 7(TLR7) and TLR8 as adjuvants would appear to be a good choice as well. TLR7 and TLR8 are toll-like receptors(TLR) present on the inner surface of endosomes. When virus particles  have been ingested by macrophages and partially destroyed, fragment molecules of their constituent building blocks are present inside the macrophages endosomes. Fragments of the single-stranded RNA that contains the COVID-19 virus’s genetic information would be among the molecules found inside such an endosome. TLR7 and TLR8 are transmembrane molecules designed to detect single-stranded RNA, like that of the coronavirus, and to signal the cell to upregulate production of inflammatory cytokines. Normally this is a good thing when trying to kill a virus. These cytokines are needed to let the rest of the immune system know that it should pay attention to this area because a viral infection is present. This is useful when designing a vaccine, to help increase the development of immunity to the inoculated pathogen.

Why is COVID-19 more likely to cause a cytokine storm than most other respiratory viruses? 

Likely wrong section: Reports regarding the role of IL-6 and TGFβ are conflicting. Cytotoxic immune cells secrete TGFβ and IL-6 after attaching to cells infected with the virus. The TGFβ draws macrophages in from the bloodstream. The IL-6 and the TGFβ together promote the macrophage to enter the M2 class of behavior – to clean debris and help with healing. Although macrophages can produce both IL-6 and TGFβ in different situations, in the instance of cytokine storm syndrome, they begin to produce TGFβ. This has been shown to cause respiratory epithelium to convert into fibroblasts and has been shown to induce macrophages to promote the deposition of fibrin. This local TGFβ is confusing, because serum levels of IL-6 appear to be high, not TGFβ. Need to do more extensive reading to solve this. Maybe someone has already solved this. IL-6 and TGFβ levels are higher in the serum of older patients. Is this related to why they are more susceptible? Is it because they are more likely to have zinc deficiencies due to impaired nutrition and adsorption?

Aury N Nagy, MD, FAANS Board Certified Neurosurgeon 

This article was made possible by the generously shared anecdotes, insights, corrections and criticisms provided by my many colleagues. I would especially like to thank the following: 

Christopher Breeden, Jason Burke, Miranda Cowan, Jeff Davidson, Michael Edwards, Effie Farnsworth, Robert Milne, M. Nafees Nagy, Shamim Nagy, Gary Skankey

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