# data[].attributes.attachment_link.title
Hyperventilation: A Possible Explanation for Long-Lasting Exercise Intolerance in Mild COVID-19 Survivors?

# data[].attributes.attachment_link.description
Since the outbreak of the coronavirus (COVID-19) pandemic, most attention has focused on containing transmission and addressing the surge of critically ill patients in acute care settings. As we enter the second phase of the pandemic, emphasis must evolve to post-acute care of COVID-19 survivors. Pe…

# data[].attributes.body
Respiratory complications Covid leaves behind

# data[].attributes.attachment_link.readable_text
Hypoxemia is a term that describes low levels of oxygen in the blood, specifically within arterial blood. The condition may indicate a problem with blood circulation or breathing and may present with symptoms such as a shortness of breath.

A doctor can measure a person's blood oxygen level through blood tests or a small device that clips onto the finger. Hypoxemia can occur for a number of reasons, which can involve underlying medical conditions such as chronic obstructive pulmonary disease (COPD), asthma, and anemia.

In this article, we will explore different types and causes of hypoxemia, how it differs from hypoxia, and how a doctor may diagnose and treat the condition.

While some people may use the terms hypoxemia and interchangeably, they are different. Though they both refer to low oxygen levels, they occur in different tissues. Specifically, hypoxemia describes low oxygen levels in the blood, while hypoxia describes low levels of oxygen in the tissues of the body.

While the conditions are different, it is possible for them to occur together. If hypoxemia is present, it may result in hypoxia, as low levels of oxygen in the blood may be unable to supply sufficient oxygen to the body's tissues. However, it is also possible for one condition to occur the other.

There are various reasons for hypoxemia to occur. Some of these :

Ventilation/perfusion mismatch

Ventilation is the air that a person breathes in and out, while perfusion is the circulation of blood. A normal ventilation/perfusion (V/Q) ratio is roughly .

A V/Q mismatch occurs when parts of the lungs receive oxygen but no blood circulation, or they receive blood circulation but no oxygen. Evidence suggests that a V/Q mismatch is the most common cause of hypoxemia.

Hypoventilation

Hypoventilation occurs when breathing is insufficient for the exchange of oxygen and carbon dioxide in the body. When hypoventilation occurs, there is a high level of carbon dioxide (CO ₂) and a low level of oxygen in the blood. As a result, hypoxemia is often present, especially during sleep.

A person who is experiencing hypoventilation may have slow and shallow breathing.
Click here to read more about hypoventilation.
Right-to-left shunt

A refers to when blood from the heart's right side enters the left side without any exchange of oxygen or CO ₂.

In normal conditions, blood that has no oxygen enters the right side of the heart and travels to the lungs to obtain oxygen. Once it has done so, the blood travels to the left side of the heart to distribute the oxygen to the rest of the body.

Diffusion impairment

Diffusion impairment occurs when there is dysfunction, or limitation, in the oxygen transport between the alveoli and the capillaries. The alveoli are sacs in the lungs where gas exchange takes place and capillaries are the blood vessels that oxygen diffuses into.

A low environmental PaO ₂ may result in hypoxemia. For example, the partial pressure of oxygen is lower at higher altitudes. This means that a person accustomed to breathing at sea level may experience hypoxemia at higher altitudes, as the amount of oxygen available is lower.

A few different conditions may cause hypoxemia. Some of these may :

Symptoms of hypoxemia can :

shortness of breath

irritability

confusion

drowsiness

rapid or irregular heartbeat

rapid and shallow breathing

blue or gray discoloration of the skin

A person should seek the advice of a medical professional for an accurate diagnosis of hypoxemia. A doctor will first carry out a physical exam, which may include looking at the skin and nails for color changes and listening to the heart and lungs.

The doctor may then carry out additional tests to determine the amount of oxygen in the blood. This may include:

Arterial blood gas test: This test involves drawing blood, then analyzing the sample in a lab to determine the level of oxygen and carbon dioxide present.

Pulse oximetry: A pulse oximeter is a device that measures how saturated the blood is with oxygen. The device normally attaches to the fingers, ears, or toes. The device shines a light through the skin and analyzes it to establish the percentage of oxygen in the blood.

Lung function tests: These are tests that analyze how well the lungs work. An example of a lung function test is spirometry. This involves blowing into a tube known as a spirometer to determine how much air a person can breathe in and out of their lungs.

As hypoxemia involves a reduction in blood oxygen levels, the aim of treatment is to the level of oxygen to a healthy level.

To do so, a person may receive oxygen therapy. This is a treatment option that provides supplemental oxygen that they can receive in a hospital or at home. There are a variety of different ways that a person may receive oxygen therapy, such as through a face mask or a tube that fits inside the nose, known as a nasal cannula.

If a person is experiencing an underlying condition that is causing hypoxemia, such as COPD or asthma, then additional treatment may be necessary to treat these conditions.

Untreated hypoxemia may lead to other complications in the body, as without adequate oxygen in the blood, the body cannot optimally function. Complications may :

brain damage and brain death

heart problems

lung complications

kidney problems

People can try a few lifestyle changes to help reduce the likelihood of hypoxemia or prevent conditions that could result in hypoxemia. This can include:

quitting smoking, as this influences ventilation

getting regular exercise

avoiding potential pollutants

preventing respiratory infections by receiving vaccines and wearing face coverings

practicing breathing exercises

taking medication as prescribed for underlying conditions that may cause hypoxemia

using supplemental oxygen if necessary

A person should seek the advice of a doctor if they experience symptoms of hypoxemia, such as heartbeat or breathing irregularities and discoloration of the skin. Early detection and treatment may help prevent the condition from progressing and causing more serious complications.

Hypoxemia describes low levels of oxygen in the bloodstream. This is different from hypoxia, which is a low level of oxygen in the body's tissues. Hypoxemia may occur for a number of reasons, such as a right-to-left shunt, a V/Q mismatch, low PaO ₂, diffusion impairment, and hypoventilation. Additionally, conditions such as COPD may also result in hypoxemia.

Symptoms of hypoxemia can include shortness of breath, confusion, a fast heartbeat, and drowsiness. A doctor may diagnose hypoxemia through a physical examination as well as tests such as an arterial blood gas analysis.

A person should contact their doctor as soon as possible if they experience symptoms of hypoxemia, as untreated hypoxemia can lead to further complications on the major organs, such as the brain and the heart.

# data[].attributes.attachment_link.title
Hypoxemia: Definition, causes, and symptoms

# data[].attributes.attachment_link.description
Hypoxemia is a term that describes low levels of oxygen in the blood, specifically within arterial blood. The condition may indicate a problem with blood circulation or breathing and may present with symptoms such as a shortness of breath. A doctor can measure a person's blood oxygen level through blood tests or a small device that clips onto the finger.

# data[].attributes.attachment_link.readable_text
Department of Physiology and Biophysics, Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel

Laboratory Medicine, Rambam Medical Center, Haifa, Israel

Department of Clinical Biochemistry Hadassah Medical Center, Hadassah Hebrew University Hospital, Mt. Scopus, Jerusalem, Israel

Department of Nephrology, Nazareth Hospital, EMMS, Nazareth and Azrieli Faculty of Medicine in Safed, Safed, Israel

The Bar-Ilan University Azrieli Faculty of Medicine in Safed, Safed, Israel

Department of Medicine, Hadassah Hebrew University Hospital, Mt. Scopus, Jerusalem, Israel

Engulfed by the grave consequences of the coronavirus disease 2019 (COVID-19) pandemic, a better understanding of the unique pattern of viral invasion and virulence is of utmost importance. Angiotensin (Ang)-converting enzyme (ACE) 2 is a key component in COVID-19 infection. Expressed on cell membranes in target pulmonary and intestinal host cells, ACE2 serves as an anchor for initial viral homing, binding to COVID-19 spike-protein domains to enable viral entry into cells and subsequent replication. Viral attachment is facilitated by a multiplicity of membranal and circulating proteases that further uncover attachment loci. Inherent or acquired enhancement of membrane ACE2 expression, likely leads to a higher degree of infection and may explain the predisposition to severe disease among males, diabetics, or patients with respiratory or cardiac diseases. Additionally, once attached, viral intracellular translocation and replication leads to depletion of membranal ACE2 through degradation and shedding. ACE2 generates Ang 1-7, which serves a critical role in counterbalancing the vasoconstrictive, pro-inflammatory, and pro-coagulant effects of ACE-induced Ang II. Therefore, Ang 1-7 may decline in tissues infected by COVID-19, leading to unopposed deleterious outcomes of Ang II. This likely leads to microcirculatory derangement with endothelial damage, profound inflammation, and coagulopathy that characterize the more severe clinical manifestations of COVID-19 infection. Our understanding of COVID-ACE2 associations is incomplete, and some conceptual formulations are currently speculative, leading to controversies over issues such as the usage of ACE inhibitors or Ang-receptor blockers (ARBs). This highlights the importance of focusing on ACE2 physiology in the evaluation and management of COVID-19 disease.

Background

Coronavirus disease 2019 (COVID-19), caused by the highly contagious coronavirus 2 (SARS-CoV-2), is initiated by invasion into host cells through viral attachment to angiotensin (Ang)-converting enzyme (ACE) 2. ACE2, expressed in numerous different tissues, serves as an anchor for specific domains on the viral spikes ( Hamming et al., 2004; Hoffmann et al., 2020; Zou et al., 2020). Additionally, ACE2, through the modulation of the renin-Ang-aldosterone system (RAS), plays an important physiologic role in the homeostasis of tissue microcirculation and inflammation ( Crackower et al., 2002; Hamming et al., 2007; Santos et al., 2008; Clarke and Turner, 2012; Datta et al., 2020). This minireview will address the role of ACE2 within the RAS, and the inter-association of ACE2 and SARS-CoV-2, with their plausible combined impact on the clinical manifestations of COVID-19 disease. We shall further address knowledge gaps that require elucidation in order to better understand the pathophysiology and clinical features of COVID-19 in order to develop effective means for disease prevention and management.

ACE2: An Important Component of RAS

Figure 1A illustrates our current understanding of the complexity of the RAS. Until recently, most clinicians were familiar with only one axis, namely renin-mediated proteolysis and conversion of angiotensinogen to the 10-amino-acid peptide Ang I, followed by a further cleavage by ACE, principally present in the lungs to form the bioactive 8-amino-acid compound Ang II ( Crackower et al., 2002; Hamming et al., 2007; Santos et al., 2008; Clarke and Turner, 2012). The COVID-19 pandemic shifted our attention to another component of RAS, namely ACE2, which plays a role in SARS-CoV-2 virulence. Ang II could be further cleaved by ACE2 to form the bioactive 7-amino-acid peptide Ang 1-7. In addition, ACE2 converts Ang I into Ang (1-9), which can be further converted to Ang 1-7 by ACE. A third pathway of Ang 1-7 generation involves neprilysin (neural endopeptidase-NEP), which converts Ang I directly into Ang 1-7 ( Tipnis et al., 2000; Crackower et al., 2002; Vickers et al., 2002; Hamming et al., 2007; Santos et al., 2008; Clarke and Turner, 2012). An alternative degradation pathway with conversion of Ang I to Ang II takes place by the proteolytic enzyme, chymase, explaining ongoing generation of Ang II in patients on ACE inhibitors ( Miyazaki and Takai, 2006).

Figure 1. Angiotensin derivatives, their targets and downstream action: (A) Balanced impact of angiotensin (Ang) II and Ang 1-7 on vascular tone and control of inflammation. (B) SARS-CoV2 infection generates Ang 1-7 depletion, likely leading to unopposed vasoconstriction and inflammation. (C) Concomitant renin-Ang-aldosterone system (RAAS) inhibition with Ang-converting enzyme (ACE) inhibitors or Ang-receptor blockers (ARBs) may restore the balance, with parallel suppression of signals mediated by Ang T ₁ receptors (AT1R) and Mas receptor (MasR).

Importantly, Ang derivatives differ by their downstream physiologic properties and are mediated by diverse signal transduction mechanisms ( Figure 1A). Ang II acts principally as a potent vasoconstrictor, pro-inflammatory, pro-fibrotic, and anti-diuretic agent. These actions are mediated by Ang II binding to Ang T ₁ receptors (AT1R) on affected cell membranes. Opposing activities may be initiated via attachment of Ang II to Ang T ₂ receptors (AT2R; Li et al., 2017). Indeed, Ang II-mediated vasoconstriction or vasodilation at the renal cortex and medulla, respectively, reflects diverse receptor distribution and activity, predominantly AT1R in the cortex and AT2R in the medulla ( Duke et al., 2003). As also shown in Figure 1A, unlike Ang II, Ang 1-7 exerts unequivocal vasodilatory, anti-inflammatory, anti-fibrotic, and natriuretic actions by binding to a G-protein-coupled Mas receptor (MasR; Li et al., 2003; Santos et al., 2008).

Thus, a tight physiologic balance exists by the opposing effects of Ang derivatives whenever this system undergoes perturbations, with the aim of preventing extreme vasoactive deviations or uncontrolled inflammation and remodeling, with Ang 1-7 serving to counterbalance the undesired adverse effects of unbridled Ang II action.

ACE2 and SARS-CoV-2 Association

Angiotensin-converting enzyme is expressed on the plasma membranes of various cell types, including alveolar and intestinal epithelia, vascular endothelial cells in the heart, kidney, and testis, and on macrophages, where it catalyzes the production of Ang 1-7 and its likely paracrine activity ( Crackower et al., 2002; Hamming et al., 2007; Santos et al., 2008; Clarke and Turner, 2012; Abassi et al., 2020c). Unfortunately, cell-membrane-bound ACE2 also serves as a binding site for the viral spike proteins of SARS-CoV-1 and SARS-CoV-2 ( Li et al., 2003; Hamming et al., 2004; Hoffmann et al., 2020; Walls et al., 2020; Wan et al., 2020; Wu et al., 2020; Zou et al., 2020). The viral attachment to ACE2 with subsequent internalization is facilitated by additional modifications and cleavage of the S1/S2 spike proteins by convertases, such as transmembrane protease serine (TMPRSS 2) and related proteases (Furin and Corin; Heald-Sargent and Gallagher, 2012; Coutard et al., 2020; Hoffmann et al., 2020; Shang et al., 2020; Walls et al., 2020), and probably by activated factor X (Xa), which was shown to cleave recombinant and pseudoviral S protein into S1 and S2 subunits ( Du et al., 2007), all exposing the fusion sites in the viral spike protein ( Figure 2).

Figure 2. Physiology of coronavirus disease 2019 (COVID 19) homing to target host cells expressing ACE2: viral spike-domains enable attachment to cell-membrane-bound ACE2. Attachment is further enabled by furin, corin, TMPRSS2, and Factor Xa. Following attachment the virus undergoes internalization and replication in host cells, a process associated with degradation of internalized ACE2. Ang 1-7 synthesis consequently declines. Unopposed Ang II action triggers inflammation which activates ADAM 17, leading to shedding of membranal ACE2, further depleting cell-bound ACE2 and local Ang 1-7 synthesis. Viral attachment to target host cells may be attenuated by its competitive binding with rising titers of circulating ACE2.

Two principal sites of SARS-CoV-2 invasion include the gastrointestinal and respiratory tracts, which express abundant ACE2. While intestinal homing is clinically more pronounced in children, manifested by gastrointestinal symptoms, the lungs conceivably serve as the principal port of entry, with viral attachment to type II alveolar cells (AT2), and to alveolar macrophages coated by membranal ACE2 ( Abassi et al., 2020c, d). Interestingly, conditions identified as predisposing to severe COVID-19 disease are characterized by enhanced pulmonary expression of ACE2. First, chronic airway disease, smoking, and pollution are associated with expansion of the population of alveolar macrophages expressing ACE2 ( Abassi et al., 2020d). Furthermore, ACE2 expression is increased in males ( La Vignera et al., 2020; Papadopoulos et al., 2020). Indeed, bioinformatics analyses revealed higher abundance of ACE2-expressing AT2 cells in men than women ( Wei et al., 2020), potentially enhancing viral susceptibility among men. In this context, testosterone has been described to induce ACE2 expression, the receptor entry of the SARS-CoV-2 infection, but also exerts protective effect against lung injury ( Kuba et al., 2005). Enhanced ACE2 is also found in diabetes ( Muniyappa and Gubbi, 2020) and heart failure ( Zisman et al., 2003; Goulter et al., 2004; Chen et al., 2020), and possibly with the administration of RAS inhibitors ( Li et al., 2017). Diabetes is also associated with increased expression of furin ( Fernandez et al., 2018). Thus, while testosterone levels decline with aging among men ( Harman et al., 2001; Feldman et al., 2002), the presence of comorbidities like obesity, diabetes mellitus, and cardiovascular diseases, possibly counterbalance the decline in viral homing capacity related to age-dependent testosterone drop ( Camacho et al., 2013; Rastrelli et al., 2015). In addition, testosterone enhances AT1R expression in male, whereas estrogen preferentially upregulates AT2R expression in females ( Chanana et al., 2020). Finally, hypogonadal males are characterized by low T cell count which may provide unrestrained environment for severe responses to SARS-CoV-2 infection ( Papadopoulos et al., 2020). In sum, it is tempting to assume that enhanced expression of ACE2 in target organs and also of other molecules permissive to viral binding to ACE2 facilitate viral invasion and augment viral load ( Figure 3), although the details of this formulation require validation in further studies.

Figure 3. A summarizing scheme of suggested COVID-17/RAS interactions: see text for details. Highlighted are factors enhancing ACE2 expression and viral binding to target host-cells, as are mechanisms leading to declining membranal ACE2 and Ang 1-7 synthesis. The impact of shedded sACE2 on tissue Ang 1-4 production and on inhibiting viral homing to target cells expressing ACE2 by means of competition require further elucidation. RAS inhibitors potentially can enhance viral invasion by enhancing ACE2 expression, yet they may attenuate the unfavorable outcome of Ang 1-7 depletion by a parallel inactivation of functionally opposing Ang II activity. Potential hazardous feed-forward loops are AT1R-mediated enhanced ACE2 shedding and intensification of viral attachment via proteases activated by vasoconstriction and ischemia, inflammation, and coagulopathy.

Unbalanced RAS in SARS-CoV 19 Disease

As illustrated in Figures 1, 2, SARS-CoV-2 invasion unbalances the RAS. Viral cellular internalization is coupled with degradation of membranal ACE2. Furthermore, circulating Ang II, combined with internalized ACE2 activates a sheddase named ADAM metallppeptidase domain 17 (ADAM 17) also called tumor necrosis factor-α-converting enzyme (TACE; Lambert et al., 2005), which in turn triggers shedding of membranal ACE2 into the circulation with the formation of soluble ACE2 (sACE2), further depleting membranal ACE2 along enhanced TNF-α production ( Figure 2). Thus, viral cellular invasion and replication, initially facilitated by ACE2 and in particular under conditions characterized by enhanced ACE2 expression, later lead to diminution of cell membrane-attached ACE2, and likely increase circulating sACE2 ( Figures 2, 3). At the microcirculatory and tissue level, this is expected to result in unbalanced paracrine action of Ang compounds, with a local depletion of Ang 1-7 leaving Ang II activity unopposed ( Figure 1B). Likely, this has a role in microcirculatory dysfunction, intense inflammation, hypercoagulability, tissue damage, and fibrosis ( Figure 3). Lung inflammation in SARS CoV-19 disease exemplifies the outcome of Ang II/Ang 1-7 imbalance: Ang II enhances vascular permeability along infiltration of neutrophils into alveolae and indirectly via induction of interleukin 8 (IL-8; Diamond, 2020). Accumulation of neutrophils and their accompanied prooxidative role lead to loss of alveolar epithelial cells and the development of ARDS. Nevertheless, this Ang II-derived lung injury is prevented by Ang 1-7 as was evident in ACE2 deficient mice ( Zou et al., 2014).

Additional adverse aspect of unrestricted Ang II action during SARS-CoV-2 infection is the increased tendency of thrombosis documented in large number of hospitalized COVID-19 patients ( Bikdeli et al., 2020; Klok et al., 2020). Although this phenomenon is multifactorial, as outlined below, AT1R activation plays an important role where it leads to enhancement of tissue factor (TF) expression on endothelial cells and sequentially initiation of clotting cascade along increased permeability and neutrophils mobilization ( Dielis et al., 2005).

Knowledge Gaps: The Missing Pieces in the Puzzle

Many sections in the preceding paragraphs are based on in vitro and animal studies, some with inconsistent and even conflicting interpretations. Furthermore, some fundamental concepts are currently being re-evaluated. For instance, previously reported ACE2 expression on vascular endothelial cells ( Hamming et al., 2004) has recently been questioned, based on the measurement of single-cell RNA ( Batlle et al., 2020). Human data based on patients infected by SARS-CoV-2 are sparse and are now being intensively studied as we write these lines. It is evident that the foregoing statements should be further examined in the human clinical scenario of COVID-19 disease.

Second, the role of altered ACE2 physiology detailed above in subsequent clinical features of the disease requires in-depth evaluation ( Essig et al., 2020). There are several hypothetical mechanisms, outlined in Figure 3, that warrant consideration. Possibly, unopposed Ang II due to depletion of cell membrane-bound ACE2 results in altered regional microcirculation and hypoxia, with the generation of reactive oxygen species and endothelial damage, glycocalyx degradation, and disseminated coagulopathy ( Abassi et al., 2020a). This may further compromise the regional microcirculation with a feed forward loop, leading to organ failure including the heart ( Abassi et al., 2020b), lungs ( Abassi et al., 2020d), and kidneys ( Batlle et al., 2020). Furthermore, intense inflammation and coagulopathy may result from unopposed Ang II and by ADAM 17-mediated activation of TNF-α/IL-6/STAT-3 pathways ( Hirano and Murakami, 2020) as well as uncontrolled heparanase activity ( Li and Vlodavsky, 2009) together with the induction of defensins ( Abu-Fanne et al., 2019). Concerning the latter, preliminary findings from our group indicate that alpha-defensin-1, released from polymorphonuclear cells as a part of the inflammatory response, plays a pivotal role in the hypercoagulopathy associated with COVID-19 disease, as its rising titers parallel increasing plasma levels of D-dimers (Higazi AAR, submitted manuscript). Regarding the interplay between Ang II and ADAM 17/TNF-α/IL-6/STAT-3 pathways, it was found that Ang II activates NF-κB and release of proinflammatory cytokines ( Dandona et al., 2007; Benigni et al., 2010). Specifically, induction of ADAM17 by Ang II initiates the conversion of interleukin-6 (IL-6Rα) to the soluble form (sIL-6Rα) along activation of signal transducer and activator of transcription 3 (STAT3) via the sIL-6Rα-IL-6 complex in various nonimmune cells including fibroblasts, endothelial cells, and epithelial cells ( Hirano and Murakami, 2020). Moreover, STAT3, essential for the NF-κB pathway, is principally stimulated by IL-6 during inflammation ( Murakami et al., 2019). Since IL-6 plays a key role in the recruitment of lymphoid cells and myeloid cells, including activated T cells and macrophages ( Murakami et al., 2019), and likely enhances defensin release (Higazi AAR, unpublished data), its elevated levels during senescence may contribute to the enhanced COVID-19 mortality in aged people and to coagulopathy. Interestingly, AT1R density is increased, while AT2R abundance declines under inflammatory conditions ( Diamond, 2020). Collectively, these results may explain proinflammatory cytokine release and hypercoagulopathy during SARS-CoV-2 infection via the associated Ang II pathway and a possible therapeutic target via the IL-6-STAT3 axis ( Diamond, 2020).

Reduced inherent expression of ACE2 in the lungs with aging, as demonstrated in rats ( Xie et al., 2006; Alghatrif et al., 2020) may reduce the risk for SARS-CoV-2 infection on the one hand, whereas its further suppression to very low levels during viral infection, on the other hand, could amplify Ang II/Ang 1-7 imbalance, leading to more profound deleterious pulmonary consequences. Conversely, younger individuals with higher inherent ACE2 expression may have a higher incidence, yet less severe SARS-CoV-2 infection, since ACE2 depletion would not be as severe as in aged patients, with Ang 1-7 generation sufficient to counteract Ang II ( Alghatrif et al., 2020). Deranged vascular reactivity will likely be affected by other mediators, such as iNOS‐ activation and intense nitric oxide production (plausibly with abundant formation of the toxic-free radical peroxynitrite), and by altered endothelial production of endothelin and prostaglandins. Notably, there are additional plausible inherent feed-forward loops in the scheme of SARS-CoV-2 infection and inflammation, including hypoxia-driven perpetuation of endothelial damage and tissue damage. Furthermore, as illustrated in Figures 2, 3, Ang II suppresses Ang 1-7 generation secondary to downregulation of membranal ACE2 via ADAM 17 activation. Moreover, Factor Xa, generated during disseminated coagulation, is expected to expose attachment sites on viral spikes and enhance viral attachment to target cells expressing ACE2 ( Du et al., 2007). Interestingly, in vitro studies illustrate that heparin interferes with ACE2 binding to the S1 viral spike protein, reducing viral internalization ( Mycroft-West et al., 2020). Thus, enhanced heparanase activity in infected patients might damage endothelial cover by heparin-like proteoglycans and further facilitate viral endothelial invasion.

Third, discussions regarding the potential impact of medications affecting RAS are currently based on inconsistent observations and educated guesses ( Essig et al., 2020). We really do not know for sure if blocking steps in the RAS cascade indeed results in enhanced ACE2 expression in humans, and whether this promotes viral attachment and invasion. On the other hand, discontinuation of RAS inhibitors might further intensify the uncontrolled action of Ang II, shown in Figure 1B, leaving it unopposed once Ang 1-7 generation is hampered. Those in favor of uninterrupted administration of RAS inhibitors would argue that, as illustrated in Figure 1C, depleting Ang II or blocking its action on AT1R [by ACE inhibitors or Ang-receptor blockers (ARBs), respectively] would balance the exhaustion of Ang 1-7 caused by viral invasion and might prevent consequent vasoconstriction ( South et al., 2020). Furthermore, it is also likely that the profile of Ang derivatives may differ in patients treated by ARBs, by ACE inhibitors or by spironolactone ( Malha et al., 2020). That is why blanket reassurance regarding continuation of RAS inhibitors during the current pandemic ( Vaduganathan et al., 2020) should be regarded with caution. A cautious approach might consider the avoidance of ACE inhibitors or ARBs during an active epidemic in non-infected and hemodynamically-stable patients in order to reduce ACE2 expression, permissive to viral attachment, but consideration of ACE inhibitors, or ARBs at advanced stages of COVID-19 disease to prevent Ang II predominance due to depleted Ang 1-7. Most of the clinical trials and data analysis are performed on adults, however potential differences between adults and children may exist, thus coronavirus-related research should be undertaken in children as well, including the impact of ACE-I and ARBs on COVID-19 evolvement among this subpopulation. Hopefully, this may provide clues for the question why children are at decreased risk of severe COVID-19 disease ( South et al., 2020). Furthermore, we have no idea about the function or malfunction of circulating sACE2 following its shedding from cell membranes. Does it exert systemic vasodilation or improve the microcirculation? Can it compete with cell-membrane-bound ACE2 ( Ciaglia et al., 2020) and reduce viral attachment to target cells as suggested in Figure 3? Nor can we tell if diverse inherent expression and activity of circulating or cell-bound ACE2 or its capacity to attach to viral spike proteins affects infection, infectivity, or susceptibility to severe and complicated disease. We also are not sufficiently knowledgeable of plausible changes in ACE2 transcription in various tissues in response to SARS-CoV-2 infection. Indeed, Rice et al. (2006) reported that up to 67% of the phenotypic variation in circulating ACE2 could be accounted for by genetic factors. These findings may partially explain the different mortality rate among the various ethnic groups, and strongly support studies of genetic analysis of ACE2 polymorphisms as a reliable approach for precision medicine in the prevention, diagnosis, and therapy of COVID-19 disease. Evidence is currently lacking as to whether levels of circulatory sACE2 may have diagnostic and prognostic implication when monitoring patients infected by SARS-CoV-2, as it does in patients with heart failure ( Epelman et al., 2008; Ortiz-Perez et al., 2013). With so many pieces of data missing, the need for vigorous clinical studies guided by physiology-based questions and hypotheses are most urgent. Such a question includes the continuation or even introduction, rather than cessation of RAS inhibitors in patients infected by SARS-CoV-2 ( Kai and Kai, 2020), or can we inhibit binding of SARS spike proteins to ACE2, for instance by antibodies, without hampering its catalytic capacities to generate Ang 1-7? Is there a role for the application of Ang 1-7 or MasR agonists or for the administration of intravenous sACE2, with an available proof of concept for such postulated approaches ( Yang et al., 2014; Hemnes et al., 2018)?

It is likely that many of the above options will be considered and examined in the near future. Meanwhile, we are challenged by epidemiologic aspects, by issues of supportive and critical care for very sick individuals, and by minimizing the risk to healthcare providers. The ultimate solution probably will be effective vaccination. Yet, until we reach this goal, studying and manipulating ACE2-viral association is a plausible approach, along with the development of effective anti-viral agents.

Data Availability Statement

The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.

Author Contributions

ZAb, AH, ZAr, KS, and SH equally participated in the design, execution of the view of point, drafted the manuscript, participated in critical discussions, and revised the manuscript. SK, SH, and ZAb prepared the figures. SH supervised the project. All authors contributed to the article and approved the submitted version.

Funding

ZAb acknowledges research support from the Israel Science Foundation Grant 544/18.

Conflict of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

References

Dielis, A. W. J. H., Smid, M., Spronk, H. M. H., Hamulyak, K., Kroon, A. A., Cate, H. T., et al. (2005). The prothrombotic paradox of hypertension. Hypertension 46, 1236-1242. doi: 10.1161/01.HYP.0000193538.20705.23

CrossRef Full Text | Google Scholar

Hoffmann, M., Kleine-Weber, H., Krüger, N., Müller, M., Drosten, C., and Pöhlmann, S. (2020). The novel coronavirus 2019 (2019-nCoV) uses the SARS-coronavirus receptor ACE2 and the cellular protease TMPRSS2 for entry into target cells. bioRxiv [Preprint]. doi: 10.1101/2020.01.31.929042v1

CrossRef Full Text | Google Scholar

Malha, L., Mueller, F. B., Pecker, M. S., Mann, S. J., August, P., and Feig, P. U. (2020). COVID-19 and the renin-angiotensin system. Kidney Int. Rep. 5, 563-565. doi: 10.1016/j.ekir.2020.03.024

CrossRef Full Text | Google Scholar

Mycroft-West, C. J., Su, D., Pagani, I., Rudd, T. R., Elli, S., Guimond, S. E., et al. (2020). Heparin inhibits cellular invasion by SARS-CoV-2: structural dependence of the interaction of the surface protein (spike) S1 receptor binding domain with heparin. bioRxiv [Preprint]. doi: 10.1101/2020.04.28.066761

CrossRef Full Text | Google Scholar

Papadopoulos, V., Li, L., and Samplaski, M. (2020). Why does COVID-19 kill more elderly men than women? Is there a role for testosterone? Andrology doi: 10.1111/andr.12868 [Epub ahead of print]

CrossRef Full Text | Google Scholar

Wei, X., Xiao, Y. -T., Wang, J., Chen, R., Zhang, W., Yang, Y., et al. (2020). Sex differences in severity and mortality among patients with COVID-19: evidence from pooled literature analysis and insights from integrated bioinformatic analysis. arXiv [Preprint]. Available at: https://arxiv.org/abs/2003.13547
Google Scholar
Wu, C., Zheng, S., Chen, Y., and Zheng, M. (2020). Single-cell RNA expression profiling of ACE2, the putative receptor of Wuhan 2019-nCoV, in the nasal tissue. medRxiv [Preprint]. doi: 10.1101/2020.02.11.20022228

CrossRef Full Text | Google Scholar

# data[].attributes.attachment_link.title
ACE2, COVID-19 Infection, Inflammation, and Coagulopathy: Missing Pieces in the Puzzle

# data[].attributes.attachment_link.description
Engulfed by the grave consequences of the coronavirus disease 2019 (COVID-19) pandemic, a better understanding of the unique pattern of viral invasion and virulence is of utmost importance. Angiotensin (Ang)-converting enzyme (ACE) 2 is a key component in COVID-19 infection.

# data[].attributes.body
Sadly many reports of adverse events after Covid mild infections and/or Covid vaccination have revealed serious health issues for mothers and babies.

# data[].attributes.attachment_link.title
COVID-19 Associated With Life-Threatening Apnea in an Infant Born Preterm: A Case Report

# data[].attributes.attachment_link.description
A pandemic linked to the new coronavirus strain (SARS-CoV-2) has been raging for several months. Pediatric populations are less impacted than adults, and critical respiratory diseases seem rare (1, 2). We report the case of an infant, who presented with life-threatening apneas at home requiring basi…

# data[].attributes.attachment_link.readable_text
Objective To quantify the risk of deep vein thrombosis, pulmonary embolism, and bleeding after covid-19.

Design Self-controlled case series and matched cohort study.

Setting National registries in Sweden.

Participants 1 057 174 people who tested positive for SARS-CoV-2 between 1 February 2020 and 25 May 2021 in Sweden, matched on age, sex, and county of residence to 4 076 342 control participants.

Main outcomes measures Self-controlled case series and conditional Poisson regression were used to determine the incidence rate ratio and risk ratio with corresponding 95% confidence intervals for a first deep vein thrombosis, pulmonary embolism, or bleeding event. In the self-controlled case series, the incidence rate ratios for first time outcomes after covid-19 were determined using set time intervals and the spline model. The risk ratios for first time and all events were determined during days 1-30 after covid-19 or index date using the matched cohort study, and adjusting for potential confounders (comorbidities, cancer, surgery, long term anticoagulation treatment, previous venous thromboembolism, or previous bleeding event).

Results Compared with the control period, incidence rate ratios were significantly increased 70 days after covid-19 for deep vein thrombosis, 110 days for pulmonary embolism, and 60 days for bleeding. In particular, incidence rate ratios for a first pulmonary embolism were 36.17 (95% confidence interval 31.55 to 41.47) during the first week after covid-19 and 46.40 (40.61 to 53.02) during the second week. Incidence rate ratios during days 1-30 after covid-19 were 5.90 (5.12 to 6.80) for deep vein thrombosis, 31.59 (27.99 to 35.63) for pulmonary embolism, and 2.48 (2.30 to 2.68) for bleeding. Similarly, the risk ratios during days 1-30 after covid-19 were 4.98 (4.96 to 5.01) for deep vein thrombosis, 33.05 (32.8 to 33.3) for pulmonary embolism, and 1.88 (1.71 to 2.07) for bleeding, after adjusting for the effect of potential confounders. The rate ratios were highest in patients with critical covid-19 and highest during the first pandemic wave in Sweden compared with the second and third waves. In the same period, the absolute risk among patients with covid-19 was 0.039% (401 events) for deep vein thrombosis, 0.17% (1761 events) for pulmonary embolism, and 0.101% (1002 events) for bleeding.

Conclusions The findings of this study suggest that covid-19 is a risk factor for deep vein thrombosis, pulmonary embolism, and bleeding. These results could impact recommendations on diagnostic and prophylactic strategies against venous thromboembolism after covid-19.

Data source

The personal identification numbers of people who tested positive for SARS-CoV-2 between 1 February 2020 and 25 May 2021 were sent from the communicable disease surveillance system, SmiNet (Public Health Agency of Sweden), to Statistics Sweden. We set the covid-19 date as the earliest from the date of disease onset, sample date, diagnosis date, or date of report to SmiNet (see supplementary table 1). Only first infections were included. Statistics Sweden identified four people who tested negative (controls) for each participant who tested positive for SARS-CoV-2, matched on age, sex, and county of residence. The index date for control participants was the corresponding date for participants who tested positive for SARS-CoV-2. The personal identification numbers for people who tested positive or negative for SARS-CoV-2 were cross linked with the Inpatient Registry (covid-19 cases: 1987-2021, controls: 1997-2021), Outpatient Registry (1997-2021), Cause of Death Registry (2020-21), Intensive Care Registry (2020-21), the Prescribed Pharmaceutical Registry from the Swedish National Board of Health and Welfare, and the Swedish Intensive Care Registry. We calculated the weighted Charlson comorbidity index for each participant. 6 7

The sample size needed to identify a clinically relevant acute effect (incidence rate ratio or risk ratio of 2) with 90% power within a risk period of 30 days was 181 events in the self-controlled case series study and 112 events in the matched cohort study. The sample size needed to identify a clinically significant effect (incidence rate ratio or risk ratio of 1.5) with 90% power in the overall 180 day risk period was 258 events in the self-controlled case series study and 354 events in the matched cohort study. Sample size calculations were performed a priori.

No data were missing in our analysis.

Deep vein thrombosis, pulmonary embolism, and bleeding classification

Outcomes were defined using ICD-9 and ICD-10 (international classification of diseases, ninth and 10th revisions, respectively) diagnosis codes for deep vein thrombosis, pulmonary embolism, and bleeding (see supplementary table 2) as reason for contact in the outpatient or inpatient registries. Depending on the analysis, we chose the first or a recurrent event. A first event was defined as a participant with no previous event between 1 January 1987 and the start of the study period for patients with covid-19, and 1 January 1997 and the start of the study period for control participants. A recurrent event was defined as a participant with a previous event during the period 1987 to day 0 (covid 19) or 1997 to day 0 (index date) for participants with covid-19 and control participants, respectively. In the analysis, we only included the first of the recurrent events occurring within 1-30 days after the covid-19 or index date.

Statistical analysis

We calculated the mean age and standard deviation for the main cohort and for participants with a first or recurrent deep vein thrombosis, pulmonary embolism, or bleeding event. For each analysis we specify the proportion of participants who were female or male. In addition, we show the proportion of participants who had the event among the whole cohort of participants with covid-19 or control participants as the absolute risk over the follow-up period of 30 days.

Matched cohort study

In the matched cohort study, 11 comparing covid-19 cases with four control participants who had not reported SARS-CoV-2 positivity to SmiNet and were matched on age, sex, and county of residence, the risk ratio and 95% confidence interval of venous thromboembolism or bleeding in the first 1-30 days after covid-19 or the index date were determined using unadjusted and adjusted conditional Poisson regression analysis. Several potential confounders were included in the adjusted risk ratio: comorbidities (weighted Charlson comorbidity index), cancer (defined as ICD-10 code C00-C97 in the patient registries within six months before and after covid-19 or the index date), surgery (before venous thromboembolism or bleeding and within 30 days before or during the specified risk period), long term anticoagulation treatment (as defined by at least two filled prescriptions from the pharmacy within 12 months before covid-19 or the index date of pharmaceuticals with anatomical therapeutic chemical codes: B01AA, B01AB, B01AE, and B01AF), and a previous event in the analyses of recurrent events. As a sensitivity analysis to investigate potential residual confounding, we included the individual groups within the Charlson comorbidity index as categorical variables in the analyses (see supplementary tables 18-20).

The risk ratios and 95% confidence intervals of outcomes were determined using the g, package gnm version 1.1-1 in R, version 4.0.2. 12 The effect of pandemic waves and severity of covid-19 (mild: not admitted to hospital; severe: admitted to hospital; critical: admitted to intensive care unit) was determined for venous thromboembolism and bleeding using participants who tested negative for SARS-CoV-2 as a baseline. We determined the risk for all venous thromboembolism or bleeding events (first or recurrent event) within 30 days after covid-19 or the index date and stratified across the different disease severity groups using controls as a baseline.

Patient and public involvement

Access to the pseudonymised historical data from our Swedish nationwide registries is restricted by law and is not publicly available. Our research question needed to be clear and focused to enable us to request the data. As our study was related to covid-19 research, there was an urgency for analysis. In addition, the learning curve to extract and analyse the data was steep. This study was conceived from an in-house perspective, and we were not funded for patient or public involvement, nor had we considered the possibility of how to involve patients or the public in other ways such as review of the manuscript or dissemination of findings. We appreciate the role of patients as partners in research and look forward to publishing future research that includes patients and members of the public.

Covid-19 as independent risk factor

Strengths and limitations of this study

The incidence of risk during the period before exposure to SARS-CoV-2 (days −3 to day 0) were found to be high for all three outcomes, driven by an increased number of events at day 0. Some of the patients with these high risks were likely infected with SARS-CoV-2 before the event. However, because including day 0 in the risk period would introduce test bias, 10 we excluded this day and incorporated it in the pre-exposure period. In addition, risks were observed to be increased during the buffer period (−30 to −4 days), probably because of a delay in testing or documentation of covid-19 diagnosis, or due to reverse causality (ie, nosocomial covid-19).

Furthermore, we compared the risk of outcomes among the different pandemic waves and found the incidences or risk to be higher in the first wave, especially during the acute phase of covid-19. This could be explained by improvements in the treatment arsenal against covid-19, especially the widespread use of thromboprophylaxis after the first wave. Additionally, we found an excess risk of outcomes in patients with more severe covid-19 admitted to hospital, but especially in those admitted to an intensive care unit. The rates for venous thromboembolism in this patient group have been shown to be high despite thromboprophylaxis. 13

We acknowledge limitations to our study. Firstly, registry based information is at risk of containing incomplete or inaccurate data. Secondly, venous thromboembolism may have been underdiagnosed in patients with covid-19. Critically ill patients may be too unstable for diagnostic evaluation of venous thromboembolism, or the evaluation might be delayed owing to contagiousness issues. Furthermore, as registry data for control participants was limited to 1997, events might have more often been falsely classified as first events in the control participants, possibly resulting in underestimation of the risks. However, overestimation of the risks might also exist owing to limited testing for covid-19, especially during the first pandemic wave. In addition, vaccine data were not available for our study. By week 17 (30 days before the end of our data), 30.3% of the population aged 12 years or older had received their first vaccine dose (Swedish Public Health Agency data). The vaccines were prioritised for elderly people primarily, therefore the distribution of vaccines was increased in elderly age groups compared with younger age groups. As we found that older people have an increased risk of deep vein thrombosis, pulmonary embolism, and bleeding, it is possible that vaccine coverage provides a protective effect in age groups older than 50 years. Potentially this could partially explain why the incidence rate ratios were lower (at least in the acute phase) during the third pandemic wave. Moreover, we acknowledge residual confounding as a limitation to our study. However, the similarity of the results between the matched cohort study and self-controlled case series analysis, which controls for all confounders that might be regarded as fixed during the study period (thus including body mass index and smoking) suggests that these are not major confounders. Furthermore, the results remained unchanged when adjusting for specific comorbidities from the Charlson comorbidity index.

Comparison with other studies

Previous studies have shown an association between thrombosis and infections. 14 15 The risk of events in our study was, however, observed to be much higher. This could be explained by several pathophysiological alterations in covid-19, such as a direct effect of the virus on endothelial cells, 16 an exaggerated inflammatory response, down regulation of angiotensin converting enzyme 2 receptors, and activation of the coagulation system. 17 Although deep vein thrombosis and pulmonary embolism traditionally belong in the venous thromboembolism spectrum of disease, the relative incidence of pulmonary embolism was much higher, which could be due to immunothrombosis (thrombosis in the pulmonary vessels from local inflammation). 18 The increased risk of bleeding could be related to endothelial dysfunction, 16 coagulopathy, 17 or disseminated intravascular coagulation. 19

Our results are in line with those of similar studies that used the self-controlled case series method to determine the association between covid-19 and thromboembolic events. A large study from England using electronic health records found that 4671 patients were admitted to hospital due to venous thromboembolism within 28 days of testing positive for SARS-CoV-2, and the incidence rate ratio for venous thromboembolism was about 14-fold increased during the first two weeks, decreasing to an increase of about eightfold during the third week and threefold during the fourth week. 20 Because the authors did not discriminate between first or recurrent events, nor did they differentiate between deep vein thrombosis and pulmonary embolism, it is difficult to compare the incidence rate ratios between their and our study. Furthermore, the English study only included people who were vaccinated against covid-19. The only other comparable nationwide self-controlled case series study to ours is from Scotland, where all people who tested positive for SARS-CoV-2 from 1 March to 5 October 2020 were included. 21 Those authors identified 179 people with deep vein thrombosis and 417 with pulmonary embolism during the study period, with incidence rate ratios increased by about 12-fold and 17-fold during the first week after covid-19, respectively. Day 0 was included in the first risk period, thereby the incidence rate ratios may be inflated as a result of test bias. 10 Although the Scottish study found an increased relative incidence of deep vein thrombosis (1.77) during the period 28 to 56 days after covid-19 that is comparable to the finding in our study (2.59) in similar periods, it was not statistically significant. Currently, no studies have used the self-controlled case series method to determine the association between covid-19 and bleeding. However, a Danish study that compared the incidence of major bleeding between participants who tested positive for SARS-CoV-2 and those who tested negative, did not find a higher incidence for those who tested positive. 22 The Danish study did not use matched controls but instead included people who probably displayed symptoms similar to those of covid-19 but who did not test positive for SARS-CoV-2. Therefore, in the Danish study it is difficult to determine if infection with SARS-CoV-2 increases the risk of bleeding compared with the background population.

Conclusions and policy implications

The present findings have major policy implications. Our findings arguably support thromboprophylaxis to avoid thrombotic events, especially for high risk patients, 23 and strengthens the importance of vaccination against covid-19. It remains to be established whether SARS-CoV-2 infection increases the risk of venous thromboembolism or bleeding more than it does for respiratory infections, such as influenza, but also whether the period of thromboprophylaxis after covid-19 should be extended. Future clinical research would be beneficial in this context.

Ethical approval

This study was approved by the Swedish Ethical Review Authority (DNR 2020-02150).

View Abstract

# data[].attributes.attachment_link.title
Risks of deep vein thrombosis, pulmonary embolism, and bleeding after covid-19: nationwide self-controlled cases series and matched cohort study

# data[].attributes.attachment_link.description
Objective To quantify the risk of deep vein thrombosis, pulmonary embolism, and bleeding after covid-19. Design Self-controlled case series and matched cohort study. Setting National registries in Sweden. Participants 1 057 174 people who tested positive for SARS-CoV-2 between 1 February 2020 and 25 May 2021 in Sweden, matched on age, sex, and county of residence to 4 076 342 control participants.

# data[].attributes.attachment_caption
Risk of thrombocytopenia and thromboembolism after covid-19 vaccination and SARS-CoV-2 positive testing: self-controlled case series study

# data[].attributes.attachment_link.title
Risk of thrombocytopenia and thromboembolism after covid-19 vaccination and SARS-CoV-2 positive testing: self-controlled case series study

# data[].attributes.attachment_link.description
Objective To assess the association between covid-19 vaccines and risk of thrombocytopenia and thromboembolic events in England among adults. Design Self-controlled case series study using national data on covid-19 vaccination and hospital admissions. Setting Patient level data were obtained for approximately 30 million people vaccinated in England between 1 December 2020 and 24 April 2021.

# data[].attributes.attachment_link.readable_text
This story is part of a larger mosaic of stories on Post-Acute Sequelae of Covid-19 (PASC), also known as Long Covid. Read part and of this series.

Comparing brain volume before and after individuals were exposed to SARS-CoV-2, this study documents ... [+]
Getty Images

Thanks to a new study from the UK we are now beginning to uncover the effects of SARS-CoV-2 infections in the brain. Comparing brain volume before and after individuals were exposed to SARS-CoV-2, this study documents significant cortical gray matter loss, equivalent to nearly 10 years of aging. Gweanaelle Douaud, the study's first author and Professor at the University of Oxford, says that infected individuals display structural "differences over time above and beyond any potential baseline differences." Most strikingly, individuals that experienced no or only mild symptoms with Covid-19 displayed specifically significant changes, but cortical damage seems to occur regardless of disease severity, age, or sex. The effect of vaccination status not yet been investigated. It may be years before the long-term consequences of these structural differences are fully understood.

Douaud et al had unique access to an extensive biomedical database, called the UK BioBank. The UK BioBank provided pre-pandemic brain scans from 785 individuals that were used as a baseline for normal size and structure relative to each participant. About three years later, in May 2021, the same population of participants returned for new brain scans. From the total cohort, 401 individuals were infected with Covid-19 between scans and the remaining served recruited as controls.

Between the first and second brain scans, individuals previously infected with Covid-19 experienced a 0.7% reduction in overall cortical gray matter on average, compared to the control group. To put that into perspective, people middle aged and beyond only lose 0.2% to 0.3% of volume per year.

Although it is clear that SARS-CoV-2 can damage the brain, how this damage occurs without directly infecting nerve cells remains a mystery. Current theories suggest there may be multiple factors contributing to these deficits. Structural abnormalities in the brain may in fact be secondary to infection occurring in other sites around the body, including the olfactory epithelium. Considering how close the nose is to the brain, this Oxford University Study considered whether losing the sense of smell may be linked to other neurological damage.

Loss of smell is a consistent clinical feature of Covid-19, with recent studies suggesting that 86% of individuals exposed to the virus may experience partial or complete loss of smell. A smaller percentage of people also develop additional neurological complications, including brain fog, fatigue, impaired cognitive function, and encephalography, characterized by impairments in brain structure and function. For some, these symptoms may continue to linger anywhere from a few months to more than a year after the initial infection. These effects, however, do not discriminate between mild and severe disease.

First, Douaud et al. found reduced gray matter in several regions associated with olfaction, consistent with symptoms of prolonged loss of smell. Individuals exposed to Covid-19 experienced the most significant neural damage to the primary olfactory cortex, or piriform cortex. This structure receives input directly from olfactory receptors that pass through the olfactory bulb, making it the first neural target for processing and perceiving odors. Reduced cortical thickness was also observed in a connected region called the orbitofrontal cortex. Illustrated in Figure 1, the orbitofrontal cortex (OFC) receives inputs from the primary olfactory cortex and is commonly referred to as the secondary olfactory cortex.

Figure 1: Schematic view of the human olfactory system. The primary and secondary olfactory cortices ... [+]
From: " A review on the neural bases of episodic odor memory: From laboratory-based to autobiographical approaches" Saive et al. 2014.

Damage to these areas may be linked to loss of smell, but there is no evidence of causation. Instead, researchers speculate that the loss of volume to brain regions associated with olfaction may be partially attributed to widespread damage to the olfactory epithelium that disrupts neural pathways and impairs function. As with many other neural systems, pathways that are not being used over a long period of time cease to exist, a process neuroscientists often call "use it or lose it" that causes brain tissues to shrink.

Surprisingly, Douaud et al. also found additional abnormalities in regions not normally associated with the sense of smell. In particular, they observed reduced gray matter volume in some regions of the limbic system, involving several structures important for producing behavioral and emotional responses. The largest differences, ranging from 0.2% to 2% reductions, were seen in the left parahippocampal gyrus and the entorhinal cortex. These regions play an important role in the hippocampal memory system, so gray matter loss could signify future memory impairments. Structures are shown below in Figure 2 for reference.

Figure 2: Illustration of the limbic system. The parahippocampal gyrus and hippocampus, which ... [+]
BruceBlaus, Wikipedia Commons

Are the differences seen in these limbic structures also linked to deficits in the olfactory system during Covid-19? Douaud et al. argues that the parahippocampal gyrus, the orbitofrontal cortex and other parts of the limbic system are in some way connected to the olfactory cortex. Since sensory inputs, including those for olfaction, are transmitted and integrated all over the brain to guide a range of behavioral responses, damage to the olfactory epithelium may also have disastrous consequences on regions of the brain not exclusively involved in olfaction. More likely, however, these and other changes in brain structure may be a consequence of a robust immune response occurring all around the brain, albeit the mechanisms underlying inflammation-induced brain damage remain unclear.

Additional exploratory analyses found gray matter loss in the amygdala, the insula which borders the temporal cortex, and the front-most portion of the cingulate gyrus, known as the anterior cingulate gyrus. Interestingly, all these regions play a role in emotion processing and regulation. More research is needed to determine whether deficits in these brain regions may be linked to mood disorders associated with long-haul Covid-19, including depression and anxiety.

Figure 3:Reference locations for amygdala, anterior cingulate cortex, and insula.
Apollo Medicine 2020

It is important to note that not every individual infected with Covid-19 will experience a reduction in brain volume, while others will experience much greater losses. Those hospitalized with Covid-19, for example, had more widespread tissue damage and atrophy, compared to those not hospitalized for infection. Beyond hospitalized vs. non-hospitalized, there was limited data from this study showing how severity of infection may contribute to these effects.

Finally, Douaud et al. asked whether these structural changes in the cerebral cortex are linked to new neurological symptoms following Covid-19 infection. They did not find significant correlations between structural changes and the prevalence of new neurological symptoms. However, does not mean that these changes will not impact brain function. They did find infected participants had a greater decline in their ability to perform a complex task, compared with the non-infected participants.
Correction: An earlier version of this article stated that the cortical damage occurs regardless of vaccination status, however the researchers have not yet been able to investigate the effect of vaccination status. Full coverage and live updates on the Coronavirus
For a vast majority of people, the regenerative properties of the olfactory bulb restores the sense of smell within a few weeks or months. What about the rest of the brain? Damage to brain cells cannot be reversed. When tissues die, cerebrospinal fluid and other biomolecules fill the excess space to maintain the integrity of the brain. Perhaps, this explains why neurological complications associated with long-haul Covid-19 show little improvement over time. Years of additional research are needed before the consequences of losing so much gray matter are fully understood. Identifying these changes now will help us to better support and treat what will be a growing class of people with cognitive impairments.

Although the loss of smell is often one of the first symptoms of Covid-19 preceding any respiratory complications, the hypothesis that the SARS-CoV-2 damages the brain when it infects cells in the olfactory epithelium remains heavily-debated. Researchers do seem confident that the virus does not directly infect brain cells. If the nose is a window to the brain, it may be time to develop new vaccines that aim to close it off from the SARS-CoV-2 virus.

# data[].attributes.attachment_link.title
A Case Of Shrunken Brains: How Covid-19 May Damage Brain Cells

# data[].attributes.attachment_link.description
This story is part of a larger mosaic of stories on Post-Acute Sequelae of Covid-19 (PASC), also known as Long Covid. Read part and of this series. Thanks to a new study from the UK we are now beginning to uncover the effects of SARS-CoV-2 infections in the brain.

# data[].attributes.headline
Moderna Patent On Lethal Cell Repair Legacy

# data[].attributes.subject
Moderna Patent On Lethal Cell Repair Legacy

# data[].attributes.attachment_link.readable_text
Knight's Campus for Accelerating Scientific Impact, University of Oregon, Eugene, OR, United States

Dr. Shroff's Charity Eye Hospital, New Delhi, India

PanTherapeutics, Lutry, Switzerland

Department of Molecular Medicine, University of Padova, Padova, Italy

Department of Physiology, Michigan State University, East Lansing, MI, United States

Department of Molecular Medicine, Morsani College of Medicine, University of South Florida (USF) Health Byrd Alzheimer's Institute, University of South Florida, Tampa, FL, United States

Division of Hematology/Oncology, Department of Medicine, University of Pittsburgh Medical Center (UPMC) Hillman Cancer Center, University of Pittsburgh School of Medicine, Pittsburgh, PA, United States

Among numerous point mutation differences between the SARS-CoV-2 and the bat RaTG13 coronavirus, only the 12-nucleotide furin cleavage site (FCS) exceeds 3 nucleotides. A BLAST search revealed that a 19 nucleotide portion of the SARS.Cov2 genome encompassing the furing cleavage site is a 100% complementary match to a codon-optimized proprietary sequence that is the reverse complement of the human mutS homolog (MSH3). The reverse complement sequence present in SARS-CoV-2 may occur randomly but other possibilities must be considered. Recombination in an intermediate host is an unlikely explanation. Single stranded RNA viruses such as SARS-CoV-2 utilize negative strand RNA templates in infected cells, which might lead through copy choice recombination with a negative sense SARS-CoV-2 RNA to the integration of the MSH3 negative strand, including the FCS, into the viral genome. In any case, the presence of the 19-nucleotide long RNA sequence including the FCS with 100% identity to the reverse complement of the MSH3 mRNA is highly unusual and requires further investigations.

Introduction

Based on a recent publication describing insertion variants of SARS-CoV-2 ( 1) we would like to bring the attention to our recent findings related to the sequence of the furin cleavage site (FCS) in SARS-CoV-2 Spike (S) protein. The SARS-CoV-2 causing the COVID-19 pandemic ( 2) has 82.3% amino acid identity to bat coronavirus SL-CoVZC45, 77.2% amino acid identity to SARS-CoV, and 96.2% genome sequence identity to bat coronavirus RaTG13. While numerous point mutation differences exist between SARS-CoV-2 and RaTG13, only one insertion and dissimilarity exceeding 3 nucleotides (nt): a 12-nucleotide insertion coding for four amino acids (aa 681-684, PRRA) in the SARS-CoV-2 S protein has been discovered. This polybasic FCS differentiates SARS-CoV-2 from other b-lineage betacoronaviruses or any other sarbecovirus ( 3). An FCS addition enhanced the infectivity of SARS Co-V-2 in 2019 ( 4). The absence of this FCS results in attenuated SARS-CoV-2 variants useful for animal vaccination, accentuating its relevance to human infection ( 5). This FCS is vital for human and ferret transmission ( 6), expands viral tropism to human cells ( 7), and is requisite for severe disease in two animal models of SARS-CoV-2 ( 8).

SARS-CoV-2 Spike Protein and MSH3

A peculiar feature of the nucleotide sequence encoding the PRRA furin cleavage site in the SARS-CoV-2 S protein is its two consecutive CGG codons. This arginine codon is rare in coronaviruses: relative synonymous codon usage (RSCU) of CGG in pangolin CoV is 0, in bat CoV 0.08, in SARS-CoV 0.19, in MERS-CoV 0.25, and in SARS-CoV-2 0.299 ( 9).

A BLAST search for the 12-nucleotide insertion led us to a 100% reverse match in a proprietary sequence (SEQ ID11652, nt 2751-2733) found in the US patent 9,587,003 filed on Feb. 4, 2016 ( 10) ( Figure 1). Examination of SEQ ID11652 revealed that the match extends beyond the 12-nucleotide insertion to a 19-nucleotide sequence: 5′-CTACGTGCCCGCCGAGGAG-3′ (nt 2733-2751 of SEQ ID11652), such that the resulting mRNA would have 3′- GAUGCACGGGCGGCUCCUC-5′, or equivalently 5′- CU CCU CGG CGG GCA CGU AG-3′ (nucleotides 23547-23565 in the SARS-CoV-2 genome, in which the four bold codons yield PRRA, amino acids 681-684 of its spike protein). This is very rare in the NCBI BLAST database.

Figure 1. The origin of the furin sequence in SARS-CoV-2. Comparison of the protein sequences at the S1/S2 junction in SARS-CoV, RaTG13, and SARS-CoV-2 demonstrating the presence of the furin cleavage site (FCS) PRRA only in SARS-CoV-2. Based on a BLAST search of the 12-nucleotide stretch coding for the FCS PRRA, a 19-nucleotide long identical sequence was identified in the patented (US 958 7003) sequence Seq ID11652. SEQ ID11652 is transcribed to a MSH3 mRNA that appears to be codon optimized for humans. This 19-nucleotide sequence including 12 nucleotides coding for the FCS PRRA, present in the human MSH3 gene might have been introduced into the SARS-CoV-2 genome by the illustrated copy choice recombination mechanism in SARS-CoV-2 infected human cells overexpressing the MSH3 gene.

The correlation between this SARS-CoV-2 sequence and the reverse complement of a proprietary mRNA sequence is of uncertain origin. Conventional biostatistical analysis indicates that the probability of this sequence randomly being present in a 30,000-nucleotide viral genome is 3.21 ×10 −11 ( Figure 2).

Figure 2. Calculations of the probability of natural occurrence of the 19nt sequence under study. The SARS-CoV-2 genome is ~30,000 nucleotides long (P1). The patented sequence is ~3,300 nucleotides long (P2). The patented library encompasses 24'712 sequences of varying lengths with median length being in the range of 3,300 nucleotides. Conventional probability calculations are given of the probability of the presence of a 19-nucleotide sequence in the human genome and in one of the patented library sequences.

The proprietary sequence SEQ ID11652, read in the forward direction, encodes a 100% amino acid match to the human mut S homolog 3 (MSH3) ( 9). MSH3 is a DNA mismatch repair protein (part of the MutS beta complex) ( 11). SEQ ID11652 is transcribed to a MSH3 mRNA that appears to be codon optimized for humans ( 12). We did not find the 19-nucleotide sequence CTCCTCGGCGGGCACGTAG in any eukaryotic or viral genomes except SARS-CoV-2 with 100% coverage and identity in the BLAST database ( Supplementary Tables 1- 3).

Discussion

MSH3 replacement with a codon-optimized mRNA sequence for human expression likely has applications in cancers with mismatch repair deficiencies. While a portion of a reverse complement sequence being present in SARS-CoV-2 could be a random coincidence, other possibilities merit consideration.

Overexpression of MSH3 is known to interfere with mismatch repair (MSH2 sequestration from the MutS alpha complex comprising MSH2 and MSH6 results in MSH6 degradation and MutS alpha depletion) ( 13), which holds virologic importance. Induction of DNA mismatch repair deficiency results in permissiveness of influenza A virus (IAV) infection of human respiratory cells and increased pathogenicity ( 14). Mismatch repair deficiency may extend shedding of SARS-CoV-2 ( 15, 16).

The absence of CTCCTCGGCGGGCACGTAG from any eukaryotic or viral genome in the BLAST database makes recombination in an intermediate host an unlikely explanation for its presence in SARS-CoV-2. A human-codon-optimized mRNA encoding a protein 100% homologous to human MSH3 could, during the course of viral research, inadvertently or intentionally induce mismatch repair deficiency in a human cell line, which would increase susceptibility to SARS-like viral infection. Infection of SEQ ID11652-MSH3-transduced human cells by a SARS-like virus could enable copy choice recombination ( 15). Replication of SARS-CoV-2 and other single stranded RNA viruses with an RNA genome of positive polarity is initiated by the synthesis of negative strand RNA in the cytoplasm of infected cells ( 17) ( Figure 1). The negative strand RNA is a template for synthesis of positive stranded RNA utilized for translation of non-structural proteins, the replication and transcription complex, or new virion capsids. Coronaviruses generate double stranded RNA at an early stage of infection through genomic replication and mRNA transcription ( 18).

Acquisition of the reverse complement FCS sequence from an overexpressed positive sense MSH3 mRNA could occur through copy choice recombination with a negative sense SARS-CoV-2 RNA intermediate ( 15), involving jumping from one template to another ( 19) ( Figure 1). The homology between SARS-CoV-2 and other known coronaviruses is discontinued and most SARS-CoV-2 sequences derive from a relatively recent common ancestor with bat RaTG13. Moreover, similarity plots (SimPlots) have identified sudden changes in sequence identity between SARS-CoV-2 and RaTG13, signaling potential recombination events, which could explain the capability of SARS-CoV-2 binding to ACE2 through its RBD, which is not the case for the RaTG13 RBD ( 15).

A criticism of this hypothesis is that the identified sequence is on the opposite strand of the open reading frame in SEQ ID11652. However, cells transfected with MSH3, which induce mismatch repair deficiency could have targeted double-stranded cDNA encoding SEQ ID11652. Such cells co-transfected with a SARS-like virus expressing RdRp could attach to this 19-nucleotide sequence ( 15) and permit integration of a fragment from the negative strand into the viral genome, including the FCS, despite being on the opposite strand of the open reading frame. Mismatch repair mechanisms have enabled integration of short fragments from antisense strands in experimental models ( 20, 21). Microhomology can direct recombination between the MSH3 and a SARS-like virus, which could take place at the 19-nucleotide sequence of interest.

The presence in SARS-CoV-2 of a 19-nucleotide RNA sequence encoding an FCS at amino acid 681 of its spike protein with 100% identity to the reverse complement of a proprietary MSH3 mRNA sequence is highly unusual. Potential explanations for this correlation should be further investigated.

Data Availability Statement

Publicly available datasets were analyzed in this study. This data can be found here: SEQ ID11652.

Author Contributions

The investigation and original draft were initiated by BA, AV, and AB. The visualization was performed by BA, AV, AB, and GP. The writing and editing were done by BA, AV, KL, GP, BU, VU, and AB. All authors contributed to the article and approved the submitted version.

Conflict of Interest

KL was employed by PanTherapeutics.

The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Publisher's Note

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

Acknowledgments

We are thankful for the contribution of Dr. Jian Ying, University of Utah, Eureka, USA to the probability analysis.

Supplementary Material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fviro.2022.834808/full#supplementary-material

References

10. Bancel S, Chakraborty T, De Fougerolles A, Elbashir SM, John M, Roy A, et al. Modified Polynucleotides for the Production of Oncology-Related Proteins and Peptides. Cambridge, MA: United States Patent. (2016).
Google Scholar
Keywords: SARS-CoV-2 spike, furin cleavage site, MSH3 gene, sequence homology, recombinability

Citation: Ambati BK, Varshney A, Lundstrom K, Palú G, Uhal BD, Uversky VN and Brufsky AM (2022) MSH3 Homology and Potential Recombination Link to SARS-CoV-2 Furin Cleavage Site. Front. Virol. 2:834808. doi: 10.3389/fviro.2022.834808

Received: 13 December 2021; Accepted: 19 January 2022;
Published: 21 February 2022.

Edited by:

Xin Yin, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences (CAAS), China

Reviewed by:

Jitao Chang, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences (CAAS), China

Copyright © 2022 Ambati, Varshney, Lundstrom, Palú, Uhal, Uversky and Brufsky. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

*Correspondence: Kenneth Lundstrom, lundstromkenneth@gmail.com

# data[].attributes.attachment_link.title
MSH3 Homology and Potential Recombination Link to SARS-CoV-2 Furin Cleavage Site

# data[].attributes.attachment_link.description
Among numerous point mutation differences between the SARS-CoV-2 and the bat RaTG13 coronavirus, only the 12-nucleotide furin cleavage site (FCS) exceeds 3 nucleotides. A BLAST search revealed that a 19 nucleotide portion of the SARS.Cov2 genome encompassing the furing cleavage site is a 100% complementary match to a codon-optimized proprietary sequence that is the reverse complement of the human mutS homolog (MSH3).

# data[].attributes.attachment_caption
The Puzzling Virus That Infects Almost Everyone

# data[].attributes.headline
CANCER triggers after Covid and/or vaccination

# data[].attributes.subject
CANCER triggers after Covid and/or vaccination

# data[].attributes.attachment_link.readable_text
Statistically speaking, the virus known as Epstein-Barr is inside you right now. It is inside 95 percent of us. It spreads through saliva, so perhaps you first caught the virus as a baby from your mother, who caught it as a baby from her mother. Or you picked it up at day care. Or perhaps from a friend with whom you shared a Coke. Or the pretty girl you kissed at the party that cold New Year's Eve.

If you caught the virus in this last scenario-as a teen or young adult-then Epstein-Barr may have triggered mono, or the "kissing disease," in which a massive immune response against the pathogen causes weeks of sore throat, fever, and debilitating fatigue. For reasons poorly understood but not unique among viruses, Epstein-Barr virus, or EBV, hits harder the later you get it in life. If you first caught the virus as a baby or young child, as most people do, the initial infection was likely mild, if not asymptomatic. Unremarkable. And so this virus has managed to fly under the radar, despite infecting almost the entire globe. EBV is sometimes jokingly said to stand for "everybody's virus." Once inside the body, the virus hides inside your cells for the rest of your life, but it seems mostly benign.

Except, except. In the decades since its discovery by the virologists Anthony Epstein and Yvonne Barr in 1964, the virus has been linked not only to mono but also quite definitively to cancers in the head and neck, blood, and stomach. It's also been linked, more controversially, to several autoimmune disorders. Recently, the link to one autoimmune disorder got a lot stronger: Two separate studies published this year make the case-convincingly, experts say-that Epstein-Barr virus is a cause of multiple sclerosis, in which the body mistakenly attacks the nervous system. "When you mentioned the virus and MS 20 years ago, people were like, Get lost ... It was a very negative attitude," says Alberto Ascherio, an epidemiologist at Harvard and a lead author of one of those studies, which used 20 years of blood samples to show that getting infected with EBV massively increases the risk of developing multiple sclerosis. The connection between virus and disease is hard to dismiss now. But how is it that EBV causes such a huge range of outcomes, from a barely noticeable infection to chronic, life-altering illness?

In the face of a novel coronavirus, my colleague Ed Yong noted that a bigger pandemic is a weirder pandemic: The sheer number of cases means that even one-in-a-million events become not uncommon. EBV is far from novel; it belongs to a family of viruses that were infecting our ancestors before they were really human. But it does infect nearly all of humanity and in rare occasions causes highly unusual outcomes. Its ubiquity manifests its weirdness. Decades after its discovery and probably millennia after those first ancient infections, we are still trying to understand how weird this old and familiar virus can be. We do little to curb the spread of Epstein-Barr right now. As the full scope of its consequences becomes clearer, will we eventually decide it's worth stopping after all?

From its very discovery, Epstein-Barr confounded our ideas of what a virus can or cannot do. The first person to suspect EBV's existence was Denis Burkitt, a British surgeon in Uganda, who had the unorthodox idea that the unusual jaw tumors he kept seeing in young children were caused by a then-undiscovered pathogen. The tumors grew fast -doubling in size in 24 to 48 hours-and were full of white blood cells or lymphocytes turned cancerous. This disease became known as Burkitt's lymphoma. Burkitt suspected a pathogen because the jaw tumors seemed to spread from area to neighboring area and followed seasonal patterns. In other words, this lymphoma looked like an epidemic.

In 1963, a biopsy of cells from a girl with Burkitt's lymphoma made its way to the lab of Anthony Epstein, in London. One of his students, Yvonne Barr, helped prepare the samples. Under the electron microscope, they saw the distinctive shape of a herpesvirus, a family that also includes the viruses behind genital herpes, cold sores, and chicken pox. And the tumor cells specifically were full of this virus. Case closed? Not yet. At the time, the idea that a virus could cause cancer was "rather remote," says Alan Rickinson, a cancer researcher who worked in Epstein's lab in the 1970s. "There was a great deal of skepticism." What's more, the virus's ubiquity made things confusing. Critics pointed out that sure, children with Burkitt's lymphoma had antibodies to EBV, but so did healthy children in Africa. So did American children for that matter, as well as isolated Icelandic farmers and people belonging to a remote tribe in the Brazilian rainforest. The virus was everywhere scientists looked, yet Burkitt's lymphoma was largely confined to equatorial Africa. What if EBV was just an innocent bystander? Why wasn't the virus causing disease anywhere else?

It was. Scientists just didn't know where to look until a stroke of luck clued them in. In 1967, a technician in a Philadelphia lab studying EBV and cancer fell ill with symptoms of mono. Because she was one of the few people who had tested negative for EBV antibodies, she had regularly donated blood for lab experiments that needed a known negative sample. When she came back after the illness, she started testing positive, highly positive. The timing suggested what we now know: EBV is the most common cause of mono.

Scientists eventually found more links between the virus and other cancers: nasopharyngeal cancer, stomach cancer, Hodgkin's lymphoma, ​​and other forms of lymphoma. In all, it plays a role in 1.5 percent of cancers globally. Those first two are cancers in the cells lining the throat and stomach, which EBV can infect. The others are in white blood cells or lymphocytes, which the virus actually specializes in infecting. In particular, EBV infects a type of lymphocyte called a B cell, each of which is born to recognize a different hypothetical enemy. If a certain B cell never finds its matching enemy, it dies as part of the body's ruthless culling of useless immune cells. If it does find a match, however, the B cell divides and transforms into memory B cells, which will remain to guard against infection for the rest of a person's life.

EBV's genius is that it co-opts this normal process. It manipulates infected B cells into thinking they have been activated, so that they turn into long-lasting memory B cells where the virus can hide for decades. (All herpesviruses in the family have this unusual ability to become latent, though they hide out in different types of cells. The chicken-pox virus, for example, uses nerve cells, sometimes coming out to cause shingles.) Occasionally, EBV emerges from its hiding place, replicating just enough to get by. If it replicates too little, it won't find another host before getting shut down by the immune system. If it replicates too much, it risks harming its current host. The virus and immune system are in constant balance, each holding the other in check. There's an "elegance with which this virus has established a long-term relationship with the host," says Sumita Bhaduri-McIntosh, an Epstein-Barr virologist and infectious-disease doctor at the University of Florida.

When this balance is interrupted, one possible result is cancer. As part of its manipulation of infected cells, EBV seems to suppress their normal dying process. And if the cell that refuses to die has other aberrant properties, then you can get cancers like Burkitt's lymphoma. "In most cases, when the virus appears in this cancer, and subsequently in other cancers, it is one part of a chain," Rickinson says. "It's obviously not the sole driver of growth." This explains why EBV doesn't cause cancer in everyone it infects, only in those unlucky enough to have also acquired the wrong set of other mutations. In the case of Burkitt's lymphoma, the cancerous cells also have a strange rearrangement of chromosomes, which scientists learned is linked to malaria infection. This accounted for the unique geographic pattern that Burkitt had observed. EBV is everywhere, but Burkitt's lymphoma was common only in places where malaria is also endemic.

Epstein-Barr became known as the first human virus linked to not just an immediate disease but also cancers that can appear years after initial infection. It challenged the traditional paradigm of viruses causing short-term illnesses that resolve and confer immunity. After all, the virus stays inside our bodies and continues to interact with our immune systems for the remainder of our lives.

Over the years, more hints of EBV's unusual abilities started appearing. The virus or the antibodies to it seemed to be disproportionately found in people suffering from autoimmune disorders such as rheumatoid arthritis, lupus, and multiple sclerosis as well as those suffering from chronic fatigue syndrome, also known as myalgic encephalomyelitis. These chronic conditions, whose biological mechanisms are even more elusive than cancer's, are particularly hard to study. While the correlations between EBV and these disorders were suggestive, they were in no way definitive. People who have these conditions might almost all have EBV, but then almost all healthy people have EBV too. "That's not a very good place to start doing epidemiology, when you have 95 percent in the control group," says Paul Farrell, an EBV researcher at Imperial College London.

The recent study from Harvard's Ascherio got around this by looking at a massive archive of serum samples taken from people over 20 years. The collection came from the Department of Defense, which stores serum from routine tests for HIV. Among the 10 million adults with samples in the repository, researchers were able to find enough people who were initially negative for EBV but then contracted it during the 20-year period. And those who did get the virus were 32 times as likely to develop multiple sclerosis as those who did not. A second study from Stanford adds a possible causation to this correlation: Some multiple-sclerosis patients have antibodies that bind both an EBV protein and a protein in the brain, which is erroneously targeted by the immune system in multiple sclerosis. This kind of cross-reaction has long been suspected in MS but only now identified. "It's just like a great volcano of information," says Rickinson about the recent studies. As with EBV-associated cancers, though, only a tiny sliver of people infected with the virus end up developing multiple sclerosis, so some other trigger or triggers must also be in play. We're only at the beginning of understanding this process.

COVID, too, has revived interest in Epstein-Barr's long-term consequences. A recent long-COVID study found EBV infection to be one of four major risk factors, suggesting that some long-COVID symptoms might be caused by reactivation of EBV when the body is weakened from fighting the coronavirus.

This association is perhaps not surprising. The debilitating fatigue associated with long COVID and other post-viral syndromes does look, in some ways, like the fatigue caused by mono. And in the 1980s, doctors noticing the similarity had begun diagnosing chronic Epstein-Barr virus syndrome in patients whose mono-like symptoms of fatigue and sore throat did not go away for months. Eventually, however, experts took Epstein-Barr out of the name and gave it the more general term of chronic fatigue syndrome, because EBV does not seem to be the sole cause of such symptoms. Chronic fatigue syndrome may have several different explanations, but the virus may still play a role in some cases even after mild infections, says Hank Balfour, a pathologist at the University of Minnesota. He has also described cases of " chronic mono," in which a severe initial EBV infection triggers mono symptoms that either linger or recur for months or even years. Mono's acute phase typically lasts for weeks, which is already unusually long for a virus but is well documented. There isn't much research on chronic mono though, and the diagnosis is not widely accepted among doctors. "It needs, I think, more attention," Balfour says. Long COVID remains a baffling consequence of the novel coronavirus, but even the long-term consequences of very common viruses like EBV are poorly understood.

As the long-term picture of EBV comes into focus, how do we think about the danger of a virus that is ubiquitous, that rarely causes serious disease but has devastating consequences when it does? We currently have no way of preventing EBV infection, short of avoiding all human interactions that might share saliva: a mother kissing her baby, a toddler doing almost anything. Vaccines have been in the works for decades; Epstein himself worked on one. The link to multiple sclerosis, many long-time researchers now hope, will revive interest in an EBV vaccine. More than a decade ago, a pharmaceutical company abandoned a vaccine candidate that successfully prevented mono but not EBV infection altogether. The result was "discouraging from a pharmacoeconomic point of view," Balfour says, because there wasn't a clear demand for a vaccine that prevented only mono. Preventing multiple sclerosis, however, might add an extra incentive.

Two new vaccine candidates, from the National Institutes of Health and Moderna, have entered or are about to enter clinical trials. A key question is whether they can do better than the old vaccine. "We would of course like to prevent infection. That's the ultimate goal, but we think even if we don't prevent infection, we can still reduce EBV-associated disease," says Jeffrey Cohen, a virologist at the NIH who works on one of the vaccines. That's because symptomatic EBV infections-such as mono-are associated with a higher likelihood of developing EBV-associated diseases, adds Balfour, who has also worked on a vaccine. However, studying how the vaccine might stop diseases that develop years later, such as cancers or multiple sclerosis, will be very hard in a typical vaccine trial. The incidences are so low, and the diseases take so long to appear, that a vaccine trial in hundreds or thousands of people over a few years is unlikely to offer much definitive evidence. Most likely, Cohen says, if the vaccines work against mono, they can be approved to prevent the disease in people who have not yet been infected by EBV. Once it's on the market and hundreds of thousands of people get it and are followed over years, then the effect on cancer or multiple sclerosis may finally become clear.

All of these recent advances make it a "fascinating time" for EBV research, says Rickinson, the biologist who once worked with the eponymous Epstein. "Unfortunately," he says, "I'm unable to pursue it myself." He recently retired from the University of Birmingham after devoting nearly 50 years to studying this enigmatic virus. It's up to the next generation now-to figure out EBV's remaining secrets and perhaps a better way of coexisting with it.

# data[].attributes.attachment_link.title
The Puzzling Virus That Infects Almost Everyone

# data[].attributes.attachment_link.description
Statistically speaking, the virus known as Epstein-Barr is inside you right now. It is inside 95 percent of us. It spreads through saliva, so perhaps you first caught the virus as a baby from your mother, who caught it as a baby from her mother. Or you picked it up at day care.

# data[].attributes.headline
Childhood Illness Reactivated By Covid & Vax

# data[].attributes.subject
Childhood Illness Reactivated By Covid & Vax

# data[].attributes.body
Covid has been revealed as a deadly trigger to reactivate old viruses, bacteria and fungus infections which people had all the way back to their childhood, but now with a more deadly spin

# data[].attributes.headline
Sudden Deaths After Even Mild Covid Or Vax

# data[].attributes.subject
Sudden Deaths After Even Mild Covid Or Vax

# data[].attributes.body
Your chances of having a heart attack, stroke or other sudden health issue are significantly higher - even if you had very mild or asymptomatic Covid.. or the vaccine

# data[].attributes.headline
Cell Repair Blocked After Covid ? Evidence

# data[].attributes.subject
Cell Repair Blocked After Covid ? Evidence

# data[].attributes.attachment_link.readable_text
doi: 10.1186/s12879-021-06500-1.

Affiliations

¹ Hull University Teaching Hospital NHS Trust, Castle Road, Cottingham, Hull, HU16 5JQ, UK. farzana.haque4@nhs.net.

² Hull York Medical School, University of Hull, Allam Medical Building, Hull, HU6 7RX, UK. farzana.haque4@nhs.net.

³ Hull University Teaching Hospital NHS Trust, Castle Road, Cottingham, Hull, HU16 5JQ, UK.

⁴ Lincoln Medical School, University of Lincoln, Lincoln, LN6 7DL, UK.

⁵ Hull York Medical School, University of Hull, Allam Medical Building, Hull, HU6 7RX, UK.

Free PMC article

Farzana Haque et al. BMC Infect Dis.

Free PMC article

doi: 10.1186/s12879-021-06500-1.

Affiliations

¹ Hull University Teaching Hospital NHS Trust, Castle Road, Cottingham, Hull, HU16 5JQ, UK. farzana.haque4@nhs.net.

² Hull York Medical School, University of Hull, Allam Medical Building, Hull, HU6 7RX, UK. farzana.haque4@nhs.net.

³ Hull University Teaching Hospital NHS Trust, Castle Road, Cottingham, Hull, HU16 5JQ, UK.

⁴ Lincoln Medical School, University of Lincoln, Lincoln, LN6 7DL, UK.

⁵ Hull York Medical School, University of Hull, Allam Medical Building, Hull, HU6 7RX, UK.

Abstract

Background: Several independent risk factors have been reported to influence viral shedding following COVID-19 infection, but the influence of host-related molecular factors has not yet been described. We report a case of a cancer patient with Lynch syndrome (hereditary nonpolyposis colorectal cancer, HNPCC) who manifested SARS-CoV-2 PCR (polymerase chain reaction) positivity for at least 54 days after contracting mild COVID-19 illness. We propose that deficient mismatch repair (MMR) may play a role in the prolonged SARS-CoV-2 RNA shedding.

Case presentation: A patient with Lynch syndrome was under surveillance for metastatic adenocarcinoma after completing palliative chemotherapy in October 2019. Between the period of April 2020 to June 2020, he was admitted multiple times to address several clinical needs mainly related to his underlying malignancy. These included progressive disease observed in the aortocaval lymph nodes leading to recurrent episodes of upper gastrointestinal bleeding, dehydration resulting in acute kidney injury and a short-lived episode of pyrexia. A SARS-CoV-2 PCR of the nasopharyngeal swab (NPS) was positive at his initial admission with mild COVID-19 symptoms. He remained positive on subsequent admissions when tested routinely for SARS-CoV-2 without demonstrating any apparent clinical features of COVID-19 infection. The MMR pathway, a component of DNA damage response (DDR), is impaired in Lynch syndrome due to an inherited genetic mutation. This pathway is also required for viral clearance from the host cells following certain RNA viral infections like influenza virus and other coronaviridae. Here we provide a current understanding of the importance of DDR deficiencies in the clearance of RNA virus and suggest how this may play a similar role in the clearance of COVID-19, as evident in our case that demonstrated persistent positivity.

Conclusion: The importance of understanding the scientific basis of extended viral shedding during the COVID-19 pandemic is now centre-stage in the establishment of robust track and trace services to allow the recovery and function of societies and economies. This patient with Lynch syndrome recovered from infection but had prolonged viral positivity, which might merit further investigation to better understand the effect of this condition on infection duration and outcome.

Keywords: Case report; Hereditary nonpolyposis colorectal cancer; Mismatch repair; SARS-CoV-2.

Conflict of interest statement

None of the authors has any conflict of interest to disclose concerning this case.

Figures

Similar articles

Lynch syndrome and Lynch syndrome mimics: The growing complex landscape of hereditary colon cancer.

Carethers JM, Stoffel EM. Carethers JM, et al. World J Gastroenterol. 2015 Aug 21;21(31):9253-61. doi: 10.3748/wjg.v21.i31.9253. World J Gastroenterol. 2015. PMID: 26309352 Free PMC article. Review.

# data[].attributes.attachment_link.keywords[]
national center for biotechnology information

# data[].attributes.attachment_link.title
Deficient DNA mismatch repair and persistence of SARS-CoV-2 RNA shedding: a case report of hereditary nonpolyposis colorectal cancer with COVID-19 infection - PubMed

# data[].attributes.attachment_link.description
The importance of understanding the scientific basis of extended viral shedding during the COVID-19 pandemic is now centre-stage in the establishment of robust track and trace services to allow the recovery and function of societies and economies. This patient with Lynch syndrome recovered from infe ...

# data[].attributes.headline
WHY DO EUROPEAN HEALTH EXPERTS WARN US ABOUTEATING MEAT?

# data[].attributes.subject
WHY DO EUROPEAN HEALTH EXPERTS WARN US ABOUTEATING MEAT?

# data[].attributes.headline
WHY DO EUROPEAN HEALTH EXPERTS WARN US ABOUT EATING MEAT?

# data[].attributes.subject
WHY DO EUROPEAN HEALTH EXPERTS WARN US ABOUT EATING MEAT?

# data[].attributes.attachment_link.provider_name
Centers for Disease Control and Prevention

# data[].attributes.attachment_link.readable_text
Data analytics

To interpret SARS-CoV-2 wastewater measurements, polymerase chain reaction (PCR)-based measurements must be converted to sample concentrations and adjusted for testing and wastewater factors, which may change from sample to sample within a wastewater system and between wastewater systems. PCR measurements must be converted to wastewater concentrations prior to submitting data to NWSS. Viral recovery and fecal normalization will be evaluated by the NWSS analytic engine as described below.

Sample concentration calculation

SARS-CoV-2 RNA is quantified using PCR technology: reverse transcription quantitative PCR (RT-qPCR), reverse transcription digital PCR (RT-dPCR), or reverse transcription droplet digital PCR (RT-ddPCR). Laboratory staff should convert concentration estimates produced by PCR software (in units of copies per reaction or copies per reaction volume) to virus concentrations per volume of unconcentrated wastewater or sludge sample. This conversion accounts for the volume of template used in the PCR (and reverse transcriptase reaction if separate), the concentration factor of nucleic acid extraction, and sample concentration processes.

Presence

Presence of viral RNA in a wastewater sample is defined for RT-qPCR measurements as a signal that crosses the threshold at a cycle number <40 during the exponential phase of amplification. For RT-dPCR or RT-ddPCR measurements, presence is defined as three or more positive droplets. If multiple assays or multiple PCR replicates are run on a sample, the virus is considered present in the sample if there is detection in any one of the assays or replicates. Viral recovery and the amount of sample processed determine the lowest detectable quantity of virus in a sample.

Matrix recovery

A matrix recovery control (also called a process control) is a non-SARS-CoV-2 virus spiked into a wastewater sample at a known concentration prior to processing. This control is used to understand viral recovery, defined as the amount of virus lost during sample processing, and is important for comparing SARS-CoV-2 concentrations in wastewater over time. Viral recovery estimates can be incorporated into SARS-CoV-2 wastewater data by dividing the measured concentration of SARS-CoV-2 by the fraction of matrix recovery control recovered. The fraction of matrix recovery control recovered is the amount of non-SARS-CoV-2 virus measured after processing divided by the amount of non-SARS-CoV-2 virus spiked into the sample before processing.

Normalization

To compare viral wastewater concentrations over time, normalize estimated viral concentrations by daily wastewater flow to account for changes in wastewater contributions. This normalization provides data in units of viral gene copies per day. To compare viral levels across sampling locations, also normalize viral concentrations by the number of people served by the sewer system, resulting in units of viral gene copies per person contributing to the sewershed per day.

If the number of people contributing to the sewershed is expected to change over the surveillance period (due to tourism, weekday commuters, temporary workers, etc.), human fecal normalization may be important for interpreting SARS-CoV-2 concentrations and comparing concentrations between sewage samples over time. Human fecal normalization targets are organisms or compounds specific to human feces that can be measured in wastewater to estimate its human fecal content. While there is no consensus method, you can normalize by human fecal content by dividing non-normalized wastewater concentrations by the human marker concentrations, resulting in a unitless ratio. This ratio may also account for viral losses in the sewage system and viral recovery through laboratory processes.

Surveillance analytics

Trends

Wastewater trend classification is the statistical analysis of changes in the normalized concentration of SARS-CoV-2 in wastewater (i.e., not by qualitative visual assessment). Trends in these wastewater data can be used to assess COVID-19 trends (reported and unreported) within the community contributing to the sewer system. Trends of SARS-CoV-2 levels in wastewater cannot be determined from fewer than three sample points (e.g., consistent weekly sampling requires 15 days of data to estimate trends). You can classify trends into categories based on the duration and direction of change in virus levels for interpretation and public health use.

Trend calculation: The distribution of SARS-CoV-2 concentrations in wastewater is important to consider when calculating trends in virus levels. Normalize concentrations prior to calculating trends to account for changes in wastewater dilution and differences in relative human waste input over time.

Trends can be calculated using linear regression with a minimum of three measurements, where the slope describes the trend.

The independent variable in the trend regression should be date, not measurement number, to estimate changes per day rather than per measurement.

As SARS-CoV-2 concentrations in wastewater are likely log-normally distributed, log-transform SARS-CoV-2 normalized concentrations prior to computing trends and other statistics.

For trends that are calculated using log10-transformed concentrations, compute the percent daily change (PDC) in virus levels from the slope as PDC = (10slope-1) × 100.

Include wastewater samples with SARS-CoV-2 levels below the limit of detection in trend calculations. This can be done by assigning the sample a value of half the assay detection limit.

Measurement variability: For more precise evaluation of wastewater data, trend calculations can incorporate the variability in each SARS-CoV-2 measurement through statistical weighting using weighted least squares regressions, which can take into account variability in the sampling, processing, and quantification steps.

Trend classification: Trends may be broadly classified by duration (short-term or sustained) and direction (increase, decrease, or plateau).

Duration: Trend classification schemes are dependent on sampling frequency. For example, short-term SARS-CoV-2 wastewater trends could be defined as trends spanning less than two weeks, and sustained trends could then be defined as trends spanning two weeks or longer. Based on a twice-weekly wastewater sampling frequency, short-term trends could then be calculated from three samples collected over an eight-day timespan, and sustained trends from the five samples collected over a 15-day timespan.

Direction: You can classify normalized SARS-CoV-2 concentration trends into "increasing," "decreasing," or "plateau" by testing trends for statistical significance. Statistical significance indicates that an increasing or decreasing trend exists, accounting for the variability in the SARS-CoV-2 data. You can also use a minimum percent daily change threshold in conjunction with statistical significance to assign trend direction.

Infection estimates

At this time, point estimates of community infection based on wastewater measurements should not be used. Such estimates depend strongly on clinical data describing the concentration of SARS-CoV-2 in feces over the course of infection and in individuals with varying levels of disease severity, and few such clinical data are currently available. As more clinical data become available, using wastewater SARS-CoV-2 data to estimate the total levels of COVID-19 (i.e., symptomatic, asymptomatic, pre-symptomatic) in a community could be a useful application of wastewater surveillance.

# data[].attributes.attachment_link.description
To interpret SARS-CoV-2 wastewater measurements, polymerase chain reaction (PCR)-based measurements must be converted to sample concentrations and adjusted for testing and wastewater factors, which may change from sample to sample within a wastewater system and between wastewater systems. PCR measurements must be converted to wastewater concentrations prior to submitting data to NWSS.