Ischemic optic neuropathy (ION) is a sudden, painless loss of vision due to an interruption of blood supply to the optic nerve. ION can be classified as anterior with disc edema (AION) or posterior without disc edema (PION). AION is typically divided into arteritic (A-AION) and non-arteritic (NA-AION) etiologies.
Recently, cases of optic neuropathy have been reported following infection with the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the virus that causes the Corona Virus Disease-19 (COVID19). Proposed mechanisms of how SARS-CoV-2 might cause ION (AION or PION) include inducing a severe inflammatory response, endothelial damage, hypercoagulable state, and hypoxemia, which leads to hypoperfusion and subsequent ischemia of the optic nerve.
Typical non-COVID19 related NA-AION is associated with risk factors: (1) structural factors which make the optic nerve head susceptible to ischemic events (e.g., small cup to disc ratio or “disc at risk”) and (2) vascular factors which predispose to acute hypoperfusion of the optic nerve head (e.g., diabetes mellitus, systemic hypertension, nocturnal arterial hypotension, ischemic heart disease, anemia). Non-arteritic posterior ischemic optic neuropathy (NA-PION) is thought to have similar vascular risk factors as NA-AION, but no structural risk has been found. Typical AION is the common presentation, while PION is rare.
Pathophysiology of COVID19-related ION
The coronavirus has been reported to cause activation of inflammatory cells (e.g. neutrophils and monocytes) and endothelial cells leading to high levels of circulating inflammatory cytokines (e.g., CRP, ferritin, IL-2, TNF-α) and excess production of pro-coagulants (e.g., tissue factor and von Willebrand factor). Extensive complement involvement and membrane attack complex-mediated microvascular endothelial cell injury have also been reported to lead to COVID19-associated coagulopathy, which can include venous, arterial, and microvascular thrombosis. COVID19 has also been reported to cause clinically significant hypoxemia.
In ION, it has been hypothesized that these factors in COVID19 (inflammatory response, hypercoagulable state, and hypoxemia) may lead to thrombosis of the blood vessels (e.g., ciliary vessels) supplying the optic nerve and subsequent ischemia of the optic nerve. However, there have been no studies to confirm this pathogenesis.
Savastano et al. reported the impact of SARS-CoV-2 infection on the microvascular network of optic nerve head in patients who recovered from COVID19. The study reported that in the patients who recovered from COVID19, there was an impairment in the blood supply to the peripapillary retinal nerve fiber layer, characterized by a reduction of radial peripapillary capillary plexus (RPCP) density. RPCP density has been previously correlated to visual acuity and visual field loss in NAION patients.
Case Reports of Presumed ION after COVID19 Infection
Vision loss OD; 5 weeks after COVID-19 symptoms and hospitalization
20/20 OD. RAPD OD. Inferior visual field loss OD. Pale optic disc with sectorial papillary edema OD
About 40% of patients with non-COVID19 related NAION will spontaneously recover some vision.
While there are no definite treatments for NAION, the underlying cause should be treated to prevent further complications. Risk factors for atherosclerosis should be controlled, including blood pressure and diabetes. Most of the recommended treatments are intended to prevent thrombosis (e.g., aspirin) or reduce the edema of the optic disc . While corticosteroids can lead to improvement in systemic symptoms and prevention of blindness in arteritic ION/giant cell arteritis (GCA), corticosteroids are not suggested for NAION. In the context of COVID19, the benefits of steroids have not been explored.
Optic neuropathy has been reported in COVID19 and the mechanisms remain ill defined although several hypotheses have been proposed including inflammatory cytokines and a transient hypercoagulable state. Many authors believe that the process is ION and both AION and PION have been reported with COVID19. Further work is necessary to confirm if the optic neuropathy is truly ischemic in origin and what potential treatments might be considered. In typical AION the major diagnostic dilemma is differentiating arteritic (i.e., giant cell arteritis) from non-arteritic AION (NAION). In the setting of COVID19 infection, the acute phase reactants (e.g., ESR, CRP, platelet count) might be elevated and mistaken for signs of GCA. Evaluation for A-AION and GCA in elderly patients including temporal artery biopsy might still be necessary however and some of the cases of AION and COVID19 in the literature may have been coincidental (GCA) and not causal. Clinicians should be aware of the possibility of ION in COVID19.
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↑ Savastano, A., Crincoli, E., Savastano, M. C., Younis, S., Gambini, G., De Vico, U., Cozzupoli, G. M., Culiersi, C., Rizzo, S., & Gemelli Against Covid-Post-Acute Care Study Group (2020). Peripapillary Retinal Vascular Involvement in Early Post-COVID-19 Patients. Journal of clinical medicine, 9(9), 2895. https://doi.org/10.3390/jcm9092895
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Authors: Jossy, Ajax; Jacob, Ninan; Sarkar, Sandip; Gokhale, Tanmay; Kaliaperumal, Subashini; Deb, Amit K IJO Ophthalmology
Neuroophthalmic manifestations are very rare in corona virus disease-19 (COVID-19) infection. Only few reports have been published till date describing COVID-19-associated neuroophthalmic manifestations. We, hereby, present a series of three cases who developed optic neuritis during the recovery period from COVID-19 infection. Among the three patients, demyelinating lesions were identified in two cases, while another case was associated with serum antibodies against myelin oligodendrocyte glycoprotein. All three patients received intravenous methylprednisolone followed by oral steroids according to the Optic Neuritis Treatment Trail ptotocol. Vision recovery was noted in all three patients, which was maintained at 2 months of the last follow up visit.
COVID-19 infection predo minantly causes a respiratory illness, but it can have a myriad of symptoms, affecting almost all organs of the body. Varied ocular manifestations including conjunctivitis, episcleritis, vascular occlusions, dacryoadenitis, mucormycosis, etc., have been reported in COVID-19 infection. Neuroophthalmic manifestations in COVID-19 infection are uncommon, but they can seldom develop either during the active course or the recovery period. Neuroophthalmic manifestations of COVID-19 infection includes optic neuritis, acute transverse myelitis, viral encephalitis, toxic encephalopathy, leukoencephalopathy, acute disseminated encephalomyelitis, diffuse corticospinal tract signs, etc. Only a handful reports of optic neuritis associated with COVID-19 infection with or without demyelinating lesions have been published. Few of them are associated with serum antibodies against myelin oligodendrocyte glycoprotein (MOG). In this report, we describe the clinical profile and treatment outcome of three patients who developed optic neuritis during recovery from COVID-19 infection.
A 16-year-old boy presented with sudden gross diminution of vision in the left eye (LE) for 3 days with headache and eyepain on extraocular movements. His past history was unremarkable. The patient had tested positive for COVID-19 infection with reverse transcription polymerase chain reaction (RT-PCR) 2 weeks prior to the incident. He was advised home isolation without any supplemental oxygen or steroids. Systemic and neurological examinations were unremarkable. On ocular examination, best-corrected visual acuity (BCVA) was 20/20 in the right eye (RE) and perception of light (PL+) in the LE, with a grade 2 relative afferent pupillary defect in the LE. Fundus examination revealed normal optic discs in both eyes with no evidence of disc edema or hyperemia [Fig. 1a and 1b]. A diagnosis of LE retrobulbar neuritis was made. Laboratory investigations, imaging, treatment received, and disease course are provided in Table 1.
A 35-year-old male presented with sudden vision loss in LE with pain on extraocular movements for 1 week. His past history was unremarkable. He was tested positive for COVID-19 infection with RT-PCR 6 months prior to the vision loss. He was advised home isolation and did not require oxygen or steroids for COVID-19. On ocular examination, BCVA was 20/20 in RE and 20/600 in LE, with grade I RAPD in LE. Fundus examination of the LE revealed edematous disc with blurred margins and peripapillary edema, which was confirmed on optical coherence tomography, while the RE fundus was normal [Fig. 2a and 2b]. A diagnosis of LE papillitis was made. Laboratory investigations, imaging, treatment, and disease course are described in Table 1.
A 38-year-old male presented with sudden gross diminution of vision and pain on extraocular movements in the LE for 5 days. The patient had a similar complaint in the LE 1 month ago. He was treated elsewhere for the same with intravenous methylprednisolone and oral prednisolone. There was symptomatic improvement in the vision within a week following the initiation of treatment. However, he noticed another similar episode of decreased vision in the LE 3 weeks later, when he presented to us. He was tested positive for COVID-19 infection with RT-PCR one-and-half month prior to the current episode. He was advised home isolation, and he also did not require oxygen or steroids for COVID-19 infection. Systemic examination was unremarkable. On ocular examination, BCVA was RE 20/20 and LE hand movements (HM+), with grade III RAPD in the LE. Fundus examination showed normal discs in both eyes [Fig. 3a and 3b]. A diagnosis of LE retrobulbar neuritis was made. Laboratory investigations, imaging findings, treatment, and disease course are described in Table 1.
Optic neuritis is an inflammatory demyelinating optic neuropathy causing acute uniocular or binocular loss of vision. Optic neuritis is mainly a clinical diagnosis based on history and examination findings. Investigations like magnetic resonance imaging, lumbar puncture, and antibodies against AQP4 and MOG help in finding the association and cause of vision loss. Once the diagnosis is established, treatment is done based on optic neuritis treatment trial (ONTT) protocol.
Neurotropism of the virus was postulated as one of the mechanisms for neuroophthalmic manifestations. Another mechanism involves molecular mimicry where the viral antigens trigger host immune response directed toward the CNS myelin proteins. All the three cases reported by us had viral prodromes and positive COVID-19 infection. It is interesting to note that all three cases had mild COVID-19 infections with no oxygen requirement or steroid use, and their recoveries were uneventful. Vision loss in all the three cases happened during the recovery period of the infections and dramatic response to steroids points toward an inflammatory disorder triggered by the viral antigen. In the third case, the patient had two similar episodes of vision loss in 2 months after the COVID-19 infection. He was tested positive for MOG antibody. MOG antibody-associated optic neuritis usually has good visual recovery with good response to steroids but shows bilaterality and recurrence. Our case also showed initial good response to systemic steroids with recurrence within 2 weeks of discontinuation of steroids. MOG antibody-associated optic neuritis in COVID-19 infection has been reported by Zhou et al., Zoric et al., Kugure et al., Sawalha et al., de Ruijter et al., Rojas-Correa et al.. Table 2 describes the details of all cases of COVID-19-associated optic neuritis. Due to the ongoing COVID-19 pandemic, we can expect more similar cases in future. So, prospective studies are warranted to establish the relationship between the viral antigen, severity of COVID-19 infection, and associated optic neuritis.
Neuro-ophthalmic manifestations are rare in COVID-19 infection, and can be seen either during the active disease phase or the recovery phase. Optic neuritis is one such rare manifestation. The three cases of optic neuritis being reported by us had mild COVID-19 infection. Two cases developed ocular symptoms and signs within the first six weeks of recovery while another case developed ocular manifestations six months after recovery from COVID-19. All the three cases showed good response to systemic steroids with significant visual recovery. Keeping the ongoing pandemic in perspective, we should, therefore, be vigilant in identifying the neuro-ophthalmic features of COVID-19 infection to prevent irreversible vision loss.
Coronaviruses are common causes of usually mild to moderate upper respiratory tract illnesses like the common cold, with symptoms that may include runny nose, fever, sore throat, cough, or a general feeling of being ill. However, a new coronavirus called Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) emerged and spread to cause the COVID-19 pandemic.
COVID-19, which means Coronavirus disease 2019, is an infectious disease that can affect people of all ages in many ways. It is most dangerous when the virus spreads from the upper respiratory tract into the lungs to cause viral pneumonia and lung damage leading to Acute Respiratory Distress Syndrome (ARDS). When severe, this impairs the body’s ability to maintain critical levels of oxygen in the blood stream—which can cause multiple body systems to fail and can be fatal.
What do we know about the effects of SARS-CoV-2 and COVID-19 on the nervous system?
Much of the research to date has focused on the acute infection and saving lives. These strategies have included preventing infection with vaccines, treating COVID-19 symptoms with medicines or antibodies, and reducing complications in infected individuals.
Research shows the many neurological symptoms of COVID-19 are likely a result of the body’s widespread immune response to infection rather than the virus directly infecting the brain or nervous system. In some people, the SARS-CoV-2 infection causes an overreactive response of the immune system which can also damage body systems. Changes in the immune system have been seen in studies of the cerebrospinal fluid, which bathes the brain, in people who have been infected by SARS-CoV-2. This includes the presence of antibodies—proteins made by the immune system to fight the virus—that may also react with the nervous system. Although still under intense investigation, there is no evidence of widespread viral infection in the brain. Scientists are still learning how the virus affects the brain and other organs in the long-term. Research is just beginning to focus on the role of autoimmune reactions and other changes that cause the set of symptoms that some people experience after their initial recovery. It is unknown if injury to the nervous system or other body organs cause lingering effects that will resolve over time, or whether COVID-19 infection sets up a more persistent or even chronic disorder.
What are the immediate (acute) effects of SARS-CoV-2 and COVID-19 on the brain?
Most people infected with SARS-CoV-2 virus will have no or mild to moderate symptoms associated with the brain or nervous system. However, most individuals hospitalized due to the virus do have symptoms related to the brain or nervous system, most commonly including muscle aches, headaches, dizziness, and altered taste and smell. Some people with COVID-19 either initially have, or develop in the hospital, a dramatic state of confusion called delirium. Although rare, COVID-19 can cause seizures or major strokes. Muscular weakness, nerve injury, and pain syndromes are common in people who require intensive care during infections. There are also very rare reports of conditions that develop after SARS-CoV-2 infection, as they sometimes do with other types of infections. These disorders of inflammation in the nervous system include Guillain-Barré syndrome (which affects nerves), transverse myelitis (which affects the spinal cord), and acute necrotizing leukoencephalopathy (which affects the brain).
Bleeding in the brain, weakened blood vessels, and blood clots in acute infection
The SARS-CoV-2 virus attaches to a specific molecule (called a receptor) on the surface of cells in the body. This molecule is concentrated in the lung cells but is also present on certain cells that line blood vessels in the body. The infection causes some arteries and veins—including those in the brain—to become thin, weaken, and leak. Breaks in small blood vessels have caused bleeding in the brain (so-called microbleeds) in some people with COVID-19 infection. Studies in people who have died due to COVID-19 infection show leaky blood vessels in different areas of the brain that allow water and a host of other molecules as well as blood cells that are normally excluded from the brain to move from the blood stream into the brain. This leak, as well as the resulting inflammation around blood vessels, can cause multiple small areas of damage. COVID-19 also causes blood cells to clump and form clots in arteries and veins throughout the body. These blockages reduce or block the flow of blood, oxygen, and nutrients that cells need to function and can lead to a stroke or heart attack.
A stroke is a sudden interruption of continuous blood flow to the brain. A stroke occurs either when a blood vessel in the brain becomes blocked or narrowed or when a blood vessel bursts and spills blood into the brain. Strokes can damage brain cells and cause permanent disability. The blood clots and vascular (relating to the veins, capillaries, and arteries in the body) damage from COVID-19 can cause strokes even in young healthy adults who do not have the common risk factors for stroke.
COVID-19 can cause blood clots in other parts of the body, too. A blood clot in or near the heart can cause a heart attack. A heart attack orInflammation in the heart, called myocarditis, can causeheart failure, and reduce the flow of blood to other parts of the body. A blood clot in the lungs can impair breathing and cause pain. Blood clots also can damage the kidneys and other organs.
Low levels of oxygen in the body (called hypoxia) can permanently damage the brain and other vital organs in the body. Some hospitalized individuals require artificial ventilation on respirators. To avoid chest movements that oppose use of the ventilator it may be necessary to temporarily “paralyze” the person and use anesthetic drugs to put the individual to sleep. Some individuals with severe hypoxia require artificial means of bringing oxygen into their blood stream, a technique called extra corporeal membrane oxygenation (ECMO). Hypoxia combined with these intensive care unit measure generally cause cognitive disorders that show slow recovery.
Diagnostic imaging of some people who have had COVID-19 show changes in the brain’s white matter that contains the long nerve fibers, or “wires,” over which information flows from one brain region to another. These changes may be due to a lack of oxygen in the brain, the inflammatory immune system response to the virus, injury to blood vessels, or leaky blood vessels. This “diffuse white matter disease” might contribute to cognitive difficulties in people with COVID-19. Diffuse white matter disease is not uncommon in individuals requiring intensive hospital care but it not clear if it also occurs in those with mild to moderate severity of COVID-19 illness.
Fortunately, people who have mild to moderate symptoms typically recover in a few days or weeks. However, some people who have had only mild or moderate symptoms of COVID-19 continue to experience dysfunction of body systems—particularly in the lungs but also possibly affecting the liver, kidneys, heart, skin, and brain and nervous system—months after their infection. In rare cases, some individuals may develop new symptoms (called sequelae) that stem from but were not present at the time of initial infection. People who require intensive care for Acute Respiratory Distress Syndrome, regardless of the cause, usually have a long period of recovery. Individuals with long-term effects, whether following mild or more severe COVID-19, have in some cases self-identified as having “long COVID” or “long haul COVID.” These long-term symptoms are included in the scientific term, Post Acute Sequelae of SARS-CoV-2 Infection (PASC).
What are possible long-term neurological complications of COVID-19?
Researchers are following some known acute effects of the virus to determine their relationship to the post-acute complications of COVID-19 infection. These post-acute effects usually include fatigue in combination with a series of other symptoms. These may include trouble with concentration and memory, sleep disorders, fluctuating heart rate and alternating sense of feeling hot or cold, cough, shortness of breath, problems with sleep, inability to exercise to previous normal levels, feeling sick for a day or two after exercising (post-exertional malaise), and pain in muscle, joints, and chest. It is not yet known how the infection leads to these persistent symptoms and why in some individuals and not others.
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Nerve damage, including peripheral neuropathy
Fatigue and post-exertional malaise
Cognitive impairment/altered mental state
Muscle, joint, and chest pain
Prolonged/lingering loss of smell (anosmia) or taste
Persistent fevers and chills
Prolonged respiratory effects and lung damage
Anxiety, depression, and stress post-COVID
How do the long-term effects of SARS-CoV-2 infection/COVID-19 relate to Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS)?
Some of the symptom clusters reported by people still suffering months after their COVID-19 infection overlap with symptoms described by individuals with myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS). People with a diagnosis of ME/CFS have wide-ranging and debilitating effects including fatigue, PEM, unrefreshing sleep, cognitive difficulties, postural orthostatic tachycardia, and joint and muscle pain. Unfortunately, many people with ME/CFS do not return to pre-disease levels of activity. The cause of ME/CFS is unknown but many people report its onset after an infectious-like illness. Rest, conserving energy, and pacing activities are important to feeling better but don’t cure the disease. Although the long-term symptoms of COVID-19 may share features with it, ME/CFS is defined by symptom-based criteria and there are no tests that confirm an ME/CFS diagnosis.
ME/CFS is not diagnosed until the key features, especially severe fatigue, post-exertional malaise, and unrefreshing sleep, are present for greater than six months. It is now becoming more apparent that following infection with SARS-CoV-2/COVID-19, some individuals may continue to exhibit these symptoms beyond six months and qualify for an ME/CFS diagnosis. It is unknown how many people will develop ME/CFS after SARS-CoV-2 infection. It is possible that many individuals with ME/CFS, and other disorders impacting the nervous system, may benefit greatly if research on the long-term effects of COVID-19 uncovers the cause of debilitating symptoms including intense fatigue, problems with memory and concentration, and pain.
Am I at a higher risk if I currently have a neurological disorder?
Much is still unknown about the coronavirus but people having one of several underlying medical conditions may have an increased risk of illness. However, not everyone with an underlying condition will be at risk of developing severe illness. People who have a neurological disorder may want to discuss their concerns with their doctors.
Because COVID-19 is a new virus, there is little information on the risk of getting the infection in people who have a neurological disorder. People with any of these conditions might be at increased risk of severe illness from COVID-19:
High blood pressure
There is evidence that COVID-19 seems to disproportionately affect some racial and ethnic populations, perhaps because of higher rates of pre-existing conditions such as heart disease, diabetes, and lung disease. Social determinants of health (such as access to health care, poverty, education, ability to remain socially distant, and where people live and work) also contribute to increased health risk and outcomes.
Can COVID-19 cause other neurological disorders?
In some people, response to the coronavirus has been shown to increase the risk of stroke, dementia, muscle and nerve damage, encephalitis, and vascular disorders. Some researchers think the unbalanced immune system caused by reacting to the coronavirus may lead to autoimmune diseases, but it’s too early to tell.
Anecdotal reports of other diseases and conditions that may be triggered by the immune system response to COVID-19 include para-infectious conditions that occur within days to a few weeks after infection:
Multi-system infammatory syndrome – which causes inflammation in the body’s blood vessels
Transverse myelitis – an inflammation of the spinal cord
Guillain-Barré sydrome (sometimes known as acute polyradiculoneuritis) – a rare neurological disorder which can range from brief weakness to nearly devastating paralysis, leaving the person unable to breathe independently
Dysautonomia – dysfunction of the autonomic nerve system, which is involved with functions such a breathing, heart rate, and temperature control
Acute disseminating encephalomyelitis (ADEM) – an attack on the protective myelin covering of nerve fibers in the brain and spinal cord
Acute necrotizing hemorrhagic encephalopathy – a rare type of brain disease that causes lesions in certain parts of the brain and bleeding (hemorrhage) that can cause tissue death (necrosis)
Facial nerve palsies (lack of function of a facial nerve) such as Bell’s Palsy
Parkinson’s disease-like symptoms have been reported in a few individuals who had no family history or early signs of the disease
Does the COVID-19 vaccine cause neurological problems?
Almost everyone should get the COVID-19 vaccination. It will help protect you from getting COVID-19. The vaccines are safe and effective and cannot give you the disease. Most side effects of the vaccine may feel like flu and are temporary and go away within a day or two. The U.S. Food and Drug Administration (FDA) continues to investigate any report of adverse consequences of the vaccine. Consult your primary care doctor or specialist if you have concerns regarding any pre-existing known allergic or other severe reactions and vaccine safety.
A recent study from the United Kingdom demonstrated an increase in Guillain-Barré Syndrome related to the Astra Zeneca COVID-19 vaccine (virally delivered) but not the Moderna (messenger RNA vaccine). Guillain-Barré syndrome (a rare neurological disorder in which the body’s immune system damages nerve cells, causing muscle weakness and sometimes paralysis) has also occurred in some people who have received the Janssen COVID-19 Vaccine (also virally delivered). In most of these people, symptoms began within weeks following receipt of the vaccine. The chance of having this occur after these vaccines is very low, 5 per million vaccinated persons in the UK study. The chance of developing Guillain-Barré Syndrome was much higher if one develops COVID-19 infection (i.e., has a positive COVID test) than after receiving the Astra Zeneca vaccine. The general sense is that there are COVID-19 vaccines that are safe in individuals whose Guillain-Barré syndrome was not associated with a previous vaccination and that actual infection is the greater risk for developing Guillain-Barré Syndrome.
There have been reports of neurological complications from other SARS-CoV-2 vaccinations. Visit the FDA COVID-19 Vaccines webpage for information about coronavirus vaccines and fact sheets for recipients and caregivers that outline possible neurological and other risks.
COVID-19 pandemic revealed several neurological syndromes related to this infection. We describe the clinical, laboratory, and radiological features of eight patients with COVID-19 who developed peripheral facial palsy during infection. In three patients, facial palsy was the first symptom. Nerve damage resulted in mild dysfunction in five patients and moderate in three. SARS-Cov-2 was not detected in CSF by PCR in any of the samples. Seven out of eight patients were treated with steroids and all patients have complete or partial recovery of the symptoms. Peripheral facial palsy should be added to the spectrum of neurological manifestations associated with COVID-19.
The ongoing COVID-19 pandemic has affected millions of people worldwide and revealed several neurological syndromes related to this infection. Anosmia/ageusia, encephalitis, encephalopathy, cerebrovascular complications, myelitis, and Guillain-Barré syndrome, among other neurological complications, occur in a significant proportion of patients (Ellul et al. 2020; Paterson et al. 2020).
Acute facial nerve palsy commonly occurs in clinical practice and is associated with considerable distress due to possible functional and esthetic sequelae (Jowett 2018). There are many potential mechanisms implicated in its occurrence, including viral infections. Herein, we review the clinical and laboratory features of eight patients with COVID-19 who developed peripheral facial palsy during the clinical course of the infection or as its first symptom.
Case series of eight patients seen from May to July 2020 with a diagnosis of COVID-19 based on positive SARS-CoV-2 RNA RT-qPCR in nasal and oropharyngeal swabs (Biomanguinhos kit (E+P1), FIOCRUZ, Brazil).
Data about the onset of facial palsy, associated clinical conditions, brain imaging, cerebrospinal fluid parameters, treatment, and outcome were recorded. Facial palsy was graded according to the House-Brackmann scale (House and Brackmann 1985). This study was approved by the Local Ethical Committee at INI/FIOCRUZ.
Among the eight patients, seven were women. All had COVID-19 diagnosis based on positive SARS-CoV-2 RNA RT-qPCR in nasal and oropharyngeal swabs. The mean age was 36 years (range 25–50 years). In three patients, facial palsy was the first symptom of COVID-19, while in the remaining five, it appeared from 2 to 10 days after onset of other clinical manifestations. All patients had mild respiratory and systemic COVID-19 symptoms, and none required hospitalization. According to the House-Brackmann grading system, nerve damage resulted in mild (grade 2) dysfunction in five patients and moderate (grade 3) in three (Table 1). The neurological examination disclosed no abnormalities in all but one patient, who had an associated ipsilateral abducent nerve palsy. Deep tendon reflexes were preserved, and no sensory abnormalities were present. Six patients underwent lumbar puncture with normal opening pressure in all cases. CSF analysis showed no inflammatory changes except for a mild protein elevation in one patient (50 mg/dl) (Table 1). SARS-Cov-2 was not detected in CSF by PCR in any of the samples. Imaging (CT scan or MRI) was normal in seven patients. In one patient, MRI showed contrast enhancement in the distal intracanalicular portion in the tympanic and mastoid segments of the left facial nerve (Fig. 1).Table 1 Clinical and laboratory manifestations of COVID-19 patients with facial palsy
Six out of seven patients were treated with oral steroids (prednisone 40–60 mg/day for 5–7 days) and one received intravenous methylprednisolone. One patient with mild manifestations received only supportive care (eye lubricant) with complete recovery 2 days later. Two patients received oral acyclovir concomitant to steroids due to possible Herpes simplex virus infection. Complete recovery occurred in five patients, while the other three still had some degree of facial weakness at the last follow-up 30 days after onset of neurological symptoms.
Infections such as HSV-1, VZV, and Lyme disease are common causes of facial paralysis (Owusu et al. 2018). The rapid expansion of COVID-19 pandemics led to the development of a growing number of neurological syndromes. Our study shows that peripheral facial palsy can occur during the clinical course of COVID-19 or anticipate other typical manifestations such as fever and respiratory symptoms.
Interestingly, all but one of our patients were women. Idiopathic facial palsy does not have a gender preference (Katusic et al. 1986). Indeed, our sample is too small to assume any conclusion, and the two other cases of isolated facial palsy in association with COVID-19 described by Goh and Casas were men (Casas et al. 2020; Goh et al. 2020).
Most patients in this study had isolated facial palsy with mild or moderate dysfunction and no other neurological findings. Except for the two described above by Goh and Casas (Casas et al. 2020; Goh et al. 2020), in all other studies, facial paralysis in COVID-19 patients occurred unilaterally or bilaterally in association with other manifestations of Guillain-Barré syndrome (Manganotti et al. 2020; Ottaviani et al. 2020; Juliao Caamaño and Alonso Beato 2020; Paybast et al. 2020; Sancho-Saldaña et al. 2020; Bigaut et al. 2020).
CSF basic parameters (cellularity, protein, and glucose levels) are usually normal in patients with idiopathic facial paralysis as observed in our series (Bremell and Hagberg 2011). SARS-CoV2 was not detected in any five cases who underwent lumbar puncture, which is consistent with a recent study that failed to show viral RNA in the CSF of COVID-19 patients with different neurological syndromes (Espíndola et al. 2020).
Possible mechanisms related to nerve damage in idiopathic facial nerve paralysis include ischemia of vasa nervorum and demyelination induced by an inflammatory process (Zhang et al. 2020). Microthrombi and other vascular changes have been consistently reported in several postmortem studies (Silberzahn et al. 1988; Nunes Duarte-Neto et al. 2020) and may be implicated in the development of facial nerve ischemia in COVID-19 patients. Direct viral damage or an autoimmune reaction toward the nerve producing inflammation would be alternative or contributing mechanisms to dysfunction.
Supportive care and oral steroids are the mainstays of treatment (Sullivan et al. 2007). Our patients had complete recovery or significant improvement in few weeks after treatment as the patient reported by Casas et al. (2020), suggesting a good outcome when peripheral facial palsy occurs in association with COVID-19.
In conclusion, peripheral facial palsy should be added to the spectrum of neurological manifestations associated with COVID-19. Most patients had an uncomplicated course with good outcome, and SARS-CoV-2 RNA could not be detected in CSF of any patient.
Manganotti P, Bellavita G, D’Acunto L et al (2020) Clinical neurophysiology and cerebrospinal liquor analysis to detect Guillain Barré syndrome and polyneuritis cranialis in COVID-19 patients: a case series. J Med Virol. https://doi.org/10.1002/jmv.26289
Acute mild respiratory SARS-CoV-2 infection can lead to a more chronic cognitive syndrome known as “COVID fog.” New findings from Fernández-Castañeda et al. reveal how glial dysregulation and consequent neural circuit dysfunction may contribute to cognitive impairments in long COVID.
More than 2 years since the first detected cases of COVID-19, the virus continues to evolve new variants, infect hundreds of millions of people, and pose both acute and chronic threats to global health. In most infections, a patient’s symptoms resolve within 2 weeks. However, recovery from initial infection is not always straightforward. In a significant fraction of patients, symptoms can persist for several months. This syndrome, which has become known as “long COVID,” may occur even following initially mild cases (that is, those not requiring hospitalization) and can include significant cognitive dysfunction.
Initial awareness of long COVID emerged from anecdotal accounts, shared by patients on social media platforms such as Twitter, Facebook, and Slack (Callard and Perego, 2021). Since then, a large number of studies have used more formal methods to catalog the nature and frequency of long COVID symptoms. Within the cognitive domain, as many as one in four patients experience a range of symptoms that have become known colloquially as “COVID fog,” which includes problems in attention, language fluency, processing speed, executive function, and memory (Becker et al., 2021).
Given the sheer scale of infections, there is a pressing need to understand how cognitive dysfunction emerges in long COVID. In this issue of Cell, a new paper by Fernández-Castañeda and colleagues (Fernández-Castañeda et al., 2022, Geraghty et al., 2019) begins to address this need. Using mice as a model, they expressed human ACE2, the viral entry receptor required for successful COVID infection. Because ACE2 expression was restricted to the trachea and lungs, these mice developed only a mild form of the disease that was limited to the respiratory system and largely cleared within a week following intranasal inoculation with SARS-CoV-2 virus.
As a starting point, the researchers noted the striking similarities between COVID fog and another cognitive syndrome known as “chemobrain.” Chemobrain (or cancer-therapy-related cognitive impairment, CRCI), is a neuroinflammatory condition that patients often experience following radiation or chemotherapy. In CRCI, elevations in neurotoxic cytokines and reactive microglia (brain-resident macrophages) lead to cascades of multicellular events that impact forms of both gray and white matter plasticity that are important for healthy cognition (Gibson and Monje, 2021).
Adopting this framework, the authors quantified changes in cytokines and microglial/macrophage reactivity following SARS-CoV-2 infection. Reminiscent of CRCI, they found that microglial/macrophage reactivity was elevated in subcortical and hippocampal white matter in mice following mild respiratory COVID. Remarkably, this elevation was persistent and still evident even 7 weeks post infection. Outside of the brain, they detected elevated cytokine levels in the cerebrospinal fluid and serum of mice. Although levels of many cytokines were altered, one in particular grabbed their attention. CCL11 remained persistently elevated in mouse cerebrospinal fluid 7 weeks post infection. Notably, elevated CCL11 levels have been causally linked to cognitive impairments observed in normal aging (Villeda et al., 2011).
Similar patterns were found in patients with COVID-19 (Figure 1 ). Reactive microglia were elevated in subcortical white matter in individuals with COVID-19 (these patients were symptomatic for COVID-19 but died from other causes). Moreover, CCL11 was elevated in plasma from patients with long COVID. Remarkably, elevated CCL11 plasma levels were only detected in those long COVID patients with cognitive symptoms. CCL11 plasma levels were unaltered in long COVID patients who did not have cognitive symptoms. These findings suggest that CCL11 plays a central role in the brain pathology contributing to COVID fog.
Acute mild respiratory COVID-19 infection can lead to a more chronic cognitive syndrome known as brain fog. Suggested pathways for brain fog include cytokine-induced activation of regional microglia, causing decreased hippocampal neurogenesis and a loss of myelinated subcortical axons.
There are several ways in which such a neuroinflammatory state might impact cognition. Previous work has shown that persistently reactive microglia in hippocampal white matter suppress hippocampal neurogenesis by blocking neuronal progenitor-cell differentiation into new granule cells (Monje et al., 2003). These newly generated neurons are important for the formation and stability of hippocampus-dependent memories. The authors found the numbers of new neurons were reduced in infected mice, and this reduction correlated with numbers of reactive microglia in the hippocampus. Remarkably, systemic administration of CCL11 recapitulated this same pattern in uninfected mice. Increased numbers of reactive microglia and decreased numbers of new neurons in these mice suggest a causal role for CCL11 in COVID fog pathology. These CCL11-induced outcomes were restricted to the hippocampus, suggesting that particular cytokines can have circuit-specific effects.
Another way in which CCL11 and/or reactive microglia might impact cognition is via effects on white matter. In CRCI, reactive microglia impair the formation of myelin-forming oligodendrocytes and myelin plasticity (Geraghty et al., 2019; Gibson et al., 2019). Similarly, the authors found reduced numbers of oligodendrocytes and their precursor cells, as well as decreased axon myelination in the subcortical white matter, several weeks following infection in mice. Although behavior was not assessed in this study, it is nonetheless reasonable to assume that such changes would alter cognitive function following mild respiratory COVID in mice, as it does in other disease contexts (Geraghty et al., 2019; Gibson et al., 2019). Since myelination regulates the speed and timing of communication between neurons, dysregulation of oligodendoglial lineage cells following infection might slow neural processing, disrupt brain synchrony, and impair cognition (Steadman et al., 2020).
The principle that inflammatory challenges may induce glial dysregulation and consequent neural circuit dysfunction is not specific to COVID-19. Many systemic infections are associated with lasting cognitive impairments. For instance, similar to COVID-19, the Spanish flu of 1918 (caused by H1N1 influenza virus infection) was associated with brain fog. In the current study, the authors compared the effects of mild respiratory SARS-CoV-2 infection with a mouse model of mild respiratory H1N1 influenza. Similar to SARS-CoV-2 infection, they found persistently elevated CCL11 in the H1N1 influenza infection. Therefore, CCL11-driven neuroinflammatory changes and cellular deficits may represent a common pathway to cognitive deficits in both mild respiratory COVID-19 and H1N1 influenza disease (as well as perhaps in other contexts, including aging).
However, different systemic infections can also have non-overlapping outcomes. For instance, whereas risk for anxiety and depression appears to be elevated in COVID-19, Spanish flu was associated with increased prevalence of psychosis (Honigsbaum, 2013). What accounts for these differences? One clue emerges from the current study. While CCL11 was elevated following SARS-CoV-2 and H1N1 influenza infection, a subset of cytokines was differentially regulated in these two conditions. It is likely that these non-overlapping cytokines (and their potentially non-overlapping circuit effects) might differentiate these diseases in terms of elevated risks for particular neuropsychiatric outcomes.
If cytokines have circuit-specific effects, should we consider cytokine profiling as a way to inform our treatment strategies for viral-induced/non-viral induced cognitive syndromes? Future research will no doubt address this and other outstanding questions. For instance, how are these effects mitigated by vaccination, administered before or after infection? And, perhaps most significantly, might interventions that block inducers of these cytokines or reset reactive microglia prove useful in treating COVID fog?
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The current report presents the case of a 76-year-old man with Parkinson’s disease (PD)who died three weeks after receiving his third COVID-19 vaccination. The patient was first vac-cinated in May 2021 with the ChAdOx1 nCov-19 vector vaccine, followed by two doses of theBNT162b2 mRNA vaccine in July and December 2021. The family of the deceased requested anautopsy due to ambiguous clinical signs before death. PD was confirmed by post-mortem exami-nations. Furthermore, signs of aspiration pneumonia and systemic arteriosclerosis were evident. However, histopathological analyses of the brain uncovered previously unsuspected findings, including acute vasculitis (predominantly lymphocytic) as well as multifocal necrotizing encephalitis of unknown etiology with pronounced inflammation including glial and lymphocytic reaction. In the heart, signs of chronic cardiomyopathy as well as mild acute lympho-histiocytic myocarditis and vasculitis were present. Although there was no history of COVID-19 for this patient, immunohistochemistry for SARS-CoV-2 antigens (spike and nucleocapsid proteins) was performed. Surprisingly, only spike protein but no nucleocapsid protein could be detected within the foci of inflammation in both the brain and the heart, particularly in the endothelial cells of small blood vessels. Since no nucleocapsid protein could be detected, the presence of spike protein must be ascribed to vaccination rather than to viral infection. The findings corroborate previous reports of encephalitis and myocarditis caused by gene-based COVID-19 vaccines.
The emergence of the severe acute respiratory syndrome coronavirus 2(SARS-CoV-2) in 2019 with the subsequent worldwide spread of COVID-19 gave rise to a perceived need for halting the progress of the COVID-19 pandemic through the rapid development and deployment of vaccines. Recent advances in genomics facilitated gene-based strategies for creating these novel vaccines, including DNA-based nonreplicating viral vectors, and mRNA-based vaccines, which were furthermore developed on an aggressively shortened timeline [1–4].The WHO Emergency Use Listing Procedure (EUL), which determines the acceptability of medicinal products based on evidence of quality, safety, efficacy, and performance, permitted these vaccines to be marketed as soon as 1–2 years after development had begun. Published results of the phase 3 clinical trials described only a few severe side effects [2,6–8]. However, it has since become clear that severe and even fatal adverse events may occur; these include in particular cardiovascular and neurological manifestations [9–13]. Clinicians should take note of such case reports for the sake of early detection and management of such adverse events among their patients. In addition , a thorough post-mortem examination of deaths in connection with COVID-19 vaccination should be considered in ambiguous circumstances, including histology. This report presents the case of a senior aged 76 years old, who had received three doses overall of two different COVID-19 vaccines, and who died three weeks after the second dose of the mRNA-BNT162b-vaccine. Autopsy and histology revealed unexpected necrotizing encephalitis and mild myocarditis with pathological changes in small blood vessels. A causal connection of these findings to the preceding COVID-19 vaccination was established by immunohistochemical demonstration of SARS-CoV-2 spike protein. The methodology introduced in this study should be useful for distinguishing between causation by COVID-19 vaccination or infection in ambiguous cases.
2. Materials and Methods
2.1. Routine Histology
Formalin-fixed tissues were routinely processsed and paraffin-embedded tissueswere cut into 5μm sections and stained with hematoxylin and eosin (H&E) for histo-pathological examination.
Immunohistochemical staining was performed on the heart and brain, using a fullyautomated immunostaining system (Ventana Benchmark, Roche). An antigen retrieval(Ultra CC1, Roche Ventana) was used for every antibody. The target antigens and dilution factors for the antibodies used are summarized in Table 1. Incubation with the primary antibody was carried out for 30 min in each case. Tissues fromSARS-CoV-2-positive COVID-19 patients were used as a control for the antibodies against SARS-CoV-2-spike and nucleocapsid (Figure 1). Cultured cells that had been transfected in vitro (see hereafter) served as a positive control for the detection of vaccine-induced spike protein expression and as a negative control for the detection of nucleocapsid protein. The slides were examined with a light microscope (Nikon ECLIPSE80i) and representative images were captured by the camera system Motic MP3.
Primary antibodies used for immunohistochemistry. Tissue sections were incubated 30min with the antibody in question, diluted as stated in the table.
Target Antigen Manufacturer Clone Dilution Incubation Time
CD3 (expressed by T-Lymphocytes) cytomed ZM-45 1:200 30 minCD68 (expressed by monocytic cells) DAKO PG-M1 1:100 30 minSARS-CoV-2-Spike subunit 1 ProSci 9083 1:500 30 minSARS-CoV-2-Nucleocapsid ProSci 35–720 1:500 30 min
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Nasal smear from a person with acute symptomatic SARS-CoV-2-infection (confirmed byPCR). Note the presence of ciliated epithelium. Immunohistochemistry for two SARS-CoV-2antigens (spike and nucleocapsid protein) revealed a positive reaction for both as to be expectedafter infection. (a) Detection of the spike protein. Positive control for spike subunit 1 SARS-CoV-2protein detection. Several ciliated epithelia of the nasal mucosa show brownish granular deposits of DAB (red arrow). Compared to nucleocapsid, the DAB-granules are fewer and less densely packed granular deposits of DAB. (b) Detection of nucleocapsid protein. Positive control for nucleocapsid SARS-CoV-2 protein detection. Several ciliated epithelia of the nasal mucosa show dense brownish granular deposits of DAB in immunohistochemistry (examples red arrows).Compared to spike detection, the granules of DAB are finer and more densely packed.Magnification: 400x.
2.3. Preparation of Positive Control Samples for the Immunohistochemical Detection of theVaccine-Induced Spike Protein
Cell culture and transfection: Ovarian cancer cell lines (OVCAR-3 and SK-OV3, CSL cell Lines Service, Heidelberg, Germany) were grown to 70% confluence in flat bottom 75cm2
cell culture flasks (Cell star) in DMEM/HAMS-F12 medium supplemented with Glutamax (Sigma-Aldrich, St. Louis, MO, USA), 10% FCS (Gibco, Shanghai, China) and Gentamycin (final concentration 20 μg/mL, Gibco), at 37 °C, 5% CO2
in a humidified cell incubator. For transfection, the medium was completely removed, and cells were incubated for 1 h with 2 mL of fresh medium containing the injection solutions directly from the original bottles, diluted 1:500 in the case of BNT162b2 (Pfizer/Biotech), and 1:100in cases of mRNA-1273 (Moderna), Vaxzevria (AstraZeneca), and Jansen (COVID-19vaccine Jansen). Then, another 15 mL of fresh medium was added to the cell cultures and cells were grown to confluence for another 3 days. Preparation of tissue blocks from transfected cells: The cell culture medium was removed from transfected cells, and the monolayer was washed twice with PBS, then trypsinized by adding 1 mL of 0.25% Trypsin-EDTA (Gibco), harvested with 10 mL of PBS/10% FCS, and washed 2× with PBS and centrifugation at 280×g for 10 min each. Cell pellets were fixed overnight in 2 mL in PBS/4% Formalin at 8 °C and then washed in PBmm Sonce. The cell pellets remaining after centrifugation were suspended in 200 μ
L PBS each,mixed with 400
L 2% agarose in PBS solution (precooled to around 40 °C), and immediately transferred to small (1 cm) dishes for fixation. The fixed and agarose-embedded cell pellets were stored in 4% Formalin/PBS till subjection to routine paraffin embedding in parallel to tissue samples.
2.4. Case Presentation and Description
2.4.1. Clinical History This report presents the case of a 76-year-old male with a history of Parkinson’s disease (PD) who passed away three weeks after his third COVID-19 vaccination. On the day of his first vaccination in May 2021 (ChAdOx1 nCov-19 vector vaccine), he experienced pronounced cardiovascular side effects, for which he repeatedly had to consult his doctor. After the second vaccination in July 2021 (BNT162b2 mRNA vaccine/Comirnaty), the family noted obvious behavioral and psychological changes(e.g., he did not want to be touched anymore and experienced increased anxiety, lethargy, and social withdrawal even from close family members). Furthermore, therewas a striking worsening of his PD symptoms, which led to severe motor impairment and a recurrent need for wheelchair support. He never fully recovered from these side effects after the first two vaccinations but still got another vaccination in December 2021.Two weeks after the third vaccination (second vaccination with BNT162b2), he suddenly collapsed while taking his dinner. Remarkably, he did not show coughing or any signs of food aspiration but just fell down silently. He recovered from this more or less, but one week later, he again suddenly collapsed silently while taking his meal. The emergency unit was called, and after successful, but prolonged resuscitation attempts
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(over one hour), he was transferred to the hospital and directly put into an artificial co-ma but died shortly thereafter. The clinical diagnosis was death due to aspirationpneumonia. According to his family, there was no history of a clinical or laboratorydiagnosis of COVID-19 in the past.2.4.2. AutopsyThe autopsy was requested and consented to by the family of the patient because ofthe ambiguity of symptoms before his death. The autopsy was performed according tostandard procedures including macroscopic and microscopic investigation. Gross braintissue was prepared for histological examination including the brain (frontal cortex,Substantia nigra, and Nucleus ruber) as well as the heart (left and right ventricular car-diac tissue).
3.1. Autopsy Findings
Anatomical Specifications: Body weight, height, and specifications of body organswere summarized in Table 2
Item Measure Body weight 60 kg Hight 175 cmHeart weight 410 g Brain weight 1560 gL iver weight 1500
Brain: A macroscopic examination of brain tissue revealed a circumscribed seg-mental cerebral parenchymal necrosis at the site of the right hippocampus. Substantianigra showed a loss of pigmented neurons. Microscopically, several areas with lacunarnecrosis were detected with inflammatory debris reaction on the left frontal side (Figure2). Staining of Nucleus ruber with H&E showed neuronal cell death, microglia, and lymphocyte infiltration (Figure 3). Furthermore, there were microglial and lymphocytic reactions as well as predominantly lymphocytic vasculitis, sometimes with mixed infiltrates including neutrophilic granulocytes (Figure 4) in the frontal cortex, para ventricu-lar, Substantia nigra, and Nucleus ruber on both sides. In some places with inflammatory changes in brain capillaries, there were also signs of apoptotic cell death within the endothelium (Figure 4). Meninges’ findings were unremarkable. The collective findings were suggestive of multifocal necrotizing encephalitis. Furthermore, chronic arterio-sclerotic lesions of varying degrees were noted in large brain vessels, which are described in detail in section “Vascular system”. Parkinson’s disease (PD): Macroscopic and histological examination of brain tissue revealed bilateral pallor of the substantia nigra with loss of pigmented neurons. In addition, pigment-storing macrophages as well as scattered neuronal necrosis with glial de bris reaction were noted. These findings were suggestive of PD, confirming the clinical diagnosis. Thoracic cavity: An examination of the chest showed a funnel-shaped chest with serial rib fractures (extending from the second to fifth ribs on the right, and from the second to sixth ribs on the left); which is a common picture of a patient who underwent cardiopulmonary resuscitation. An endotracheal tube was properly inserted. There was evidence of regular placement of a central venous catheter in the left femoral vein. There was evidence of regular placement of an arterial catheter in the left radial artery. The
urinary catheter was inserted as well. There was a 9 cm long skin scar on the front of theright shoulder.
Lungs: Macroscopical lung examination revealed cloudy secretion and purulentspots with notably brittle parenchyma. The pleura showed bilateral serous effusion,amounting to 450 mL of fluid on the right side and 400 mL on the left side. Bilateralmucopurulent tracheobronchitis was evident with copious purulent secretion in the tra-chea and bronchi. Bilateral chronic destructive pulmonary emphysema was detected.Bilateral bronchopneumonia was noted in the lower lung lobes at multiple stages of de-velopment and lobe-filling with secretions and fragile parenchyma. Furthermore, chronicarteriosclerotic lesions of varying degrees were noted, which are described in detail in thesection “Vascular system”.Heart: Macroscopic cardiac examination revealed manifestations of acute andchronic cardiovascular insufficiency, including ectasia of the atria and ventricles. Fur-thermore, left ventricular hypertrophy was noted (wall thickness: 18 mm, heart weight:410 g, body weight: 60 kg, height: 1.75 m). There was evidence of tissue congestion(presumably due to cardiac insufficiency) in the form of pulmonary edema, cerebraledema, brain congestion, chronic hepatic congestion, renal tissue edema, and pituitarytissue edema. Moreover, there was evidence of shock kidney disorder. Histological ex-amination of the heart revealed mild myocarditis with fine-spotted fibrosis and lym-pho-histiocytic infiltration (Figure 5). Furthermore, there were chronic arterioscleroticlesions of varying degrees, which are described in detail under “Vascular system”. Inaddition to these, there were more acute myocardial and vascular changes in the heart.They consisted of mild signs of myocarditis, characterized by infiltrations with foamyhistiocytes and lymphocytes as well as hypereosinophilia and some hypercontraction ofcardiomyocytes. Furthermore, mild acute vascular changes were observed in the capil-laries and other small blood vessels of the heart. They consisted of mild lym-pho-histiocytic infiltrates, prominent endothelial swelling and vacuolation, multifocalmyocytic degeneration and coagulation necrosis as well as karyopyknosis of single en-dothelial cells and vascular muscle cells (Figure 5). Occasionally, adhering plasma coag-ulates/fibrin clots were present on the endothelial surface, indicative of endothelialdamage (Figure 5).Vascular system (large blood vessels): The pulmonary arteries showed ectasia andlipidosis. The kidney showed slight diffuse glomerulosclerosis and arteriosclerosis withrenal cortical scars (up to 10 mm in diameter). The findings are suggestive of generalizedatherosclerosis and systemic hypertension. Major arteries including the aorta and its branches as well as the coronary arteries showed variable degrees of arteriosclerosis andmild to moderate stenosis. Furthermore, examination revealed mild nodular arterioscle-rosis of cervical arteries. Ascending aorta, aortic arch, and thoracic aorta showed mod-erate, nodular, and partially calcified arteriosclerosis. The cerebral basilar artery showedmild arteriosclerosis. Nodular and calcified arteriosclerosis were of high grade in theabdominal aorta and iliac arteries and moderate grade with moderate stenosis in theright coronary arteries. Coronary artery examination showed variable degrees of arteri-osclerosis and stenosis more on the left coronary arteries. The left anterior descendingcoronary artery (the anterior interventricular branch of the left coronary artery; LAD)showed high-grade and moderately stenosed arteriosclerosis. The arteriosclerosis andstenosis of the left circumflex artery (the circumflex branch of the left coronary artery)were mild.
Mild cerebral basal artery sclerosis. High-grade nodular and calcified arteriosclerosis of the abdominal aorta and the iliac arteries. Moderate stenosed arteriosclerosis of the right coronary artery. Lymphocytic periarteritis was detected as well
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Frontal brain. Already in the overview image (
a), prominent vacuolations with increased parenchymal cellularity are evident, indicative of degenerative and inflammatory processes. At higher magnification (b acute brain damage is visible with diffuse and zonal neuronal and glial cell death, activation of microglia, and inflammatory infiltration by granulocytes and lymphocytes.1: neuronal deaths (cells with red cytoplasm); 2: microglial proliferation; 3: lymphocytes. H&Estain. Magnification 40× ) and 200× (b).
Brain, Nucleus ruber. In the overview image (a0 note pronounced focal necrosis with increased cellularity, indicative of ongoing inflammation and glial reaction. At higher magnification (b, death of neuronal cells is evident and associated with an increased number of glial cells. Note activation of microglia and presence of inflammatory cell infiltrates, predominantly lymphocytic. 1: neuronal death with hypereosinophilia and destruction of cell nucleus with signs of karyolysis (nuclear content being distributed into the cytoplasm 2: microglia (example); 3:lymphocyte (example). H&E stain. Magnification 40× ( and 400× (b).
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Heart left ventricle. (
): Mild lympho-histiocytic myocarditis.Pronounced interstitialedema (7) and mild lympho-histiocytic infiltrates (2 + 4). Signs of cardiomyocytic degeneration (5)with cytoplasmic hypereosinophilia and single contraction bands. (
): Arteriole with signs of acutedegeneration and associated inflammation, associated by lymphocytic infiltrates (2) within thevascular wall, endothelial swelling and vacuolation (3), and vacuolation of vascular myocytes withsigns of karyopyknosis (1). Within the vascular lumen (
Colon: the elongated sigmoid colon was elongated with fecal impaction.-
Kidneys: slight diffuse glomerulosclerosis and arterio-sclerosis, renal corticalscars (up to 10 mm in diameter), bilateral mild active nephritis and urocystitis aswell as evidence of shock kidney disorder.-
Liver: slight lipofuscinosis.-
Spleen: mild acute splenitis.-
Stomach: mild diffuse gastric mucosal bleeding.-
Thyroid gland: bilateral nodular goiter with chocolate cysts (up to 0.5 cm indiameter).-
Prostate gland: benign nodular prostatic hyperplasia and chronic persistentprostatitis.
3.3. Immunohistochemical Analyses
Immunohistochemical staining for the presence of SARS-CoV-2 antigens (spike protein and nucleocapsid) was studied in the brain and heart. In the brain, SARS-CoV-2spike protein subunit 1 was detected in the endothelia, microglia, and astrocytes in thenecrotic areas (Figures 6 and 7). Furthermore, spike protein could be demonstrated in theareas of lymphocytic periarteritis, present in the thoracic and abdominal aorta and iliac branches, as well as a cerebral basal artery (Figure 8). The SARS-CoV-2 subunit 1 was found in macrophages and in the cells of the vessel wall, in particular the endothelium(Figure 9), as well as in the Nucleus ruber (Figure 10). In contrast, the nucleocapsid pro-tein of SARS-CoV-2 could not be detected in any of the corresponding tissue sections(Figures 11 and 12). In addition, SARS-CoV-2 spike protein subunit 1 was detected in the cardiac endothelial cells that showed lymphocytic myocarditis (Figure 13). Immuno-histochemical staining did not detect the SARS-CoV-2 nucleocapsid protein (Figure 14)
3.4. Autopsy-Based Diagnosis
The 76-year-old deceased male patient had PD, which corresponded to typicalpost-mortem findings. The main cause of death was recurrent aspiration pneumonia. Inaddition, necrotizing encephalitis and vasculitis were considered to be major contribu-tors to death. Furthermore, there was mild lympho-histiocytic myocarditis with fi-ne-spotted myocardial fibrosis as well as systemic arteriosclerosis, which will have alsocontributed to the deterioration of the physical condition of the senior.The final diagnosis was abscedating bilateral bronchopneumonia (J18.9), Parkin-son’s disease (G20.9), necrotic encephalitis (G04.9), and myocarditis (I40.9).Immunohistochemistry for SARS-CoV-2 antigens (spike protein and nucleocapsid)revealed that the lesions with necrotizing encephalitis as well as the acute inflammatorychanges in the small blood vessels (brain and heart) were associated with abundant de-posits of the spike protein SARS-CoV-2 subunit 1. Since the nucleocapsid protein ofSARS-CoV-2 was consistently absent, it must be assumed that the presence of spike pro-tein in affected tissues was not due to an infection with SARS-CoV-2 but rather to thetransfection of the tissues by the gene-based COVID-19-vaccines. Importantly, spikeprotein could be only demonstrated in the areas with acute inflammatory reactions(brain, heart, and small blood vessels), in particular in endothelial cells, microglia, andastrocytes. This is strongly suggestive that the spike protein may have played at least acontributing role to the development of the lesions and the course of the disease in thispatient.
This is a case report of a 76-year-old patient with Parkinson’s disease (PD) who diedthree weeks after his third COVID-19 vaccination. The stated cause of death appeared to be a recurrent attack of aspiration pneumonia, which is indeed common in PD [14,15].However, the detailed autopsy study revealed additional pathology, in particular necrotizing encephalitis and myocarditis. While the histopathological signs of myocarditis were comparatively mild, the encephalitis had resulted in significant multifocal necrosis and may well have contributed to the fatal outcome. Encephalitis often causes epileptic seizures, and the tongue bite found at the autopsy suggests that it had done so in this case. Several other cases of COVID-19 vaccine-associated encephalitis with status epilepticus have appeared previously [16–18].The clinical history of the current case showed some remarkable events in correlation to his COVID-19 vaccinations. Already on the day of his first vaccination in May2021 (ChAdOx1 nCov-19 vector vaccine), he experienced cardiovascular symptoms, which needed medical care and from which he recovered only slowly. After the second vaccination in July 2021 (BNT162b2 mRNA vaccine), the family recognized remarkable behavioral and psychological changes and a sudden onset of marked progression of hisPD symptoms, which led to severe motor impairment and recurrent need for wheel chairs upport. He never fully recovered from this but still was again vaccinated in December2021. Two weeks after this third vaccination (second vaccination with BNT162b2), he suddenly collapsed while taking his dinner. Remarkably, he did not show any coughing or other signs of food aspiration but just fell from his chair. This raises the question of whether this sudden collapse was really due to aspiration pneumonia. After intense resuscitation, he recovered from this more or less, but one week later, he again suddenlycollapsed silently while taking his meal. After successful but prolonged resuscitation at-tempts, he was transferred to the hospital and directly set into an artificial coma but died shortly thereafter. The clinical diagnosis was death due to aspiration pneumonia. Due to his ambiguous symptoms after the COVID-vaccinations the family asked for an autopsy. Based on the alteration pattern in the brain and heart, it appeared that the small blood vessels were especially affected, in particular, the endothelium. Endothelial dysfunction is known to be highly involved in organ dysfunction during viral infections, as it induces a pro-coagulant state, microvascular leak, and organ ischemia [19,20]. This is also the case for severe SARS-CoV-2 infections, where a systemic exposure to the virus and its spike protein elicits a strong immunological reaction in which the endothelial cells play a crucial role, leading to vascular dysfunction, immune-thrombosis, and inflammation .Although there was no history of COVID-19 for this patient, immunohistochemistry for SARS-CoV-2 antigens (spike and nucleocapsid proteins) was performed. Spike pro-tein could be indeed demonstrated in the areas of acute inflammation in the brain (par-ticularly within the capillary endothelium) and the small blood vessels of the heart. Re-markably, however, the nucleocapsid was uniformly absent. During an infection with thevirus, both proteins should be expressed and detected together. On the other hand, thegene-based COVID-19 vaccines encode only the spike protein and therefore, the presenceof spike protein only (but no nucleocapsid protein) in the heart and brain of the currentcase can be attributed to vaccination rather than to infection. This agrees with the pa-tient’s history, which includes three vaccine injections, the third one just 3 weeks beforehis death, but no positive laboratory or clinical diagnosis of the infection.Discrimination of vaccination response from natural infection is an important ques-tion and had been addressed already in clinical immunology, where the combined ap-plication of anti-spike and anti-nucleocapsid protein-based serology was proven as auseful tool . In histology, however, this immunohistochemical approach has not yet been described, but it is straightforward and appears to be very useful for identifying thepotential origin of SARS-CoV-2 spike protein in autopsy or biopsy samples. Where addi-tional confirmation is required, for instance in a forensic context, rt-PCR methods might be used to ascertain the presence of the vaccine mRNA in the affected tissues [23,24].Assuming that, in the current case, the presence of spike protein was indeed driven by the gene-based vaccine, then the question arises whether this was also the cause the accompanying acute tissue alterations and inflammation. The stated purpose of the gene-based vaccines is to induce an immune response against the spike protein. Such an immune response will, however, not only results in antibody formation against the spike protein but also lead to direct cell- and antibody-mediated cytotoxicity against the cells expressing this foreign antigen. In addition, there are indications that the spike protein on its own can elicit distinct toxicity, in particular, on pericytes and endothelial cells of blood vessels [25,26]
While it is widely held that spike protein expression, and the ensuing cell and tissuedamage will be limited to the injection site, several studies have found the vaccinemRNA and/or the spike protein encoded by it at a considerable distance from the injec-tion site for up to three months after the injection [23,24,27–29]. Biodistribution studies inrats with the mRNA-COVID-19 vaccine BNT162b2 also showed that the vaccine does notstay at the injection site but is distributed to all tissues and organs, including the brain. After the worldwide roll-out of COVID-19 vaccinations in humans, spike proteinhas been detected in humans as well in several tissues distant from the injection site(deltoid muscle): for instance in heart muscle biopsies from myocarditis patients ,within the skeletal muscle of a patient with myositis  and within the skin, where itwas associated with a sudden onset of Herpes zoster lesions after mRNA-COVID-19vaccination .The underlying diagnosis in this patient was Parkinson’s disease, and one may askwhat role, if any, this condition had played in the causation of the encephalitis, and themyocarditis detected at post-mortem examination. PD had been long-standing in thecurrent case, whereas the encephalitis was acute. Conversely, there is no plausiblemechanism and no case report of PD causing secondary necrotizing encephalitis. On theother hand, numerous cases have been reported of autoimmune encephalitis and en-cephalomyelitis after COVID-19 vaccination [12,31]. Autoimmune diseases in organsother than the CNS have been reported as well, for example, a striking case of a patientwho after mRNA vaccination suffered multiple autoimmune disorders all at once—acutedisseminated encephalomyelitis, myasthenia gravis, and thyroiditis . In the case re-ported here, it may be noted that the spike protein was primarily detected in the vascularendothelium and sparsely in the glial cells but not in the neurons. Nevertheless, neuronalcell death was widespread in the encephalitic foci, which suggests some contribution ofimmunological bystander activation, i.e., autoimmunity, to the observed cell and tissuedamage.A contributory role of PD in the development of cardiomyopathy is indeed docu-mented and cannot be ruled out with absolute certainty. However, inflammatory myo-cardial changes with pathological alterations in small blood vessels as seen in the currentcase are uncommon. Instead, the most prominent cause of cardiac failure in PD patientsis rather due to cardiac autonomic dysfunction [33,34]. PD seems well to be significantlyassociated with increased left ventricular hypertrophy and diastolic dysfunction . Inthe current case, ventricular dilatation and hypertrophy were present but seem ratherrelated to manifest signs of chronic hypertension. In contrast, myocardial inflammatoryreactions had been well-linked to gene-based COVID-19 vaccinations in numerous cases[9,35–37]. In one case, the spike protein of SARS-CoV-2 could also be demonstrated byimmunohistochemistry in the heart of vaccinated individuals .
Numerous cases of encephalitis and encephalomyelitis have been reported in con-nection with the gene-based COVID-19 vaccines, with many being considered causally related to vaccination [31,38,39]. However, this is the first report to demonstrate the presence of the spike protein within the encephalitic lesions and to attribute it to vac-cination rather than infection. These findings corroborate a causative role of thegene-based COVID-19 vaccines, and this diagnostic approach is relevant to potentially vaccine-induced damage to other organs as well
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Authors: University of Zurich Summary: 6, 2022:Science Daily
As the Covid-19 pandemic evolves, the issue of post-infection consequences is growing in significance. Does Long Covid impact previously healthy young adults? Although this group is of great societal importance, representing the next generation and the backbone of the workforce, the intermediate-term and long-term effects of SARS-CoV-2 infections have scarcely been researched in this population. Available original research tends to focus on sufferers who were hospitalized, the elderly or those with multiple morbidities, or restricts evaluations to a single organ system.
Long Covid implications in young Swiss military personnel
A new study, funded by the Swiss Armed Forces, and conducted under the leadership of Patricia Schlagenhauf, Professor at the Epidemiology, Biostatistics and Prevention Institute of the University of Zurich (UZH), has now evaluated possible Long Covid implications in young Swiss military personnel. The study, published in the journal Lancet Infectious Diseases, was done between May and November 2021 with 29 female and 464 male participants with a median age of 21. 177 participants had confirmed Covid-19 more than 180 days prior to the testing day, and the control group was made up of 251 SARS-CoV-2 serologically negative individuals. Unlike other studies the novel test battery also evaluated cardiovascular, pulmonary, neurological, ophthalmological, male fertility, psychological and general systems.
Despite overall recovery also sequelae after recent infections remain
The findings show that young, previously healthy, non-hospitalized individuals largely recover from mild infection and that the impact of the SARS-CoV-2 virus on several systems of the body is less than that seen in older, multi-morbid or hospitalized patients. However, the study also provided evidence that recent infections — even mild ones — can lead to symptoms such as fatigue, reduced sense of smell and psychological problems for up to 180 days, as well as having a short-term negative impact on male fertility. For non-recent infections — more than 180 days back — these effects were no longer significant.
Specific constellation carries risk of developing metabolic disorders
For those with non-recent infections, however, the study — which had a long follow-up — provided evidence of a potentially risky constellation: “Increased BMI, high cholesterol and lower physical stamina is suggestive of a higher risk of developing metabolic disorders and possible cardiovascular complications,” says principal investigator Patricia Schlagenhauf. “These results have societal and public-health effects and can be used to guide strategies for broad interdisciplinary evaluation of Covid-19 sequelae, their management, curative treatments, and provision of support in young adult populations.”
Significant landmark study points the way
The study, conducted in collaboration with clinics at the University Hospital Zurich and Spiez Laboratory, is novel in that it quantitatively evaluated multi-organ function using a sensitive, minimally invasive test battery in a homogenous group of people several months after a Covid-19 infection. A valuable facet of the study was the control group, serologically confirmed to have had no SARS-CoV-2 exposure. “This combination of a unique test battery, a homogenous cohort and a control group make this a very powerful, landmark study in the evidence base on Long Covid in young adults,” says Schlagenhauf.
Jeremy Werner Deuel, Elisa Lauria, Thibault Lovey, Sandrine Zweifel, Mara Isabella Meier, Roland Züst, Nejla Gültekin, Andreas Stettbacher, Patricia Schlagenhauf. Persistence, prevalence, and polymorphism of sequelae after COVID-19 in unvaccinated, young adults of the Swiss Armed Forces: a longitudinal, cohort study (LoCoMo). The Lancet Infectious Diseases, 2022; DOI: 10.1016/S1473-3099(22)00449-2
COVID-19 is primarily known as a respiratory disease caused by SARS-CoV-2. However, neurological symptoms such as memory loss, sensory confusion, severe headaches, and even stroke are reported in up to 30% of cases and can persist even after the infection is over (long COVID). These neurological symptoms are thought to be produced by the virus infecting the central nervous system, however we don’t understand the molecular mechanisms triggering them. The neurological effects of COVID-19 share similarities to neurodegenerative diseases in which the presence of cytotoxic aggregated amyloid protein or peptides is a common feature. Following the hypothesis that some neurological symptoms of COVID-19 may also follow an amyloid etiology we identified two peptides from the SARS-CoV-2 proteome that self-assemble into amyloid assemblies. Furthermore, these amyloids were shown to be highly toxic to neuronal cells. We suggest that cytotoxic aggregates of SARS-CoV-2 proteins may trigger neurological symptoms in COVID-19.
The disease caused by viral infection with severe acute respiratory syndrome (SARS)-COV-2 is known as COVID-19 and whilst predominantly a respiratory disease affecting the lungs it has a remarkably diverse array of symptoms. These include a range of moderate to severe neurological symptoms reported in as many as 30% of patients, which can persist for up to 6 months after infection1. These symptoms include memory loss, sensory confusion (e.g., previously pleasant smells become fixed as unpleasant), cognitive and psychiatric issues, severe headaches, brain inflammation and haemorrhagic stroke1,2,3,4,5. COVID-19-related anosmia and phantosmia have been shown to correlate with a persistence of virus in the olfactory mucosa and in the olfactory bulb of the brain, and with persistent inflammation; however, negative evidence for continuing viral replication has also been shown for long-term anosmia6. Furthermore, there is evidence that SARS-CoV-2 is neuroinvasive with either the full virus7,8 or viral proteins8 being found in the CNS of mouse models and the post-mortem brain tissue of COVID-19 patients. Whilst the neuroinvasiveness of SARS-CoV-2 is apparent the molecular origin of the associated neurological symptoms is as yet unknown, although they are similar to hallmarks of amyloid-related neurodegenerative diseases such as Alzheimer’s (AD)9,10, and Parkinson’s11. For instance, impaired olfactory identification ability and mild cognitive impairment have also been reported in the early stages of AD and prodromal AD12.
A number of in vitro studies have shown that proteins from SARS-CoV-2 can detrimentally affect a variety of cell types including kidney, liver and immune cells13,14. Furthermore, experiments on brain organoids show that SARS-CoV-2 can infect neuronal cells resulting in cell death15. Combined these papers point to a potential cytotoxic cause of neurological symptoms in COVID-19.
Proteins from the Zika virus16 and also the coronavirus responsible for the SARS outbreak in 2003 (SARS-COV-1)17 have been shown to contain sequences that have a strong tendency to form amyloid assemblies. As the proteome of SARS-CoV-1 and SARS-CoV-2 possess many similarities18, we propose amyloid nanofibrils formed from proteins in SARS-CoV-2 may be implicated in the neurological symptoms in COVID-19. Therefore, amyloid-forming proteins from the SARS-CoV-2 virus in the CNS of COVID-19 infected patients could have similar cytotoxic and inflammatory functions to amyloid assemblies that are the molecular hallmarks of amyloid-related neurodegenerative diseases such as AD (Aβ, Tau) and Parkinson’s (α-synuclein). The worst-case scenario given the present observations is that of the progressive neurological amyloid disease being triggered by COVID-19. To the authors’ knowledge, there has so far been no documented example of this; however, it has been noted that up-regulation of Serum amyloid A protein driven by inflammation in COVID-19 seems like a probable trigger for the systemic (non-neurological) amyloid disease AA amyloidosis19, which is already known to be a concomitant of inflammatory disease in general.
If the proteome of SARS-COV-2 does contain amyloid-forming sequences, this raises the question, what is their function? It is known that viral genomes evolve rapidly and are highly constrained by size; therefore, every component is typically functional either to help the virus replicate or to impede the host immune system. To this end, there are several potential roles for amyloid assemblies in pathogens generally20 and specifically in coronaviruses such as SARS-CoV-2. The simplest is that amyloid is an inflammatory stimulus21, and proinflammatory cytokines can up-regulate the expression of the spike protein receptor ACE-2 such that intercellular transmissibility of SARS-CoV-2 is increased. Alternatively Tayeb-Fligelman et al.22 found that the nucleocapsid protein in SARS-CoV-2, which is responsible for packaging RNA into the virion, contains a number of highly amyloidogenic short peptide sequences within its intrinsically disordered regions22. It has been shown that the self-assembly of these peptides is enhanced in the presence of viral RNA, during liquid–liquid phase separation (LLPS is an important stage in the viral replication cycle)23,24. These findings suggest amyloids may play an important role in RNA binding and packaging during the viral replication cycle. Finally, it is also possible that amyloid assemblies in coronaviruses might have a role in inhibiting the action of the host antiviral response similar to a discussed role for amyloid in other viruses. Pham et al.25 observed that amyloid aggregates from murine cytomegalovirus can interfere with RIPK3 kinase activation and potentially inhibit its antiviral immune signalling capabilities.
In this study, we choose to focus on a selection of proteins from the SARS-CoV-2 proteome known as the open reading frames (ORFs). These ORF proteins were chosen as they have no obvious roles in viral replication26, perhaps freeing them up to have yet uncharacterised roles in disrupting the host antiviral responses. By sequence and length, they appear to be largely unstructured, making them good candidates for amyloid formation in vivo. We performed a bioinformatic screening of the ORF proteins to look for potential amyloidogenic peptide sequences. This analysis was used to select two sub-sequences, one each from ORF6 and ORF10, for synthesis. The synthesised peptides were both found to rapidly self-assemble into amyloid assemblies with a variety of polymorphic morphologies. Cytotoxicity assays on neuronal cell lines showed these peptide assemblies to be highly toxic at concentrations as low as 0.0005% (0.04 mg mL−1).
Since commencing this work, others have found that ORF6 is the most cytotoxic single protein of the SARS-CoV-2 proteome, showing localisation to membranes when overexpressed in human and primate immune cell lines13. In contrast, ORF10 has been reported as an unimportant gene with very low expression and no essential role in virus replication26; however, the functions of immune suppression or inflammation promotion via amyloid formation would be non-essential, if present, and should not necessarily require transcription in large volumes, making ORF10 an intriguing second candidate for the present study. It is also interesting that ORF10 and ORF8 are the only two coded proteins present in SARS-CoV-2 which do not have a homologue in SARS-CoV-127, perhaps suggesting a unique amyloid etiology for COVID-19. While long-term consequences from SARS-CoV-1 infection were severe, including tiredness, depression, and impaired respiration, few or zero unequivocally neurological post-viral symptoms were recorded from the (admittedly quite small) set of documented cases28.
Results and discussion
Amyloid aggregation prediction algorithms identified two short peptides from ORF6 and ORF10 that are likely to form amyloids
Figure 1 shows selected output from bioinformatics tools applied to predict the amyloidogenicity of peptide sequences within larger polypeptides. Application of the ZIPPER tool to ORF6 provides more than ten choices of six-residue windows of the sequence predicted to be highly amyloidogenic (Fig. 1a), while ORF10 shows only three such highly amyloidogenic sequence windows (Fig. 1b). To narrow down our search for candidate peptides we also used the TANGO algorithm, for ORF6 there are two regions that are predicted to be highly aggregation prone, I14LLIIMR and D30YIINLIIKNL. The region I14-R20 overlaps almost perfectly with the hexapeptide I14LLIIM identified by ZIPPER. The region 30–40 also contains multiple hits in ZIPPER, but as this study was limited to two candidate peptides we chose ILLIIM as our first candidate as it closely resembles the sequence ILQINS from Hen Egg White Lysozyme that has previously been seen to be highly amyloidogenic (the mutation TFQINS in human lysozyme is disease-linked)29,30,31. Looking now at the TANGO plots for ORF10 the main aggregation-prone sequence is residues F11TIYSLLLC, although there are no high stability hexapeptides in this sequence predicted by ZIPPER. The octapeptide R24NYIAQVD was chosen due to its zwitterionic residue pair R-D which should strongly enhance interpeptide association, despite being too far apart in the sequence to trigger the highly local bioinformatics algorithms. Encouragingly ZIPPER also predicts the hexapeptide NYIAQV contained within RNYIAQVD to be highly amyloidogenic. Based on the outputs from ZIPPER and TANGO and also on the experience in making and studying amyloid, we selected RNYIAQVD and ILLIIM to be synthesised and their amyloid-forming capability investigated.
Nanoscale imaging reveals both peptide sequences self-assemble into polymorphic amyloid assemblies
Atomic force microscopy (AFM) imaging of the two peptide assemblies revealed that both peptides assembled at 37 °C almost immediately at 1 mg mL−1 (Supplementary Figs. 1 and 2) into a highly polymorphic mixture of nanofibrous and crystalline structures (Supplementary Fig. 3). For both peptides, the dominant polymorph was needle-like crystalline assemblies as seen in Fig. 2. In an attempt to ensure any observed polymorphism was not due to a heterogeneous mixture of insoluble peptide seeds we added warm PBS (90 °C) to the lyophilised peptides, and maintained this elevated temperature for at least 3 h in order dissolve as much of the monomeric peptide as possible. Self-assembly was subsequently initiated by slowly reducing the temperature, using a previously developed protocol32. This method produced less polymorphism resulting in the needle-like crystalline polymorph being overwhelmingly dominant, however a number of twisted fibrillar polymorphs were still present for RNYIAQVD (Fig. 2i and Supplementary Fig. 4a). To facilitate repeatable quantitative analysis of the biochemical and biophysical properties of the assemblies we used the slow cooling assembly method for all further experiments.
AFM and transmission electron microscopy (TEM) imaging of assemblies formed at either 1 or 5 mg mL−1 for 24 h revealed that assemblies from both peptides tend to stack on top of each other forming multi-laminar structures (Fig. 2a–d and Supplementary Figs. 5 and 6). Evidence of lateral assembly of the needles was also observed but this appears to happen more frequently in the ILLIIM assemblies (Fig. 2g) compared to RNYIAQVD. ILLIIM tends to form very large (2–3 μm in width) multi-laminar crystalline assemblies (Fig. 2g), whereas RNYIAQVD predominantly forms long linear needle-like structures. The apparent lower tendency of RNYIAQVD to form large two-dimensional lateral assemblies can be explained by the polymorphism seen in this peptide, which does not promote translational symmetry (i.e., extended crystals). Figure 2i and Supplementary Fig. 4a both clearly show that in addition to the flat needle-like crystals seen elsewhere, RNYIAQVD can also form non-planar partially twisted fibrillar assemblies. This polymorphism observed in RNYIAQVD assemblies may reduce the ability of the crystals to laterally associate and stack into multi-laminar species, simply because of a mismatch in planarity between two adjacent assemblies, the molecular basis for this polymorphism is briefly discussed in the next section of the manuscript. AFM was further used to investigate the height of the individual assemblies of both ILLIIM and RNYIAQVD. Figure 2a, b shows a line section through a multi-laminar RNYIAQVD assembly with two distinct layers with a step height of 5.5 nm between each layer. Similarly for ILLIIM (Fig. 2c and Supplementary Fig. 5), we see multi-laminar stacking with individual step heights that vary between 4 and 12 nm. Turning to RNYIAQVD we see single crystals with step heights varying between 5 and 20 nm (Supplementary Fig. 6). Together this heterogeneous distribution of crystal heights provides further evidence for the polymorphic nature of both the ILLIIM and RNYIAQVD assemblies.
Quantitative analysis of the distribution of assembly heights and contour lengths was performed using the freely available software tool FiberApp33. The analysis of assembly height distribution was taken from the z-axis (height) of the AFM images, both peptide assemblies show a heterogeneous distribution of fibril heights due to the previously observed tendency of both assemblies to form polymorphic multi-laminar stacks (Fig. 2f, h). Analysis of the distribution of the contour length of the two assemblies showed a biphasic distribution of lengths for both fibrils with two broad sub-populations centred around 1 and 3 µm (Fig. 2j, l). The sub-population at 3 µm was seen to be much larger for the RNYIAQVD peptide (Fig. 2j) compared to the ILLIIM (Fig. 2l). This population of longer fibrils correlates with the observation from Fig. 2 that for RNYIAQVD longer, thinner assemblies are favoured (self-assembly via fibril extension) over the wider shorter assemblies more commonly seen for ILLIIM (assembly via lateral association of protofilaments). Analysis of the persistence length of the fibrils (Supplementary Fig. 7) showed that whilst both peptides formed very straight linear assemblies, the persistence length of RNYIAQVD (λ = 41.92 µm) is greater than that of ILLIIM (λ = 31.96 µm).
To further investigate the polymorphic nature of the assembly of these peptides, we investigated the structures formed from 1:1 mixtures of the two peptides. Interestingly when mixed prior to assembly the peptides form a wide range of polymorphic structures exceeding that of either peptide assembled individually. Supplementary Fig. 8 shows a selection of some of the polymorphs formed, especially interesting are the large flat structures with well-defined edges that seem almost to interlock (Supplementary Fig. 8c). Such well-ordered 2D crystals were never observed for either peptide individually, and provide clear evidence of co-crystallisation. At this stage we have no evidence for the biological relevance of this co-crystallisation; however, as the ORF proteins from which these peptides are identified are themselves very small proteins (ORF6 is 61 amino acids in length), it is feasible that these small proteins may undergo similar co-crystallisation during their viral replication cycle facilitating an as yet unknown biological function.
X-ray scattering, spectroscopic characterisation, fluorescent microscopy and molecular modelling confirm the amyloid nature of the assemblies
Figure 3a shows the radially averaged 1D small-angle X-ray scattering (SAXS) plots for ILLIIM and RNYIAQVD at the lower concentration studied (at the higher concentration, sedimentation made recording X-ray scattering spectra impossible). In the central part of the scattering curve, the ILLIIM assemblies produced a slope with a q−2 dependence which is consistent with the form factor of an infinite 2D surface30, and is most likely arising due to the broader lateral dimensions observed by AFM and TEM for ILLIIM compared to RNYIAQVD. RNYIAQVD, however, displays a q−4 dependence in the central part of the scattering curve, appearing more towards the high-q limit. Porod’s law indicates that q−4 scaling (at high q but still less than 0.1 Å−1) is consistent with any aggregates having sharp surfaces but does not otherwise specify shape34. The data from the SAXS plots provide supporting evidence that the laterally associated amyloid assemblies seen by AFM and TEM are not artefacts induced either by the dehydration (AFM), applying vacuum conditions (TEM) or the mica (AFM) or carbon (TEM) substrates used, but a genuine structure observed also in bulk.
Figure 3b shows the CD spectra of mature assemblies, of both peptides. Assemblies of ILLIIM display a quite simple spectrum indicating the dominance of β-sheets, with a minimum between 225 and 230 nm and a strong maximum at 205 nm29. The CD spectra of RNYIAQVD possess a well-defined minimum at 203 nm and a distinct shoulder at around 215 nm.
To further investigate the predicted secondary structure of both peptide assemblies we employed the secondary structure analysis software BeStSel (Supplementary Table 1)35,36. As expected from the classic shape of the spectra the predicted secondary structure of ILLIIM at 5 mg mL−1 is exclusively made up of β-sheets (41.8%) and β-turns (58.2%). At these high concentrations, the composition of these β-sheets is shown to be exclusively left twisted, whilst at lower concentrations (1 mg mL−1) a more complex mixture of right, left and non-twisted (relaxed) β-sheets are predicted. At both concentrations, the CD spectra of RNYIAQVD again suggest the secondary structure is dominated by β-sheets; however, now they appear to be exclusively in the form of higher energy right-twisted β-sheets, similar to that observed in the highly strained structure of other amyloidogenic ultra-short peptides30. This additional strain introduced by β-sheets opposing the left-handed chirality seen in natural amino acids may explain the additional polymorphism and twisted microstructures seen in the AFM and TEM images of the RNYIAQVD assemblies (Fig. 2i and Supplementary Fig. 4a)29. The BeStSel fitting algorithm predicted the remainder of the RNYIAQVD secondary structure is composed of α-helical structure and further backbone conformations that could not be assigned (Supplementary Table 1). Part-helical CD spectra do not necessarily imply helical structure, especially considering that a single octapeptide cannot literally be 19% helix (two residues). Backbone conformation as reported by CD correlates through sheet structure to the assembled tertiary structure but no single level of organisation exclusively dictates any other, this is especially true in the case of coupling the twist of a peptide strand to the overall twist of the aggregate, which can relax to meet surface and shape-driven constraints through intersheet and interchain as well as intrachain degrees of freedom29.
To further investigate the conformation of the amyloid assemblies formed we utilised the conformation-specific antibody A11 and the fluorescent probe thioflavin T (ThT). The former binds specifically to non-fibrillar amyloid oligomers and the latter is a commonly used molecular probe that becomes highly fluorescent when binding to amyloid assemblies37. As expected, when ILLIIM and RNYIAQVD assemblies were stained with ThT both demonstrated clearly visible fluorescent emission at 590 nm, providing further evidence of their amyloid nature (Supplementary Fig. 9a, b). Conversely, A11 exhibited no positive binding; specifically, the level of fluorescence observed was similar to the background staining seen in the negative controls, as confirmed by similar fluorescent intensities for both assemblies (Supplementary Fig. 9e, f) and the negative controls (Supplementary Fig. 9d, h). Higher levels of A11 binding were seen for the positive control that consisted of phenylalanine assemblies known to form oligomeric species38 (Supplementary Fig. 9g), with fluorescent intensities for these assemblies over 4 times greater than for ILLIIM or RNYIAQVD. Combined these data confirm the amyloid nature of the two ORF peptide fragments and suggest that non-fibrillar oligomeric amyloid species are absent.
The amyloid nature of the two assemblies is further confirmed by the wide-angle X-ray scattering (WAXS) spectra (Fig. 3d) of the peptide assemblies which possessed a number of strongly diffracting Bragg peaks. Both peptides have a clear peak at 1.38 Å−1 corresponding to a d-spacing of 4.6 Å which is indicative of an amyloid assembly composed of extended β-sheets39. It is worth noting that the apparent intrastrand spacing of ILLIIM assemblies is very slightly lower than the typically reported values (4.7–4.8 Å). This may be explained by the BeStCell analysis of the ILLIIM assemblies at 1 mg mL−1, which suggests that the β-sheets in these assemblies are composed of a complex mixture of left-handed, strained right-handed and relaxed β-sheets (Supplementary Table 1); therefore, it is perhaps not surprising that the observed average intersheet spacing very slightly differs from that which is commonly reported. Furthermore, Lomont et al.40 report that the observed intrastrand spacing from a range of amyloid crystal structures can vary by as much as 0.45 Å. ILLIIM also has a very strong Bragg reflection at 0.58 Å−1 (11 Å) corresponding to a typical intersheet spacing given moderately bulky hydrophobic sidechains forming a steric zipper. RNYIAQVD has a number of well-defined Bragg peaks between 0.3 and 0.75 Å−1 that are consistent with a mixture of first and second-order reflections corresponding to an amyloid-like 3D symmetry. Typical reflections arising from the combinations of the longer two axes of the unit cell of short peptide amyloid crystals arise in the 0.3–0.75 Å−1 region with a qualitatively similar appearance to the pattern from RNYIAQVD, although in this case the peaks could not be individually assigned.
Discovery of sub-Å resolution structures from solution WAXS is highly challenging; however, given the simple nature of the scattering from the ILLIIM system, it was possible to produce an atomistic model matching the positions of the observed peaks, although not their sharpness. Physically, peak sharpness increases with the ordering length scale, indicating that some structures in the solution were larger than could be managed in the simulation. The sheet-like shape factor and the presence of peaks at roughly 2π/4.6 and 2π/11 Å−1 indicate assembly in solution dominated by the hydrogen bonding axis (with the typical parallel β sheet period of ≈ 4.7 Å) and by a sidechain-sidechain hydrophobic zipper interface. A metastable candidate structure of size 6 × 50 × 1 peptides was constructed following this geometry (see Methods) and found to reproduce the observed WAXS and to fully exclude water at the steric zipper (Fig. 4). The q−2 dependence of ILLIIM scattering at low q in solution (Fig. 3a) is consistent with a 2D sheet-like structure similar to that produced in the modelling. Initial assembly into sheets is also consistent with the eventual formation of multi-laminar structures as shown in the AFM (Fig. 2), as well as with the tendency of ILLIIM in particular to form lateral assemblies of needle microcrystals (Fig. 2g, k vs. e, i). Atomistic details of the interaction of the 2D sheet-like oligomer structure of Fig. 4 with neuronal cell membrane are difficult to predict and would be an interesting subject for further work. However, the juxtaposition of hydrophobic sidechains with polar termini in the ILLIIM fragment (or with titratable residues in the longer fragment E13ILLIIMR, which unfortunately could not be synthesised) has a length of approximately 10 Å, comparable to the polar-hydrophobic-polar length scale of 40 Å for the two leaflets of the eukaryotic cell membrane, indicating a potential for planar aggregates of, in particular, four sheets in thickness (four peptides end-to-end, linked in the middle by salt bridges) to disrupt the cell membrane.
The ThT stain, which becomes highly fluorescent upon binding to β-sheet rich amyloid assemblies was used to assess the assembly kinetics of both ILLIIM and RNYIAQVD (Fig. 3c). Both peptides show rapid kinetics with significant assembly occurring almost instantaneously and reaching a plateau after 30–60 min. Longer amyloidogenic polypeptides typically show a distinct lag phase in their assembly kinetics; however, this was not observed in these sequences. This apparent lack of a lag phase in the assembly kinetics behaviour is typical of amyloidogenic short peptides, which have been previously seen to assemble very rapidly29,41. This is highly likely to be due to a lack of additional non-amyloidogenic amino acid sequences acting as a kinetic barrier to amyloid formation. The ThT signal for ILLIIM at 5 mg mL−1 plateaus at about 300 a.u., this is slightly stronger than the maximum signal generated from mature fibrils of the somewhat homologous peptide ILQINS, which was around 250 a.u29,30,31,42, suggesting that the amyloidogenicity of the two peptides is comparable. RNYIAQVD, whilst showing similar ThT values at low concentrations (1 mg mL−1), generated a ThT signal nearly 3 times as large at 5 mg mL−1 suggesting that the assembly of this peptide is highly concentration dependent. For reasons yet unknown, it seems that RNYIAQVD appears to reach a maximum ThT value and then begin to drop, this can be seen at both concentrations but is most obvious at the higher concentration. This could be due to a reversible self-assembly as seen in other functional amyloids39,41,43,44, or to self-quenching of the amyloid bound aromatic ThT molecules, or simply to a reduction of exposed ThT-binding sites as larger aggregates with a lower surface area to volume ratio come to dominate the solution.
Cytotoxicity of both studied peptides also began to drop slightly (without statistical significance) at the highest concentrations tested (vide infra, Fig. 5), together with the drop in ThT response this supports the existence of a kinetically or thermodynamically available aggregate structure with reduced ‘amyloid activity’. This is reminiscent of strongly amyloid correlated diseases such as AD, where the toxicity of amyloid can vary dramatically, with the relationship between the amount of amyloid deposited to the progress of the disease being idiosyncratic and highly non-linear45. Combined the CD spectroscopy (Fig. 3b), the ThT spectroscopy (Fig. 3c) and confocal microscopy (Supplementary Fig. 9), the presence of the Bragg peaks corresponding to the intra- and inter-β-sheet spacings (Fig. 3d) and the molecular modelling (Fig. 4) confirm beyond doubt the β-sheet rich, amyloid nature of these two fragments.
ILLIIM and RNYIAQVD peptide assemblies are both highly toxic towards the neuroblastoma cell line SH-SY5Y
Given the physical evidence and the discussions referred to in the introduction of various means by which SARS-CoV-2 and other viral infections could enhance their fitness (to the detriment of the host) by the production of amyloidogenic peptides, we hypothesised that the SARS-CoV-2 viral transcript fragments ILLIIM and RNYIAQVD are toxic to human neurons. This is in particular supported by the previously reported neuroinvasive capabilities of SARS-CoV-27,8, the noted similarities of the symptoms to a (hopefully transient form of) AD5 and the previous detection of amyloid assemblies driven by other viruses20. To investigate this further we performed a number of cytotoxicity assays of the two peptide sequences against a human-derived neuroblastoma cell line (SH-SY5Y) often used as a model cell line for studying Parkinson’s and other neurodegenerative diseases46. Using an MTT assay we found that cells grown in the presence of both peptide assemblies possessed much lower viability after 48 h incubation. Concentrations as low as 0.04 and 0.03 mM (for RNYIAQVD and ILLIIM, respectively) were seen to reduce the viability of cultured cells after 48 h to <50% (IC50) compared to the cells cultured without the peptides (Fig. 5a, b). This toxicity in relation to concentration is similar to that reported for Aβ4247 although expression levels and time-scales (sudden for COVID versus chronic for AD) are likely to be very different.
To gain further insight into the mechanism of cell death occurring in the peptide exposed cells, we performed a detailed flow cytometry analysis using the apoptotic stain Annexin V and the viability dye 7-AAD. Figure 5c shows representative flow cytometry plots; cells can be identified as viable (bottom left quadrant), viable but undergoing early apoptosis (bottom right), non-viable and necrotic (top left) or non-viable due to late-stage apoptosis (top right). The percentages of cells in these quadrants are roughly equal for all conditions tested except in the case of late-stage apoptosis where we see a large increase in the cells exposed to the peptide assemblies (a 6.25-fold increase for RNYIAQVD at 2.5 mg mL−1). Quantification over a range of concentrations showed that on average cells exposed to both ILLIIM and RNYIAQVD had a 3–5-fold increase in late-stage apoptosis compared to SH-SY5Y cells cultured in the absence of peptide assemblies (Fig. 5d, e). No evidence of increasing necrosis was seen in any of the samples, suggesting that the amyloid assemblies are triggering programmed cell death via an apoptotic pathway. This triggering of late-stage apoptosis in the cells was more pronounced for ILLIIM than for RNYIAQVD, showing statistically significant increases in apoptotic cells at concentrations as low as 0.04 mg mL−1 for ILLIIM compared to 0.15 mg mL−1 for RNYIAQVD. This increase in apoptosis down to low concentration provides convincing evidence, especially for ILLIIM, that the amyloid aggregates are responsible for this toxicity, as at these low concentrations we would expect very little un-assembled peptide to exist. The mechanisms of cell death in neurodegenerative diseases are complex and can vary between different diseases48, and here we provide evidence that induction of apoptosis may be an important mechanism of neuronal death in COVID-19. Intriguingly, the conserved protein ORF6 from SARS-CoV-1 (not SARS-CoV-2) has previously been shown to induce apoptosis49. Furthermore, we performed a series of cell counting experiments and demonstrated that after 48 h incubation we saw statistically significant decreases in cell number for both peptides at concentrations as low as 0.04 mg mL−1 for ILLIIM and 0.32 mg mL−1 for RNYIAQVD. These results confirm that in addition to the cytotoxic nature of the peptide assemblies, they significantly reduce cell number especially in the case of ILLIIM. The significant increase in apoptosis and reduction in cell number seen for ILLIIM correlates with the work of Lee et al. who have previously shown that the ORF6 protein (that contains the ILLIIM sequence) is the most cytotoxic protein in the proteome of SARS-CoV-213. Combined with our data, this suggests that this toxicity might be due to the amyloidogenic nature of this short protein.
Previous research has shown that the polymorphism, size distribution and the morphology of amyloid aggregates can have a large influence on their cytotoxicity. Marshall et al.50 showed that a range of crystal-forming assemblies formed from short peptide sequences show surprisingly little toxicity to the same neuroblastoma cell line used in this study. Our TEM and AFM images (Fig. 2) confirm that the assemblies formed by the sequences identified from ORF6 and ORF10 look very similar to the assemblies in Marshall et al.50 but the SARS-CoV-2-related peptides are significantly more toxic, suggesting a specific mechanism of toxicity for these assemblies. Xue et al.51 showed that shorter amyloid assemblies from a range of different proteins/peptides have increased the ability to disrupt the bilayer of unilamellar vesicles and provide a greater cytotoxic effect on neuroblastoma cells. Mocanu et al.52 showed a dose-dependent cytotoxic effect in epithelial cells for long-thin lysozyme amyloid fibrils, and a threshold dependent mechanism for the larger laterally associated fibrils. We see similar effects to both Xue et al. and Mocanu et al. suggesting that the observed toxicity of the assemblies may be related to their aspect ratio. We observed that ILLIIM assemblies are both more toxic, wider (Fig. 2h) and shorter than their RNYIAQVD counterparts (Fig. 2k), this is shown schematically in Fig. 6. Similarly to Mocanu et al.52 we see that the long-thin RNYIAQVD fibrils show a clear dose-dependent increase in apoptosis (Fig. 5e), and the laterally associated ILLIIM fibrils show similarly high levels of apoptosis induction at all concentrations above a threshold of 0.04 mg mL−1 (Fig. 5d).
There is a wealth of literature suggesting that in neurodegenerative diseases like Alzheimer’s and Parkinson’s amyloid oligomers are the main toxic culprits and mature amyloid fibrils are a more inert assembly end-point. This is seemingly at odds with our data; however, there have also been multiple studies that show mature assemblies can also display significant toxicity37,53,54. Alternatively, it may be the nature of the amyloids species seen here that differs from amyloids in neurodegenerative diseases; the amyloids seen here appear to be largely crystalline (especially in the case of ILLIIM). Previous work has shown that amyloid crystals are deeper in the free energy landscape compared to twisted protofilaments and amyloid ribbons30,55, representing a global energy minima. AFM and TEM data have shown that these stable amyloid crystals are the dominant polymorph for ILLIIM (Fig. 2c, g, k) and that RNYIAQVD shows examples of higher energy (partially) twisted fibrils (Fig. 2i and Supplementary Figs. 4a and 6). Therefore, we hypothesise that the low energy ILLIIM crystalline assemblies are more slowly metabolised and cells are exposed for longer timeframes to the cytotoxic effect compared to RNYIAQVD assemblies. To date, there have been few investigations into the toxicity of amyloid crystals compared to other more commonly reported amyloid species. For the reasons above, the toxic nature of these amyloid assemblies warrants further investigations into the potential presence of amyloid aggregates from SARS-CoV-2 in the CNS of COVID-19 patients, and the potential role of amyloids in the neurological symptoms observed.
In conclusion, using a bioinformatics approach we identified two strongly amyloidogenic sub-sequences from the ORF6 and ORF10 sections of the SARS-COV-2 proteome. Nanoscale imaging, X-ray scattering, molecular modelling, spectroscopy and kinetic assays revealed that these self-assembled structures are amyloid in nature, and screening against neuronal cells revealed that they are highly toxic (approximately as toxic as the toxic amyloid assemblies in AD) to a cell line frequently used as a neurodegenerative diseases model. The neuroinvasive nature of SARS-COV-2 has been established previously7,8; therefore, it is entirely plausible that amyloid assemblies either from these ORF proteins or other viral proteins could be present in the CNS of COVID-19 patients. The cytotoxicity and protease-resistant structure of these assemblies may result in their persistent presence in the CNS of patients post-infection that could partially explain the lasting neurological symptoms of COVID-19, especially those that are novel in relation to other post-viral syndromes such as that following the original SARS-CoV-1. The outlook in relation to triggering of progressive neurodegenerative disease remains uncertain. Given the typically slow progress of neurodegenerative disease if such a phenomenon exists, it will most probably take some time to become evident epidemiologically.
Amyloid prediction algorithms
The online amyloid prediction algorithms TANGO and ZIPPER were used to predict peptide sequences with a tendency to form β-rich amyloid assemblies. TANGO is an algorithm that predicts aggregation nucleating regions in unfolded polypeptide chains56. It works on the assumption that the aggregating regions are buried in the hydrophobic core of the natively folded protein. ZIPPER is an algorithm that predicts hexapeptides within larger polypeptide sequences that have a strong energetic drive to form the two complementary β-sheets (known as a steric zipper) that give rise to the spine of an amyloid fibril57. Both methods are physically motivated but rely on statistically determined potentials.
Self-assembly of peptides
NH2-ILLIIM-CO2H and Ac-RNYIAQVD-NH2 (>95% pure) were purchased from GL Biochem Ltd (Shanghai, China). Ideally, it would have been preferred to have both peptides capped (N-terminus: Acetyl and C-terminus: Amide), as they would better represent small fragments of a larger peptide sequence. Due to the fact ILLIIM contains no charged sidechains, synthesising capped sequences to high purity would have been very challenging, therefore only the RNYIAQVD sequence remained capped and the ILLIIM sequence had regular carboxyl and amino termini. To ensure that all peptide seeds were fully dissolved before self-assembly was initiated the peptides were solubilised in warmed PBS (90 °C) at either 1 or 5 mg mL−1 The warmed peptide solutions were vortexed vigorously and held at 90 °C for 3 h to ensure maximum dissolution. After the second round of vortexing, the peptide suspensions were cooled slowly. This protocol has been previously used to maximise a homogenous starting population of monomeric peptide32. Alternatively, self-assembly was carried out at a constant temperature of 37 °C without pre-solubilising the peptides in hot PBS.
Atomic force microscopy (AFM)
AFM imaging was performed on a Bruker Multimode 8 AFM and a Nanoscope V controller. Tapping mode imaging was used throughout, with antimony (n)-doped silicon cantilevers having approximate resonant frequencies of 525 or 150 kHz and spring constants of either 200 or 5 Nm−1 (RTESPA-525, Bruker or RTESPA-150, Bruker). No significant differences were observed between cantilevers. 50 µL aliquots of the peptide (either at 1 or 5 mg mL−1) were drop cast onto freshly cleaved muscovite mica disks (10 mm diameters) and incubated for 20 min before gently rinsing in MQ water and drying under a nitrogen stream. All images were flattened using the first order flattening algorithm in the nanoscope analysis software and no other image processing occurred. Statistical analysis of the AFM images was performed using the open-source software FiberApp33 from datasets of no less than 900 fibres.
Transmission electron microscopy (TEM)
Copper TEM grids with a formvar-carbon support film (GSCU300CC-50, ProSciTech, Qld, Australia) were glow discharged for 60 s in an Emitech k950x with k350 attachment. Then, 5 µL drops of sample suspension were pipetted onto each grid, allowed to adsorb for at least 30 s and blotted with filter paper. Two drops of 2% uranyl acetate were used to negatively stain the particles with excess negative stain removed by blotting with filter paper after 10 s each. Grids were then allowed to dry before imaging. Grids were imaged using a Joel JEM-2100 (JEOL (Australasia) Pty Ltd) transmission electron microscope equipped with a Gatan Orius SC 200 CCD camera (Scitek Australia).
Small- and wide-angle X-ray scattering (SAXS/WAXS)
SAXS/WAXS experiments were performed at room temperature on the SAXS/WAXS beamline at the Australian Synchrotron. Peptide assemblies in PBS prepared at both 1 and 5 mg mL−1 were loaded into a 96-well plate held on a robotically controlled x–y stage and transferred to the beamline via a quartz capillary connected to a syringe pump. Data from the 5 mg mL−1 assemblies were discarded due to sedimentation of the assemblies preventing reliable sample transfer into the capillaries. The experiments used a beam wavelength of λ = 1.03320 Å−1 (12.0 keV) with dimensions of 300 µm × 200 µm and a typical flux of 1.2 × 1013 photons per second. 2D diffraction images were collected on a Pilatus 1M detector. SAXS experiments were performed at q ranges between 0.002 and 0.25 Å−1 and WAXS experiments were performed at a q range between 0.1 and 2 Å−1. These overlapping spectra provide a total q range of 0.002–2.2 Å−1. Spectra were recorded under flow (0.15 mL min−1) to prevent X-ray damage from the beam. Multiples of approximately 15 spectra were recorded for each time point (exposure time = 1 s) and averaged spectra are shown after background subtraction against PBS in the same capillary.
Circular dichroism spectroscopy
CD spectroscopy was performed using an AVIV 410-SF CD spectrometer. Spectra were collected between 190 and 260 nm in PBS using 1 mm quartz cuvettes with a step size of 0.5 nm and 2 s averaging time. Data were analysed using the BeStSel (Beta Structure Selection) method of secondary structure determination35.
Atomistic models were constructed using the Nucleic Acid Builder58. Simulations were run in explicit water (TIP3P59) using the ff15ipq forcefield60 and the pmemd time integrator61. In order to hold the unit cell geometry to values consistent with the observed scattering, the alpha carbon of the central residue of each chain was subjected to a restraining force with spring constant 2 kcal mol−1 Å−2. Periodic boundaries were applied to the system such that it formed a truncated octahedron, which was relaxed during equilibration to a volume of 10,310 nm3, giving a density of 0.973 reference with 323,535 water molecules. The system state after 10 ns of equilibration was stripped of water molecules more than 10 Å from any non-hydrogen solute atom, and passed to CRYSOL3 for calculation of orientationally averaged scattering profile given the example state (including the ordered waters from the explicit solvent shell, and also including an approximate treatment of ordered water beyond this shell)42,62.
Thioflavin T amyloid kinetic assays
Peptide assemblies were made up to concentrations of 1 or 5 mg mL−1 suspensions containing 25 µM ThT in PBS. The first fluorescence measurement (t = 0) was recorded immediately after sample preparation. All the samples were then stored at room temperature and fluorescence intensity was recorded at different time points. Measurements were performed in triplicate using a ClarioStar fluorimeter equipped with a 96-well plate reader (excitation wavelength: 440 nm, emission wavelength: 482 nm).
Cell line and cultures
Human-derived neuroblastoma cells (SH-SY5Y, ATCC Product Number: CRL-2266) were cultured in DMEM-F12 (Invitrogen) medium supplemented with 10% (v/v) foetal calf serum (FCS), 100 UmL−1 penicillin and 100 µgmL−1 streptomycin (Invitrogen, Carlsbad, CA). Cells were cultured at 37 °C in a humidified atmosphere containing 5% CO2.
Immunofluorescent and thioflavin T microscopy
ThT staining was performed by incubating the amyloid assemblies with a 25 µM solution of ThT in PBS for 15 min, in an 8-well Labtek II chamber (Nunc). For the antibody stain the same assemblies were incubated in a 1:200 dilution of the A11 polyclonal antibody raised in rabbit (Invitrogen, REF: AHB0052, LOT:VF299837) in 2% BSA in PBS for 1 h. Following this the wells were carefully washed in PBS and a solution of the 2° antibody (Goat-Anti Rabbit IgG-Alexa Fluor 647, Product A21244, Lot #: 1871168, Molecular Probes) at 1:1000 dilution in PBS was incubated with the peptides for 1 h. Finally the assemblies were once again washed in PBS before being imaged via laser scanning confocal microscopy using a FV3000 microscope (Olympus) and 60× objective lens (1.35 NA Oil Plan Apochromat) using the following settings: ThT channel λex = 450 nm, λem = 490 nm, Alexa Fluora 647 channel λex = 650 nm, λem = 665 nm. The same imaging settings were used for all samples and the negative controls (peptide assemblies + 2° antibody only) were used to determine the level of background fluorescence. For positive controls, amyloid assemblies of phenylalanine were used under concentrations known to readily form oligomeric amyloid assemblies38, which were shown here to bind strongly to the A11 antibody (Supplementary Fig. 9e, f).
Cell viability assay
Cells were seeded into 96-well plates at 1 × 105 cells per mL and incubated for 24 h to ensure good attachment to the surface. A stock solution of peptide assemblies (10 mg mL−1) was serially diluted into DMEM-F12 media 2.5–0.02 mg mL−1 or 3.3–0.027 mM for ILLIIM and 2.45–0.02 mM for RNYIAQVD) and seeded onto the SH-SY5Y cells and incubated for 48 h, cell viability was determined using 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) (Sigma-Aldrich) as described previously63. Equivalent MTT assays were performed on cells cultured in the same ratios of PBS to media, but in the absence of peptide, assemblies to confirm that the culture conditions were non-toxic (Supplementary Fig. 10). Absorbance readings of untreated control wells in 100% cell culture media were designated as 100% cell viability. Statistical analysis was performed by one-way ANOVA tests with Tukey comparison in the software GraphPad (Prism) ***p < 0.001. Flow cytometry assays to determine cell viability were performed in a similar manner to the MTT assays. Briefly, to determine the effect of the peptides on cellular viability SH-SY5Y cells were cultured in the presence of the peptides for 48 h, harvested and stained with the apoptosis stain Annexin V for 10 min on ice (Cat No. 550474, BD Biosciences, 5 µL in 100 µL of 2% FCS in PBS). Samples were diluted with 300 µL of 2% FCS in PBS and stained with the viability dye 7-AAD (559925 BD Biosciences, 5 µL per sample) and analysed using flow cytometry (FACS Aria III; BD Biosciences). Cell counts were performed manually using a hemocytometer, with tryphan blue to differentiate non-viable cells.
All code used in this study is either free (NAB 1.3, pymol 2, TANGO 2.2 and ZIPPER) or commercially available (pmemd 19, CRYSOL3 3.0.3).
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N.P.R. would like to acknowledge The La Trobe Institute of Molecular Sciences (LIMS) for the receipt of a Nicholas Hoogenraad fellowship, and the CASS foundation for partially funding this work through a philanthropic grant (#10053, ‘Determining the role of protein aggregation in COVID-19’). N.P.R. would also like to acknowledge that Fig. 6 was created using Biorender.com. The authors thank Dr Susi Seibt for assistance on the SAXS/WAXS beamline at the Australian Synchrotron. This research was undertaken, in part, on the SAXS/WAXS beamline at the Australian Synchrotron, part of ANSTO. Molecular dynamics calculations made use of the HPC service of the University of Luxembourg64. The project was part-funded by grant C20/MS/14588607 of the Fonds Nationale de la Recherche, Luxembourg.
Authors: University of Otago July 12, 2022: Science Daily
Summary: Researchers have uncovered how post-viral fatigue syndromes, including Long COVID, become life-changing diseases and why patients suffer frequent relapses.
Arising commonly from a viral infection, Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS), is known to cause brain-centred symptoms of neuroinflammation, loss of homeostasis, brain fog, lack of refreshing sleep, and poor response to even small stresses.
Long-COVID has similar effects on people and is believed to also be caused by neuroinflammation.
Lead author Emeritus Professor Warren Tate, of the University of Otago’s Department of Biochemistry, says how these debilitating brain effects develop is poorly understood.
In a study published in Frontiers in Neurology, he and colleagues from Otago, Victoria University of Wellington and University of Technology Sydney, developed a unifying model to explain how the brain-centred symptoms of these diseases are sustained through a brain-body connection.
They propose that, following an initial viral infection or stressor event, the subsequent systemic pathology moves to the brain vianeurovascular pathways or through a dysfunctional blood-brain barrier. This results in chronic neuroinflammation, leading to a sustained illness with chronic relapse recovery cycles.
The model proposes healing does not occur because a signal continuously cycles from the brain to the body, causing the patient to relapse.
The creation of this model is not only important for the “huge research effort ahead,” but also to provide recognition for ME/CFS and Long COVID sufferers.
“These diseases are very closely related, and it is clear the biological basis of Long COVID is unequivocally connected to the original COVID infection — so there should no longer be any debate and doubt about the fact that post viral fatigue syndromes like ME/CFS are biologically based and involve much disturbed physiology,” Emeritus Professor Tate says.
This work will enable best evidence-based knowledge of these illnesses, and best management practices, to be developed for medical professionals.
“Patients need appropriate affirmation of their biological-based illness and help to mitigate the distressing symptoms of these very difficult life-changing syndromes which are difficult for the patients to manage by themselves.
“This work highlighted that there is a susceptible subset of people who develop such syndromes when exposed to a severe stress, like infection with COVID-19, or the glandular fever virus Epstein Barr, or in some people with vaccination that is interpreted as a severe stress.
“What should be a transient inflammatory/immune response in the body to clear the infection, develop immunity and manage the physiological stress, becomes chronic, and so the disease persists.”
Warren Tate, Max Walker, Eiren Sweetman, Amber Helliwell, Katie Peppercorn, Christina Edgar, Anna Blair, Aniruddha Chatterjee. Molecular Mechanisms of Neuroinflammation in ME/CFS and Long COVID to Sustain Disease and Promote Relapses. Frontiers in Neurology, 2022; 13 DOI: 10.3389/fneur.2022.877772
Acute disseminated encephalomyelitis (ADEM) is a demyelinating disease, and there are some data that link this event with various vaccinations. We report a young female admitted to the hospital with headache, fever, back pain, nausea, vomiting, and urinary retention. Two weeks prior, she received the first dose of SARS-CoV-2 mRNA vaccine. Brain and spinal cord magnetic resonance imaging (MRI) showed distinctive for ADEM widespread demyelinating lesions. The patient was successfully treated with methylprednisolone.
Acute disseminated encephalomyelitis is a central nervous system demyelinating disease that usually affects the cerebral hemispheres, brainstem, cerebellum, and spinal cord and thus typically presents with multifocal neurologic symptoms. It is widely considered as a monophasic disease with a very rare recurrent variant (multiphasic disseminated encephalomyelitis, MDEM).1
Viral infections appear to trigger approximately up to three-quarters of ADEM cases, which manifests in a rapid onset of multifocal neurological deficits. On the other hand, for many years, there have been, and still, there are concerns that vaccinations may cause autoimmune demyelination. Articles published several years ago suggested that about 5% of ADEM events are associated with immunization for varicella, rabies, measles, mumps, rubella, influenza, hepatitis B, Japanese B encephalitis, diphtheria, pertussis, and tetanus.2 A quite recent report also claims that 61% of ADEM cases developed symptoms 2–31 days after vaccination.3 However, the results of another decent study in which data almost from 64 million vaccine doses have been explored did not show a significant association between ADEM and prior immunization.4 The evidence for a causal link between specific acute demyelinating events and any vaccine has been deemed inconclusive by the Institute of Medicine in the United States.5 ADEM can be classified as an adverse event following immunization (AEFI), defined as any untoward medical occurrence that follows immunization and does not necessarily have a causal relationship with the usage of the vaccine.6 Vaccinations are crucial for the slowdown of the spread of the COVID-19 pandemic, so AEFI monitoring and reporting is an essential part of the strategy against SARS-CoV-2.
A 19-year-old female was admitted to the hospital with complaints of 3-day severe headache, fever (37.5°C), back and neck pain with nausea and vomiting. She also noticed urinary retention last 2 days. Besides atopic dermatitis and depression, she had no significant medical history, upper respiratory infection, or diarrhea. Neurological examination showed nuchal rigidity, bilateral Babinski signs without other neurological deficit symptoms.
Two weeks prior, she received the first dose of SARS-CoV-2 mRNA vaccine (Moderna COVID-19 Vaccine, ModernaTX, Inc. USA).
Brain magnetic resonance imaging (MRI) showed multiple, poorly demarcated, hyperintense lesions in T2-weighted and fluid-attenuated inversion recovery (FLAIR) images located in both brain hemispheres, pons, the medulla oblongata, and cerebellum. Few of them were contrast-enhanced lesions. Cervical and thoracic MRI revealed a widespread hyperintense area in T2-weighted and FLAIR images extended from medulla oblongata to Th11 segment with overlapping few contrast-enhancing lesions (Fig. 1).
Upon hospitalization, the cerebrospinal fluid (CSF) white blood cell (WBC) count was 294 × 106/L (reference range, 0–5 × 106/L): 91% lymphocytes (reference range, 40%–80%), 8% monocytes (reference range 15%–45%), 1% neutrophils (reference range 0%–6%); protein levels were 648 mg/L (reference range, 200–400 mg/L) and red blood cell (RBC) count was 77/µL. CSF was negative for antibodies to major pathogens and cultures of bacteria and fungi; genome sequencing also revealed no pathogens (Neisseria meningitidis, Streptococcus pneumoniae, group B streptococcus, Haemophilus influenzae, Listeria monocytogenes, HSV1, HSV2, VZV, CMV, EBV, HHV6). The reverse transcription real-time PCR (RT-PCR) and the antigen test were used to detect active SARS-CoV-2 infections.
Oligoclonal bands in blood and CSF were negative, and blood antibodies, including anti-aquaporin-4 and anti-myelin oligodendrocyte glycoprotein, were also negative. Empirical therapy with ceftriaxone and acyclovir was started. After establishing a diagnosis of ADEM, patient was treated with methylprednisolone (MPS). The therapeutic plasma exchange (TPE) was also initiated, but the procedure has been stopped because of allergic reactions. The clinical status improved after MPS. Control lumbar puncture was done 12 days after the first one; CSF WBC count was 61 × 106/L and protein levels were 338 mg/L. She was discharged from the hospital without any symptoms except a mild headache. On follow-up after 40 days, she complained of only mild headache.
We are in a worldwide SARS-CoV-2 pandemic, COVID-19 infection could be complicated by many neurological symptoms and disorders: Guillain-Barre syndrome, encephalopathy, cerebrovascular diseases, meningitis, neuralgia, ataxia, or epileptic crisis.7, 8 In the literature, there are also reports of patients with ADEM associated with confirmed COVID-19 disease.9–17
Our patient manifested a typical radiological pattern for ADEM with extensive, diffuse demyelinating lesions in the brain and along all cervical and thoracic spinal cord. The CSF examination results were specific for ADEM with pleocytosis and the absence of oligoclonal bands. However, clinical urinary retention without lower limbs motor or sensory deficits is quite rare.
Panicker reported a bigger group of 61 patients with ADEM, where lower urinary tract dysfunction was found in 20 (33%) of them (16 patients had urinary retention), but mostly in patients with lower paraparesis.18
In the literature, we found only two cases of patients with ADEM following SARS-CoV-2 vaccination; two women both received inactivated SARS-CoV-2 vaccine of Sinovac (Vero Cells, Beijing Institute of Biological Products Co., Ltd., Beijing, China).19, 20
The first woman revealed symptoms 2 weeks after vaccination; she presented somnolence and memory decline and improved after steroids and iv immunoglobulins therapy. The second one was admitted to the hospital after the first tonic-clonic seizure one month after vaccination, she had typical scattered, demyelinating lesions in the brain, but because of lack of encephalopathy, it was called ADEM-like presentation.
The reported cases do not impair the importance of COVID-19 vaccinations with global SARS-CoV-2 pandemic, where the risk of neurologic complications, hospitalization, and even death of infected patients is still prevailing. But the clinicians should be aware of the potential implications of this rare neurological condition.