Embalmers are finding long, rubbery clots inside of corpses since the implementation of Covid vaccines.
According to one Alabama embalmer who has been treating corpses for over 20 year, the strange fibrous clots emerged in May of 2021, shortly after the Covid vaccines first became available to the public.
“It wasn’t until May or June of last year that I started to say, ‘something is really different about the blood’ and then later in September, I took my first picture, since I couldn’t come out with just one piece of evidence because what if it’s just a fluke?” Alabama embalmer Richard Hirschmann told 1819 News. “Now, I have been gathering evidence and I have pictures of over 100 cases. And it’s not stopping. It’s not slowing down.”
Embalmers are finding long, rubbery clots inside of corpses since the implementation of Covid vaccines.
According to one Alabama embalmer who has been treating corpses for over 20 year, the strange fibrous clots emerged in May of 2021, shortly after the Covid vaccines first became available to the public.
“It wasn’t until May or June of last year that I started to say, ‘something is really different about the blood’ and then later in September, I took my first picture, since I couldn’t come out with just one piece of evidence because what if it’s just a fluke?” Alabama embalmer Richard Hirschmann told 1819 News. “Now, I have been gathering evidence and I have pictures of over 100 cases. And it’s not stopping. It’s not slowing down.”
The Epoch Times spoke to Richard Hirschmann and other embalmers who have all documented the same rubbery clots in corpses starting in 2021.
“In 20 years of embalming, I had never seen these white fibrous structures in the blood, nor have others in my field. In the past year, I have seen these strange clots in many different individuals, and it doesn’t seem to matter what they die of, they often have similar substances in their blood. This makes me very concerned because if something is wrong in the blood, it begs the question: is something causing people to die prematurely?” Hirschman told the Epoch Times.
Hirschmann said he has noticed that the blood in people’s bodies has changed in the last two years.
Mr. Hirschmann said he cannot confirm that the blood clots are caused by the Covid vaccines, but it is his hope that the clots are investigated.
Have all documented the same rubbery clots in corpses starting in 2021.
Several embalmers across the country have been observing many large, and sometimes very long, “fibrous” and rubbery clots inside the corpses they treat, and are speaking out about their findings.
Numerous embalmers from different states confirmed to The Epoch Times that they have been seeing these strange clots, starting from either 2020 or 2021.
It’s not yet known if the cause of the new clot phenomenon is COVID-19, vaccines, both, or something different.
The Epoch Times received videos and photos of the anomalous clots, but could not upload them due to the level of gore.
Mike Adams, who runs an ISO-17025 accredited lab in Texas, analyzed clots in August and found them to be lacking iron, potassium, magnesium, and zinc.
Adams’s lab uses inductively coupled plasma mass spectrometry (ICP-MS), triple quadrupole mass spectrometer, and liquid chromatography-mass spectrometry, usually testing food for metals, pesticides, and glyphosate.
“We have tested one of the clots from embalmer Richard Hirschman, via ICP-MS. Also tested side by side, live human blood from an unvaccinated person,” Adams told The Epoch Times.
He found that the clots are lacking key elements present in healthy human blood, such as iron, potassium, and magnesium, suggesting that they are formed from something other than blood.
Adams is joining analytic forces with more doctors and plan to invest out of their own pocket in equipment in order to further determine their composition and probable causation.
The string-like structures differ in size, but the longest can be as long as a human leg and the thickest can be as thick as a pinky finger.
Neurological manifestations are a significant complication of coronavirus disease (COVID-19), but underlying mechanisms aren’t well understood. The development of animal models that recapitulate the neuropathological findings of autopsied brain tissue from patients who died from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection are critical for elucidating the neuropathogenesis of infection and disease. Here, we show neuroinflammation, microhemorrhages, brain hypoxia, and neuropathology that is consistent with hypoxic-ischemic injury in SARS-CoV-2 infected non-human primates (NHPs), including evidence of neuron degeneration and apoptosis. Importantly, this is seen among infected animals that do not develop severe respiratory disease, which may provide insight into neurological symptoms associated with “long COVID”. Sparse virus is detected in brain endothelial cells but does not associate with the severity of central nervous system (CNS) injury. We anticipate our findings will advance our current understanding of the neuropathogenesis of SARS-CoV-2 infection and demonstrate SARS-CoV-2 infected NHPs are a highly relevant animal model for investigating COVID-19 neuropathogenesis among human subjects.
Multiple and continuing reports demonstrate a substantial number of patients with coronavirus disease 2019 (COVID-19) develop new-onset neurological symptoms. Several case reports have, in fact, identified neurological complications as the initial presentation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, particularly among those who develop stroke1,2,3. Among the more urgent COVID-19-associated neurological presentations, stroke, meningoencephalitis, and hemorrhagic necrotizing encephalopathies have been associated with more severe disease2,4,5,6; however, even comparatively mild neurological symptoms, such as dizziness or unresolving headache4,7, may be indicative of neuropathological processes in the context of infection and disease. Notably, individuals across the lifespan, with and without significant comorbidities, and with all disease severities, including asymptomatic patients, have suffered the variety of reported neurological manifestations8.
While damage to the central nervous system (CNS) of patients with COVID-19 is increasingly evident, the neuropathogenesis remains unclear. Here, we provide a comprehensive assessment of brain pathology associated with SARS-CoV-2 infection in two non-human primate (NHP) models of infection with varied disease severity. This work reveals neuroinflammation, brain hypoxia, microhemorrhages, and pathology consistent with hypoxic-ischemic injury with rare infection of brain vasculature in SARS-CoV-2 infected NHPs and provides key insights into SARS-CoV-2-associated neuropathogenesis. Our findings are consistent with those reported on autopsied brain of human subjects who died with SARS-CoV-2 infection. Additional molecular analyses on brain from our animal models suggest reduced oxygen to the CNS may contribute significantly to injury in the context of infection. Importantly, animals that did not develop acute respiratory distress syndrome (ARDS) demonstrated neuropathology that may lead to long-term neurological symptoms of post-acute sequelae of COVID-19 (PASC), or “long COVID”.
Significant inflammation in brain
Eight adult NHPs, including four Rhesus macaques (RM), 13–15 years of age, and four wild-caught African green monkeys (AGMs), approximately 16 years of age, were inoculated with the 2019-nCoV/USA-WA1/2020 strain of SARS-CoV-29 via a multi-route mucosal or aerosol challenge (Table 1). Two animals of each species were inoculated via aerosol and two by multi-route exposure. Multi-route mucosal exposure included conjunctival, nasal, pharyngeal, and intratracheal routes. Control animals included two RMs, approximately 18–22 years of age, and two AGMs, approximately 17 years of age. Control animals were mock-infected through multi-route mucosal exposure of the same growth media used for virus propagation. All study animals underwent the same clinical tests and procedures.Table 1 Study animals.Full size table
All animals exposed to SARS-CoV-2 developed infection within the first week of exposure, as demonstrated by the detection of the viral nucleocapsid (N) mRNA in nasal swabs taken within 3–7 days after challenge (Table 1). No differences in infection were noted between the two inoculation strategies. Further verification of infection is seen through detection of the virus by immunohistochemistry (IHC) in lung (Supplementary Data Fig. 1). Additional detailed findings in lung and clinical measures have been previously reported10.
All animals survived to study endpoint, except for AGM1 and AGM2. At 8 days post infection, AGM1 was found recumbent and marginally responsive to stimuli. This animal also presented with dyspnea/tachypnea (respiratory rate of 72 breaths per minute), hypothermia (<32.2 °C), and hypoxemia [blood oxygen saturation (SpO2) = 77%] and was euthanized. At 22 days post infection, shortly before its scheduled study endpoint, AGM2 developed severe tachypnea, hypothermia, and hypoxemia, with a respiratory rate of 96 breaths per minute and SpO2 = 77% and was subsequently euthanized.
Seven regions of the CNS, including frontal, parietal, occipital, and temporal lobes, basal ganglia, cerebellum, and brainstem were collected at necropsy from all animals and investigated for neuroinflammation through histopathological and immunohistochemical methods. A summary of the neuropathological findings is included in Table 2.Table 2 CNS Pathology and Summary of Findings.Full size table
Neuroinflammation was seen in all study animals but was greater in those with SARS-CoV-2, as compared to age-matched mock-infected controls (Fig. 1). The pan-microglial protein, ionized calcium-binding adapter molecule 1 (Iba-1), was upregulated in the context of infection and revealed morphological alterations indicative of microglial activation, with retracted, thickened processes and a large cell body (Fig. 1b, d). Occasional, small perivascular cuffs were observed in infected (Fig. 1f, h) but not control animals (Fig. 1e, g). In contrast, nodular lesions were seen more frequently than cuffs and were present in both infected (Fig. 1j, l) and mock-infected (Fig. 1i, k) animals, however, these appeared larger in the context of infection.
To further characterize microglial activation, tissues were investigated for the MHC class II cell surface receptor, HLA-DR (Fig. 1m–p). Similar to findings in brain of aged human subjects11, microglial expression of HLA-DR was observed in animals without SARS-CoV-2 infection (Fig. 1m, o). Expression was also seen in brain of infected animals (Fig. 1n, p); however, this did not appear greater than those that were mock-infected. HLA-DR did highlight nodular lesions in all animals, which were larger in infection, as seen with Iba-1.
Additional evidence of increased neuroinflammation in infection was seen through glial fibrillary acidic protein (GFAP) IHC, which was upregulated in infected animals (Fig. 1r, t), as compared to age-matched controls (Fig. 1q, s). GFAP immunopositivity revealed astrocytic hypertrophy in the context of aging, suggestive of astrocyte activation, however, this was more pronounced in infection, which also displayed significant loss of individual astrocytic domains.
Neuronal injury and apoptosis
Hematoxylin and eosin (H&E; Fig. 2) staining revealed marked changes in neuronal morphology, which was most often observed in cerebellum and brainstem (Fig. 2b–d). Neuronal degeneration was characterized by pyknotic and karyorrhectic nuclei with shrunken cytoplasm and vacuolation in the surrounding neuropil (Fig. 2b–d). The cerebellum contained several regions of degenerate Purkinje neurons that exhibited cellular blebs and debris and cytoplasmic vacuoles (Fig. 2b, c). Contiguous with areas of degenerate Purkinje cells, neurons and glia within the molecular and granular layers appeared pyknotic with condensed, basophilic nuclei (Fig. 2b). Similar morphologic changes were noted in glial cells adjacent to apoptotic neurons in the brainstem (Fig. 2d). In both brainstem and cerebellum, neurons are seen at various stages of nuclear dissolution (Fig. 2b–d). Degeneration of Purkinje cells was further confirmed with FluoroJade C (Fig. 2e, f).
Given the prominent morphologic changes noted within Purkinje cells, we sought to identify the mechanisms underlying these degenerative changes by investigating all brain regions for the presence of cleaved caspase 3, the activated form of this key executioner of apoptosis. Cleaved caspase 3 was seen in at least one CNS region from all infected animals except AGM1, which did not have any positive cells (Fig. 2i, Supplementary Data Fig. 2). Three animals, RM1, AGM3, and AGM4 showed positivity in more than one brain region, while RM2 had cleaved caspase 3 positive cells in all regions examined (Fig. 2i; Supplementary Data Fig. 2). In cerebellum, cytoplasmic and nuclear-cleaved caspase 3 was predominantly restricted to cells within and proximal to the Purkinje cell layer (Fig. 2g). Other CNS regions, including brainstem, had foci of cleaved caspase 3 positivity (Fig. 2h). In comparison to infected animals, mock-infected controls showed little-to-no positivity (Fig. 2i; Supplementary Data Fig. 2). Unbiased quantitation revealed a statistically significant difference in cleaved caspase 3 positivity between infected and mock-infected animals in all brain regions investigated (Fig. 2i). When stratified by species, statistical significance was not achieved by Mann–Whitney U Test, which is likely due to the low number of each species (Supplementary Data Fig. 2). Interestingly, cleaved caspase 3 was not detected in any CNS region examined from AGM1, who was euthanized at 8 days post infection due to advanced illness. This may suggest programmed cell death in the CNS occurs later in the disease process.
While vacuolation was at times observed in the cerebellar gray and white matter (Supplementary Data Fig. 3a, b), significant demyelination was not a major finding in this study. Luxol Fast Blue (LFB) did reveal localized myelin pallor, suggestive of oligodendrocyte injury and/or loss, in the cerebellum of RM3 and occipital lobe of AGM3 (Supplementary Data Fig. 3c, d).
Microhemorrhages, as suggested by the presence of erythrocyte extravasation into the brain parenchyma, were identified in all study animals and seen with and without ischemic injury of adjacent tissues, characterized by localized/regional tissue pallor (Fig. 3a–f). Although the number of bleeds varied, all animals were observed to have at least one. Infected animals appeared to have larger bleeds than mock-infected controls, with more dense accumulation of red blood cells on the parenchymal side of the blood vessel (Fig. 3, compare a–d with e, f). Quantitation of microhemorrhages was determined on Axio Scan.Z1 (Zeiss) scanned slides and HALO software (Indica Labs, v2.3.2089.70 and v3.1.1076.405) and normalized by tissue area (Fig. 3g). The whole brain showed a higher increase in the number of microbleeds in infection which reached statistical significance in the basal ganglia (Fig. 3h and Supplementary Data Fig. 4).
Accumulation of cerebral microhemorrhages occurs with aging and are seen most frequently in deep brain structures, including brainstem, basal ganglia, and cerebellum12. This may be due to age-associated decrease in arterial elasticity and increased blood pressure on brain microvasculature, as well as other risk factors for vascular injury, such as diabetes and dyslipidemia. Vascular injury can promote thrombosis, or blood clot formation within a blood vessel, which may aid in stopping the brain microbleed or, conversely may underlie microhemorrhages and result in more serious brain injury by impeding the flow of blood in the brain, leading to stroke. To assess the potential contribution of thrombosis to microhemorrhage development in SARS-CoV-2 infection, we examined all brain regions for luminal accumulation of the platelet glycoprotein, CD61 (aka, integrin b-3). This revealed multiple blood vessels with aggregated platelets in both infected and mock-infected animals, which were seen with and without associated microbleeds (Fig. 4a–d). Microhemorrhages without CD61 accumulation were also observed (Fig. 4e, f). Quantitation of total brain microhemorrhages with and without associated CD61 positivity revealed a greater frequency without thrombi (CD61 positivity) in the context of infection, apart from AGM5 who had many bleeds without visible thrombi (Fig. 4g, h). These findings suggest that in the context of infection, leakage of blood vessels without vascular damage/injury occurs more frequently.
Chronic hypoxemia/brain hypoxia
Microhemorrhages and ischemia appear to play a central role in neuronal injury observed in this study. The brain is a highly metabolic organ with a limited capacity for energy storage. Due to the significant energy demands of the brain and neurons, a prolonged reduction in blood flow and concomitant reduction in oxygen and glucose can be detrimental to neuronal vitality, in addition to the resulting neurotoxicity of erythrocyte breakdown products and inflammation. Of particular interest is the finding that AGM1, who was found recumbent and minimally responsive to stimuli at 8 days post infection, had a substantial number of microbleeds in the cerebellum, basal ganglia, and brainstem (Table 2). These findings suggest AGM1 suffered multiple acute microhemorrhages that may have contributed to her rapid decline. Alternatively, AGM1’s rapid pulmonary decline may have promoted end stage microhemorrhages. The timing of acute microhemorrhages in the disease process is unclear and warrants further investigation.
In addition to localized ischemic injury, all infected animals experienced variations in SpO2 that fluctuated between 89 and 99% but stayed below 95% for most over the study course (Fig. 5a). Correspondingly, blood carbon dioxide (CO2) ranged from 24 to 33 mEq/L, remaining above the physiological range for most of the study animals (Fig. 5b). While these levels are not immediately alarming, they may suggest mild hypoxemia and impaired gas exchange in the lungs. As such, chronic hypoxemia may contribute to impairment of the endothelium and/or neurovascular unit leading to increased vascular permeability. The brain requires aerobic metabolism of glucose for ATP production and any prolonged or intermittent reductions of blood O2 may contribute to localized CNS hypoxia and energy failure. Even minor reductions in oxygen may promote injury, particularly among neurons, which appear to have suffered the greatest insult in this study. In support of this notion, large regions of Purkinje cells, which are especially vulnerable to hypoxic insult13,14, as well as cells in their immediate proximity, appear degenerate or committed to undergoing apoptosis.
To assess brain tissue for evidence of hypoxia, we performed IHC against the oxygen-regulated alpha subunit of hypoxia inducible factor-1 (HIF-1a), which is upregulated and stabilized under hypoxic conditions. For this analysis, only basal ganglia, brainstem, and cerebellum were investigated because our earlier studies demonstrated these brain regions had the greatest injury/pathology. This study demonstrated marked upregulation of HIF-1a in brain of infected animals, as compared to mock-infected controls (Fig. 5f–m). Areas of intense positivity, suggestive of HIF-1a accumulation, were predominantly seen in and around blood vessels, which extended into the brain parenchyma in infection (Fig. 5g, i, k, m). Areas of HIF-1a positivity were noted in mock-infected animals but were less intense than that seen in brain of infected animals and/or did not extend appreciably into the parenchyma (Fig. 5f, h, j, l). Non-biased quantitation of HIF-1a intensity [optical density (OD)] around blood vessels, which excluded the blood vessel lumen, revealed a statistically significant increase in HIF-1a by cells comprising the vasculature and neighboring parenchymal cells of infected animals, as compared to controls, in brainstem (Fig. 5c, *p = 0.0154) and basal ganglia (Fig. 5d, **p = 0.0016) but not cerebellum (Fig. 5e, p = 0.0940). Our approach for quantifying HIF-1a expression around the vasculature, while excluding the blood vessel lumen, is shown in Supplementary Data Figs. 5 and 6. Statistical significance was only retained in the basal ganglia when stratified by species (RMs *p = 0.049, AGMs *p = 0.034; Supplementary Data Fig. 7).
Rare virus in brain-associated endothelium
The potential for direct virus involvement in CNS pathology was explored through IHC and RNAscope analyses of all brain regions. Using an antibody against SARS-CoV-2 nucleocapsid protein (SARS-N), IHC studies revealed rare virus infection in brain that, when seen, appeared to be restricted to the vasculature (Fig. 6a). Sparse virus was detected most frequently within the basal ganglia, cerebellum, and/or brainstem and seen less often within the temporal, parietal, and occipital lobes (Table 2). This was verified further through in situ hybridization (ISH) analyses, employing RNAscope Technology with enhanced signal amplification. Using an anti-sense probe to the viral spike protein RNA (SARS-S), cytoplasmic positivity was seen in brain of infected animals but not in mock-infected controls (Fig. 6c–h; Supplementary Data Fig. 8). The specificity of the probe used in these studies is demonstrated in lung, which only showed positivity in the context of infection (Supplementary Data Fig. 8).
The single-label studies suggested SARS-CoV-2 infection in brain is limited to the brain vasculature and appeared to be restricted to endothelial cells. Suspected endothelial cell infection is supported by colocalization of SARS-N with von Willebrand factor (vWF; Fig. 6i–k). A blood vessel in close proximity to that shown in Fig. 6i–k but without detectable virus is included to demonstrate the specificity of the SARS-N antibody (Fig. 6l–n).
Using a highly sensitive CRISPR-based fluorescent detection system (CRISPR-FDS)15, virus was not identified in the cerebrospinal fluid (CSF) (Fig. 6o), consistent with most findings among human subjects, except in rare cases of encephalitis16,17,18. In contrast, this method detected limited viral RNA in whole brain, frozen at the time of necropsy, that was largely representative of our IHC/IF findings (Fig. 6o). Similar to our findings in fixed tissues, virus was more frequently observed in basal ganglia, cerebellum, and brainstem. CRISPR-FDS analysis also revealed viral RNA in the frontal lobe of one animal, AGM1, which was not convincingly seen by IHC/IF for this region in any study animal. This may reflect differences in sampling error that is inherently present in the two methods, where the amount of tissue used for the CRISPR-FDS studies is greater than that used in IHC, and/or extracerebral virus that may have been present in the blood vessel lumen.
Together, our findings demonstrate scarce SARS-CoV-2 infection in brain-associated endothelial cells in deep brain structures of NHPs, even in the absence of severe disease or overt neurological symptoms.
Neurological manifestations are commonly seen in the context of SARS-CoV-2 infection but are highly varied and range in severity from impaired smell and/or taste to stroke2,19. As such, the mechanisms underlying SARS-CoV-2-associated neurological complications are likely complex. Relevant animal models of infection and CNS involvement that reflect human disease are critical for elucidating these mechanisms, as well as identifying and/or developing effective therapeutic strategies. In our two models of aged NHPs infected with SARS-CoV-2, we found evidence of prominent neuroinflammation, microhemorrhages with and without associated microthrombi, and neuronal injury and death consistent with hypoxic-ischemic injury but without substantial virus detection in brain. Our findings are largely in line with those reported in autopsy studies of individuals who died from infection20,21,22,23,24,25,26. Like human disease, reactive astrocytes and microglia were a common feature, seen throughout the entirety of the brain in infected animals. This appeared greater in basal ganglia, brainstem, and cerebellum, which contained the majority of cuffs and nodular lesions observed. Lymphocyte infiltrate, which has been reported in human brain22,24, was not observed in any brain region investigated from our study animals. This may reflect a shorter time with severe disease in our animal model. Additional life-saving efforts were not made for animals that developed serious disease (e.g., ARDS), as would be done with humans, and were quickly euthanized to minimize pain and suffering of the animal. It is worth noting that autopsy reports of significant lymphocyte infiltration into the CNS or COVID-associated encephalitis are relatively few and may be a less common complication of disease8.
Our findings of hypoxic-ischemic injury in brain of NHPs are also in agreement with autopsy studies of brain from human subjects21,27. This may arise from chronic, peripheral hypoxemia, as well as reduced cerebral blood flow due to acute microhemorrhages. The brain is a highly metabolic organ and requires aerobic metabolism of glucose for adenosine triphosphate (ATP) production. Any prolonged or chronic intermittent reductions of blood SpO2 may contribute to localized CNS hypoxia and energy failure. Even minor, but sustained, reductions in oxygen may promote injury, particularly among neurons, which appear to have suffered the greatest insult in this study. In support of this notion, large stretches of Purkinje cells, which are especially vulnerable to hypoxic insult13,14, as well as cells within their immediate proximity, appear degenerate and/or committed to undergoing apoptosis. Areas of injured neurons at various stages of nuclear dissolution were noted in other brain regions, including brainstem. Moreover, neuronal injury did not appear to be a direct consequence of virus infection, as only limited virus was convincingly detected in brain vasculature and did not appear to involve parenchymal cells. Instead, neuronal injury and death most likely occur as a result of energy failure, which is an early consequence of hypoxic-ischemic events. Multiple microhemorrhages, microinfarcts, and hypoxemia appear to play a role in neuronal injury and death observed in these animals.
Consistent with a hypoxic environment, we detected upregulation/stabilization of HIF-1a in infected animals that localized to the brain vasculature and was significantly greater than mock-infected controls in the deep brain regions assessed. This was observed in all infected animals, regardless of disease severity, suggesting reduced brain oxygen may be a common complication of infection. While the mechanism is not yet elucidated, chronic hypoxemia, as well as an exaggerated and prolonged immune response likely play an important role. Indeed, several inflammatory mediators and growth factors have been reported to stabilize and/or promote expression of HIF-1a, including nitric oxide, interleukin 1b, and tumor necrosis factor-a28,29,30.
Interestingly, we did not observe HIF-1a upregulation in cerebellar Purkinje cells in any animal. This may be due to the kinetics of HIF-1a expression, which has been shown in a mouse model of chronic hypoxia to peak in Purkinje cells at 4–5 h and return to normoxic levels after 9–12 h in a continual hypoxic environment31. These findings suggest that any potential upregulation and/or stabilization of HIF-1a in Purkinje cells had returned to normal levels by the time the animals were euthanized. It is also likely that degenerate Purkinje cells no longer produce HIF-1a. Our conflicting findings in the brain vasculature may be due to continued exposure of these cells to peripheral factors that promote HIF-1a stabilization and/or expression.
A direct role for the virus in HIF-1a upregulation cannot be ruled out, however, the negligible frequency of SARS-CoV-2 infected cells seen in the CNS compartment argues against the virus being a significant factor. A recent RNAseq analysis, however, found increased HIF-1a mRNA in peripheral blood mononuclear cells (PBMC) acquired from SARS-CoV-2 infected human subjects, as compared to healthy, non-infected controls32. Additional in vitro analyses suggested SARS-CoV-2 ORF3a protein induces HIF-1a production in transfected cells, as well as several cytokines upregulated in the context of infection32. How this translates to HIF-1a expression in vivo, however, remains unclear.
In agreement with most reports of living subjects and those who died from COVID-198, we did not detect virus in CSF and found only minimal virus in the brain that appeared to be limited to the vasculature, suggestive of hematological dissemination of virus to the brain. Infection of pericytes, perivascular macrophages, and/or cells within the brain parenchyma cannot be ruled out but was not convincingly demonstrated in these studies. Instead, virus appeared to be restricted to the endothelium, which is consistent with a previous study of human biopsy tissues that demonstrated the principal receptor for SARS-CoV-2, angiotensin-converting enzyme 2 (ACE2), is expressed by endothelial cells throughout the body, including brain33,34. More recently, a large autopsy series out of Mount Sinai demonstrated robust ACE2 expression by brain vasculature in patients who died from SARS-CoV-2 infection20. This may suggest a greater vulnerability of the brain to infection in the context of severe disease but was not observed in NHPs that developed ARDS in this study. One autopsy report identified virus in a subset of cranial nerves22, however, these were not available for investigation. Additionally, the olfactory bulb, which was not recovered from our study animals, may also be an important site for virus entry into the CNS and requires additional investigation.
Notably, the animals in this study were of advanced age, which is associated with a higher risk for the development of cerebrovascular disease among infected patients8. Indeed, aging, itself, is the greatest risk factor for cerebrovascular disease, due, at least in part to age-related changes of cerebral vascular structure and/or function that contribute to reduced cerebral blood flow, which may be further compounded by underlying vascular pathology35,36. This may predispose the aging vasculature to cerebrovascular events, particularly in the context of prolonged systemic inflammation and hypoxemia, which have been shown to contribute to increased vascular permeability through microglia and astrocyte responses37,38.
Here, we show substantial pathological changes in brain of SARS-CoV-2 infected NHPs that are compatible with autopsy and imaging reports of infected human subjects. Additionally, our pathological investigation suggests a significant role for brain hypoxia in the neuropathogenesis of COVID-19, including animals without severe disease. It is reasonable to anticipate that similar findings may occur among human subjects, particularly those with continuing neurological symptoms after recovery from infection39,40,41. For example, an increasing number of retrospective neuroimaging reports have reported cerebral microhemorrhages in critically ill patients with COVID-1942,43,44. Many patients, however, including those who do not require hospitalization, report comparatively milder neurological symptoms that are not evaluated through neuroimaging. As such, neuropathology among these individuals remains unclear but likely contributes to lingering neurocognitive difficulties reported by a number of convalesced/convalescing patients45 and warrants further investigation. This further increases the significance of NHPs as a viable model for elucidating the mechanisms that underlie SARS-CoV-2-associated neuropathology that are translatable to human disease, as neuropathogenesis can be more closely examined in animals that do not experience mortal disease. Additionally, neuropathological complications may contribute to worsening disease among infected patients. For example, damage to the brainstem, which modulates the respiratory cycle by regulating inspiratory and expiratory muscle activity, may contribute to worsening respiratory distress and failure in patients with COVID-19. Additional studies, employing relevant animal models, are warranted and likely to reveal important insight into human disease.
While SARS-CoV-2 neuropathogenic processes are poorly understood, this work reveals infected NHPs are a viable animal model for understanding the neuropathogenesis and potential long-term consequences of infection. We also provide important insight into the mechanisms underlying CNS disease, which was seen even in the absence of severe respiratory disease and may suggest that vascular leakage and hypoxic brain injury is a common complication of SARS-CoV-2 infection and COVID-19. Neuronal degeneration and activation of caspase 3 observed in this study supports this notion and indicates non-reversible neuronal injury may be significant to individuals suffering from PASC. Finally, our findings and conclusions presented herein suggest the need for long-term neurological follow-up of persistently symptomatic convalescent patients.
Ethics and biosafety statement
All animal studies were approved by the Tulane University Institutional Animal Care and Use Committee and carried out in the Regional Biocontainment Laboratory at the Tulane National Primate Research Center (TNPRC) within an animal biosafety level 3 facility. The TNPRC is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). All animals were cared for in accordance with the Institute for Laboratory Animal Research Guide for the Care and Use of Laboratory Animals, 8th edition. The Tulane University Institutional Biosafety Committee (IBC) approved all procedures for sample handling, inactivation, and removal from BSL3 containment.
Animal study design
A total of twelve NHPs, including six Indian-origin RMs (ages 13–21 years) and six AGMs of Caribbean origin (all approximately 16–17 years of age), were included in this study. Four of each species was inoculated with SARS-CoV-2 strain 2019-nCoV/USA-WA1/2020 (MN985325.1) and two of each species were mock-infected with culture media used for virus propagation (Table 1). The viral strain used was isolated from the first confirmed SARS-CoV-2 case in the United States and deposited by the Centers for Disease Control9. All animals underwent the same procedures and biological sampling.
All RMs were acquired from the TNPRC specific pathogen-free breeding colony and confirmed negative for simian type D retrovirus (SRV), simian immunodeficiency virus (SIV), simian T cell lymphotropic virus type 1 (STLV1), measles virus (MV), Macacine herpesvirus 1 (MHV1/B virus), and tuberculosis (TB). The AGMs were wild-caught and also confirmed negative for SRV, SIV, STLV, MV, and TB. The AGMs were housed at the Center for over a year before assignment to this study. All animals were tested and found negative for SARS-CoV-2 (antibody and virus) prior to experimental infection.
Two routes of virus exposure, multi-route mucosal and aerosol, were employed to mimic major routes of infection among humans. Two animals from each species were randomly subjected to the different routes of exposure for a total of four animals in each species challenge group. Multi-route exposure included conjunctival, nasal, pharyngeal, and intratracheal routes for a cumulative dose of 3.61 × 106 PFU (plaque-forming unit). Animals exposed to virus by aerosol received an approximate inhaled dose of 2 × 103 TCID50 (50% tissue culture infectious dose). Study animals were euthanized for necropsy at 24–28 days post infection unless humane endpoints required euthanasia at an earlier time (Table 1). Postmortem examination was performed by a board-certified veterinary pathologist (R.V.B.).
Quantification of Nasal Swab SARS-CoV-2 subgenomic nucleocapsid mRNA (sg-N mRNA)
Nasal swab specimens were collected in 200 µL DNA/RNA Shield (Zymo Research) and extracted for viral RNA (vRNA) using the Quick-RNA Viral kit (Zymo Research). Viral RNA Buffer (Zymo) was dispensed directly to the swab in the DNA/RNA Shield (Zymo). A modification to the manufacturers’ protocol was to insert the swab directly into the spin column to centrifugate, allowing all the solution to cross the spin column membrane. The vRNA was eluted (45 µL), from which 5 µL was added to a 0.1 mL fast 96-well optical microtiter plate format (Thermo Fisher) for a 20 µL RT-qPCR reaction. The RT-qPCR reaction used TaqPath 1-Step Multiplex Master Mix (Thermo Fisher) along with the following primers and probe: Forward primer: (sgm-N FOR) 5′-CGATCTCTTGTAGATCTGTTCTC-3′; Probe: (sgm-N PRB) 5′-FAM TAACCAGAATGGAGAACGCAGTGGG-BHQ1-3′; Reverse primer: (sgm-N REV) 5′-GGTGAACCAAGACGCAGTAT-3′. The reaction master mix was added using an X-Stream repeating pipette (Eppendorf) to the microtiter plates. Loaded plates were covered with optical film (Thermo Fisher), vortexed, and pulse centrifuged. The RT-qPCR reaction employed the following program: UNG incubation at 25 °C for 2 min, RT incubation at 50 °C for 15 min, and an enzyme activation at 95 °C for 2 min, followed by 40 cycles of denaturation at 95 °C for 3 s and annealing at 60 °C for 30 s. Fluorescence signals were detected with an Applied Biosystems QuantStudio 6 Sequence Detector. Data were captured and analyzed with Sequence Detector Software v1.3 (Applied Biosystems). Equivalent viral copy numbers were calculated by plotting Cq values obtained from unknown (i.e., test) samples against a standard curve representing known viral copy numbers. The limit of detection of the assay was ten copies per reaction volume. A 2019-nCoV positive control (IDTDNA) was analyzed in parallel with every set of test samples to verify the RT-qPCR master mix and reagents were prepared correctly. A non-template control was included in the qPCR to ensure there was no cross-contamination between reactions.
IHC was performed on 5 µm zinc formalin-fixed paraffin-embedded (FFPE) brain sections46. Sections were deparaffinized in xylenes and rehydrated through an ethanol series ending in distilled water. Heat-mediated antigen retrieval was carried out in a vacuum oven with Tris-EDTA buffer (10 mM Trizma base, 1 mM EDTA, 0.05% Tween 20, pH 9.0) or sodium citrate buffer (10 mM sodium citrate, 0.05% Tween 20, pH 6.0). All washes were performed using tris buffered saline containing Tween 20 (TTBS; 0.1 M Trizma base, 0.15 M NaCl, 0.1% Tween 20, pH 7.4). Following antigen retrieval, tissues were blocked with 20% normal horse or goat serum. Endogenous biotin was blocked with Avidin-Biotin Solution (Vector Labs). Titrated primary antibodies included anti-cleaved caspase 3 (rabbit polyclonal, 1:250, Abcam, ab2302), anti-von Willebrand factor (vWF, rabbit EPR12010, 1:62.5, Abcam, ab179451), anti-HIF-1a (mouse mgc3, 1:1600, Abcam, ab16066), anti-CD61 (rabbit RM382, 1:125, Invitrogen, MA5-33041), anti-ionized calcium-binding adapter molecule 1 (Iba-1, goat polyclonal, 1:200, Abcam, ab5076), anti-GFAP (rabbit EPR1034Y, 1:500, Abcam, ab68428), anti-HLA-DR (mouse TAL.1B5, 1:400, Novus, NB600989), and anti-SARS-CoV-2 nucleocapsid (rabbit polyclonal, 1:125, Novus, NB100-56576). Tissues were incubated with primary antibody overnight at room temperature and detected using the appropriate biotinylated secondary antibody (1:200, Vector Labs, BA-1100, BA-2000, BA-9500) and alkaline phosphatase-Vector Red according to manufacturer instructions (Vector Labs). Tissues were counterstained with Mayer’s hematoxylin and coverslipped.
Double labeling of 5 µm FFPE brain tissue was performed by sequential application of primary antibodies with their corresponding secondary47. SARS-CoV-2 nucleocapsid was detected with Alexa Fluor 555 goat anti-rabbit IgG (1:500, Invitrogen, A21428). Von Willebrand factor was detected with Alexa Fluor 488 goat anti-rabbit IgG (1:500, Invitrogen, A11008). Controls consisted of brain tissue incubated in blocking buffer only, tissue incubated with one primary and the corresponding secondary antibody, and tissue incubated with fluorophore-conjugated secondaries only. Tissues were coverslipped with Vectashield® HardSet™ Antifade mount with DAPI (Vector Labs).
In situ hybridization (RNAscope)
ISH was carried out on 5 µm FFPE tissues using RNAScope® Multiplex Fluorescent V2 Assay Kit (Advanced Cell Diagnostics), according to the manufacturer’s directions. Briefly, sections were deparaffinized in xylenes and dried, followed by incubation with hydrogen peroxide. Heat-mediated antigen retrieval was carried out in a steamer with the provided kit buffer. A hydrophobic barrier was drawn around the tissues before treatment with the kit-provided protease reagent and hybridized with the V-nCoV2019-S probe (Advanced Cell Diagnostics) in a HybEZ oven (Advanced Cell Diagnostics). All washes were performed with the kit wash buffer. Signal amplification was accomplished with three successive AMP solutions and HRP channel (Advanced Cell Diagnostics) and visualized with Opal 570 (1:1000, Akoya Biosciences). Autofluorescence was quenched with TrueVIEW® Autofluorescence Quenching Kit (Vector Labs). Positive and negative control tissues and tissues without probe exposure were included in every run to ensure the specificity of staining and assess background.
Hematoxylin and eosin
Deparaffinized and rehydrated slides were taken through Hemalast and hematoxylin, followed by differentiator and bluing solutions. After which, slides were dehydrated in 95% EtOH and stained with eosin. Stained slides were dehydrated, cleared, and coverslipped.
Luxol fast blue
Slides were deparaffinized and rehydrated through 95% EtOH, then incubated in warmed 0.1% LFB solution. Afterward, slides were washed, dipped in 0.05% lithium carbonate, differentiated in 70% EtOH, and rinsed. Following a check under microscope, the slides were oxidized in 0.5% periodic acid solution, then immersed in Schiff’s reagent before rinsing, dehydration, clearing, and coverslipping.
Five micrometers FFPE tissues were immersed in 0.06% KMNO4 for 10 min and washed. Tissues were then immersed in 0.0002% FluoroJade C (Histo-Chem) containing 0.1% acetic acid in the dark for 20 min, counterstained with 4′,6-diamidino-2-phenylindole (DAPI), washed, and dried at 60 °C. Cleared tissues were coverslipped with DPX mount (Sigma).
Imaging and quantitation
Slides were scanned with the Axio Scan.Z1 digital slide scanner (Zeiss). Brightfield images were acquired using HALO (Indica Labs, v2.3.2089.70 and v3.1.1076.405). Fluorescent images were acquired on a Leica DMi8 automated confocal microscope, model SP8, equipped with a Leica imaging software application suite X model LAS X, software v220.127.116.1125 and an Olympus IX73 inverted microscope with cellSens Dimension 3 software v3.1. Colocalization images were created in Photoshop (Adobe, v21.2.0) by overlaying the same image acquired through the appropriate fluorophore filter. Presented images were subjected to brightness, contrast, and/or darken midtones enhancement in Photoshop, applied to the entire image to reduce background.
Threshold and multiplex analyses were performed with HALO algorithms for non-biased quantitation of proteins of interest, without processing. For active caspase 3 hematoxylin-stained nuclei were used to quantify the number of cells and Vector Red intensity above a rigorous threshold accounted for the cells positive. Quantitation of HIF-1a was performed using an area quantification algorithm for Vector Red intensity. Annotations were drawn to outline blood vessel-associated parenchymal stain based on the algorithm results. The annotated area was analyzed for OD of Vector Red staining. The average OD within the annotated area was calculated in HALO per tissue section.
Microhemorrhages were independently counted and annotated within HALO on seven distinct 5 µm CD61-immunostained regions of the CNS from all infected and control animals by two individuals. Counts were normalized by area of each tissue section. Microhemorrhages were defined by the presence of blood vessels with red blood cell extravasation (>10 red blood cells on the parenchymal side of an unbroken blood vessel). Normalized microhemorrhage counts were plotted for each specific brain region and total regions investigated. CD61+ aggregates within blood vessels were counted and annotated on HALO on seven distinct 5 µm sectioned regions of the CNS from all infected and control animals by two individuals. Blood vessel-associated CD61+ thrombi were defined by aggregated CD61 stained platelets within a vessel.
RNA isolation from whole tissues
Dissected frontal lobe, basal ganglia, cerebellum, and brainstem were collected fresh and immediately frozen at necropsy. One milliliter of Trizol LS (Thermo Fisher) was added to 100 mg of thawed tissue and homogenized in gentleMACS M tubes using a gentleMAC Dissociator (Miltenyi Biotec). The resulting lysate was then centrifuged at 3000 × g for 5 min and supernatant transferred into a 2 mL microcentrifuge tube. An equal volume of ethanol (95–100%) was added to the sample in Trizol LS (1:1) and mixed well. The resulting mixture was transferred to a Zymo-Spin III CG Column in a 2 mL collection tube (Zymo) and centrifuged for 30 s. The column was washed with RNA Wash Buffer (Zymo), followed by treatment with DNase I for 30 min to remove residual genomic DNA. The column was washed with RNA Wash Buffer (Zymo) and RNA eluted with 45 μL of DNase/RNase-free water (Thermo Fisher).
CRISPR-based fluorescent detection system (CRISPR-FDS)
CRISPR-FDS reaction was carried out with the following steps15. Isolated RNA samples were mixed with one-step RT-PCR mix containing 2× PlatinumTM SuperFiTM RT-PCR Master Mix (Thermo Fisher), forward primer (10 μM), reverse primer (10 μM), SuperScriptTM IV RT Mix (Thermo Fisher), and nuclease-free water. Samples were then incubated in a T100 thermocycler (Bio-Rad) using a cDNA synthesis protocol, immediately followed by a DNA amplification protocol. CRISPR-FDS reactions were performed as follows: a sample RT-PCR reaction was transferred to a 96-well half-area plate and mixed with CRISPR reaction mixture containing 10X NEBuffer™ 2.1, gRNA (300 nM), EnGen® Lba Cas12a (1 μM), fluorescent probe (10 μM), and nuclease-free water. After incubation at 37 °C for 20 min in the dark, fluorescence signal was detected using SpectraMax i3x Multi-Mode Microplate Reader (Molecular Devices). A positive sample was defined as any specimen with a CRISPR-FDS signal that was greater than the cut-off threshold of 3.6 × 106 photoluminescence (PL) intensity (arb. units).
Kolmogorov-Smirnov normality test, Mann–Whitney U test, and Student’s unpaired two-tailed t-tests were performed with GraphPad Prism software, v9.0.2. When separated by species the number of controls was below the detectable limit for the Kolmogorov–Smirnov normality test. Data were defined as gaussian or non-gaussian based on the overall distribution. P values ≤ 0.05 were considered significant.
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Coronavirus disease-19 (COVID-19) is an ongoing global pandemic caused by the “severe acute respiratory syndrome coronavirus 2” (SARS-CoV-2), which was isolated for the first time in Wuhan (China) in December 2019. Common symptoms include fever, cough, fatigue, dyspnea and hypogeusia/hyposmia. Among extrapulmonary signs associated with COVID-19, dermatological manifestations have been increasingly reported in the last few months.
The polymorphic nature of COVID-19-associated cutaneous manifestations led our group to propose a classification, which distinguishes the following six main clinical patterns: (i) urticarial rash, (ii) confluent erythematous/maculopapular/morbilliform rash, (iii) papulovesicular exanthem, (iv) chilblain-like acral pattern, (v) livedo reticularis/racemosa-like pattern, (vi) purpuric “vasculitic” pattern. This review summarizes the current knowledge on COVID-19-associated cutaneous manifestations, focusing on clinical features and therapeutic management of each category and attempting to give an overview of the hypothesized pathophysiological mechanisms of these conditions.Keywords: COVID-19, Cutaneous manifestations, SARS-CoV-2Go to:
In December 2019, a novel zoonotic RNA virus named “severe acute respiratory syndrome coronavirus 2” (SARS-CoV-2) was isolated in patients with pneumonia in Wuhan, China. Since then, the disease caused by this virus, called “coronavirus disease-19” (COVID-19), has spread throughout the world at a staggering speed becoming a pandemic emergency . Although COVID-19 is best known for causing fever and respiratory symptoms, it has been reported to be associated also with different extrapulmonary manifestations, including dermatological signs . Whilst the COVID-19-associated cutaneous manifestations have been increasingly reported, their exact incidence has yet to be estimated, their pathophysiological mechanisms are largely unknown, and the role, direct or indirect, of SARS-CoV-2 in their pathogenesis is still debated. Furthermore, evidence is accumulating that skin manifestations associated with COVID-19 are extremely polymorphic . In this regard, our group proposed the following six main clinical patterns of COVID-19-associated cutaneous manifestations in a recently published review article: (i) urticarial rash, (ii) confluent erythematous/maculopapular/morbilliform rash, (iii) papulovesicular exanthem, (iv) chilblain-like acral pattern, (v) livedo reticularis/racemosa-like pattern, (vi) purpuric “vasculitic” pattern (shown in Fig. Fig.1)1) . Other authors have attempted to bring clarity in this field, suggesting possible classifications of COVID-19-associated cutaneous manifestations [4, 5, 6]. Finally, distinguishing nosological entities “truly” associated with COVID-19 from cutaneous drug reactions or exanthems due to viruses other than SARS-CoV-2 remains a frequent open problem.
Clinical features of COVID-19-associated cutaneous manifestations.
Herein, we have striven to provide a comprehensive overview of the cutaneous manifestations associated with COVID-19 subdivided according to the classification by Marzano et al. , focusing on clinical features, histopathological features, hypothesized pathophysiological mechanisms and therapeutic management.Go to:
Clinical Features and Association with COVID-19 Severity
It is well known that urticaria and angioedema can be triggered by viral and bacterial agents, such as cytomegalovirus, herpesvirus, and Epstein-Barr virus and mycoplasma. However, establishing a cause-effect relationship may be difficult in single cases [7, 8]. Urticarial eruptions associated with COVID-19 have been first reported by Recalcati  in his cohort of hospitalized patients, accounting for 16.7% of total skin manifestations. Urticaria-like eruptions have been subsequently described in other cohort studies. Galván Casas et al.  stated that urticarial rash occurred in 19% of their cohort, tended to appear simultaneously with systemic symptoms, lasted approximately 1 week and was associated with medium-high severity of COVID-19. Moreover, itch was almost always present . Freeman et al.  found a similar prevalence of urticaria (16%) in their series of 716 cases, in which urticarial lesions predominantly involved the trunk and limbs, relatively sparing the acral sites. As shown in Table Table1,1, urticaria-like signs accounted for 11.9% of cutaneous manifestations seen in an Italian multicentric cohort study on 159 patients [unpubl. data]. Urticarial lesions associated with fever were reported to be early or even prodromal signs of COVID-19, in the absence of respiratory symptoms, in 3 patients [11, 12, 13]. Therefore, the authors of the reports suggested that isolation is needed for patients developing such skin symptoms if COVID-19 infection is suspected in order to prevent possible SARS-CoV-2 transmission [11, 12, 13]. COVID-19-related urticaria occurred also in a familial cluster, involving 2 patients belonging to a Mexican family of 5 people, all infected by SARS-CoV-2 and suffering also from anosmia, ageusia, chills and dizziness . Angioedema may accompany COVID-19-related urticaria, as evidenced by the case published in June 2020 of an elderly man presenting with urticaria, angioedema, general malaise, fatigue, fever and pharyngodynia . Urticarial vasculitis has also been described in association with COVID-19 in 2 patients .
Prevalence of different clinical patterns in the main studies on COVID-19-associated cutaneous manifestations
First author (total size of study population)
Number of patients with urticarial rash (%)
Number of patients with confluent erythematous/maculopapular/morbilliform rash (%)
Number of patients with papulo-vesicular exanthem (%)
Number of patients with chilblain-like acral pattern (%)
Number of patients with livedo reticularis/racemosa-like pattern (%)
Number of patients with purpuric “vasculitic” pattern (%)
Histopathological studies of urticarial rashes are scant. In a 60-year-old woman with persistent urticarial eruption and interstitial pneumonia who was not under any medication, Rodriguez-Jiménez et al.  found on histopathology slight vacuolar interface dermatitis with occasional necrotic keratinocytes curiously compatible with an erythema multiforme-like pattern. Amatore et al.  documented also the presence of lichenoid and vacuolar interface dermatitis, associated with mild spongiosis, dyskeratotic basal keratinocytes and superficial perivascular lymphocytic infiltrate, in a biopsy of urticarial eruption associated with COVID-19 (Fig. (Fig.22).
Histopathological features of the main cutaneous patterns associated with COVID-19. a Urticarial rash. b Confluent erythematous maculopapular/morbilliform rash. c Chilblain-like acral lesions. d Purpuric “vasculitic” pattern.
Shanshal  suggested low-dose systemic corticosteroids as a therapeutic option for COVID-19-associated urticarial rash. Indeed, the author hypothesized that low-dose systemic corticosteroids, combined with nonsedating antihistamines, can help in managing the hyperactivity of the immune system in COVID-19, not only to control urticaria, but also to improve possibly the survival rate in COVID-19.Go to:
Clinical Features and Association with COVID-19 Severity
Maculopapular eruptions accounted for 47% of all cutaneous manifestations in the cohort of Galván Casas et al. , for 44% of the skin manifestations included in the study by Freeman et al. , who further subdivided this group of cutaneous lesions into macular erythema (13%), morbilliform exanthems (22%) and papulosquamous lesions (9%), and for 30.2% of the cutaneous manifestations included in the unpublished Italian multicentric study shown in Table Table1.1. The prevalence of erythematous rash was higher in other studies, like that published by De Giorgi et al.  in May 2020, in which erythematous rashes accounted for 70% of total skin manifestations. In the series by Freeman et al. , macular erythema, morbilliform exanthems and papulosquamous lesions were predominantly localized on the trunk and limbs, being associated with pruritus in most cases. In the same series, these lesions occurred more frequently after COVID-19 systemic symptoms’ onset . The clinical picture of the eruptions belonging to this group may range from erythematous confluent rashes to maculopapular eruptions and morbilliform exanthems. Erythematous lesions may show a purpuric evolution  or coexist from the beginning with purpuric lesions . Erythematous papules may also be arranged in a morbilliform pattern . In a subanalysis of the COVID-Piel Study  on maculopapular eruptions including also purpuric, erythema multiforme-like, pityriasis rosea-like, erythema elevatum diutinum-like and perifollicular eruptions, morbilliform exanthems were the most frequent maculopapular pattern (n = 80/176, 45.5%) . This study showed that in most cases lesions were generalized, symmetrical and started on the trunk with centrifugal progression. In the same subanalysis, hospital admission due to pneumonia was very frequent (80%) in patients with a morbilliform pattern . In this group, the main differential diagnoses are represented by exanthems due to viruses other than SARS-CoV-2 and drug-induced cutaneous reactions.
Histopathology of erythematous eruptions was described by Gianotti et al. , who found vascular damage in all the 3 cases examined. A clinicopathological characterization of late-onset maculopapular eruptions related to COVID-19 was provided also by Reymundo et al. , who observed a mild superficial perivascular lymphocytic infiltrate on the histology of 4 patients. In contrast, Herrero-Moyano et al.  observed dense neutrophilic infiltrates in 8 patients with late maculopapular eruptions. The authors of the former study postulated that this discrepancy could be attributable to the history of new drug assumptions in the series of Herrero-Moyano et al.  (Fig. (Fig.22).
The management of confluent erythematous/maculopapular/morbilliform rash varies according to the severity of the clinical picture. Topical corticosteroids can be sufficient in most cases , systemic corticosteroids deserving to be administered just in more severe and widespread presentations.Go to:
Clinical Features and Association with COVID-19 Severity
COVID-19-associated papulovesicular exanthem was first extensively reported in a multicenter Italian case series of 22 patients published in April 2020 . In this article, it was originally described as “varicella-like” due to resemblance of its elementary lesions to those of varicella. However, the authors themselves underlined that the main clinical features of COVID-19-associated papulovesicular exanthem, namely trunk involvement, scattered distribution and mild/absent pruritus, differentiated it from “true” varicella. In this study, skin lesions appeared on average 3 days after systemic symptoms’ onset and healed after 8 days, without scarring sequelae . The exact prevalence of papulovesicular exanthems is variable. Indeed, in a cohort of 375 patients with COVID-19-associated cutaneous manifestations , patients with papulovesicular exanthem were 34 (9%), while they were 3 out of 52 (5.8%), 1 out of 18 (5.5%) and 2 out of 53 (4%) in the cohorts published by Askin et al. , Recalcati  and De Giorgi et al. , respectively. In the Italian multicentric study shown in Table Table1,1, papulovesicular rash accounted for 18.2% of skin manifestations. Furthermore, even if papulovesicular exanthem tends to involve more frequently the adult population, with a median age of 60 years in the study by Marzano et al. , also children may be affected . Galván Casas et al.  reported that vesicular lesions generally involved middle-aged patients, before systemic symptoms’ onset in 15% of cases, and were associated with intermediate COVID-19 severity. Fernandez-Nieto et al.  conducted a prospective study on 24 patients diagnosed with COVID-19-associated vesicular rash. In this cohort, the median age (40.5 years) was lower than that reported by Marzano et al. , and COVID-19 severity was mostly mild or intermediate, with only 1 patient requiring intensive unit care support. In our cohort of 22 patients, a patient was hospitalized in the intensive care unit and 3 patients died . Vesicular rash, which was generally pruritic, appeared after COVID-19 diagnosis in most patients (n = 19; 79.2%), with a median latency time of 14 days . Two different morphological patterns were found: a widespread polymorphic pattern, more common and consisting of small papules, vesicles and pustules of different sizes, and a localized pattern, less frequent and consisting of monomorphic lesions, usually involving the mid chest/upper abdominal region or the back .
Mahé et al.  reported on 3 patients with typical COVID-19-associated papulovesicular rash, in which the histological pattern of skin lesions showed prominent acantholysis and dyskeratosis associated with the presence of an unilocular intraepidermal vesicle in a suprabasal location. Based on these histopathological findings, the authors refused the term “varicella-like rash” and proposed a term which was more suitable in their view: “COVID-19-associated acantholytic rash.” Histopathological findings of another case of papulovesicular eruption revealed extensive epidermal necrosis with acantholysis and swelling of keratinocytes, ballooning degeneration of keratinocytes and signs of endotheliitis in the dermal vessels . Acantholysis and ballooned keratinocytes were found also by Fernandez-Nieto et al.  in 2 patients.
The differential diagnosis with infections caused by members of the Herpesviridae family has been much debated. Tammaro et al.  described the onset of numerous, isolated vesicles on the back 8 days after COVID-19 diagnosis in a Barcelonan woman and reported on 2 patients from Rome presenting with isolated, mildly pruritic erythematous-vesicular lesions on their trunk, speculating that these manifestations might be due to viruses belonging to the Herpesviridae family. On the other hand, classic herpes zoster has been reported to complicate the course of COVID-19 .
The controversy regarding the role of herpesvirus in the etiology of papulovesicular exanthems fuelled an intense scientific debate. Indeed, some authors raised the question whether papulovesicular exanthem associated with COVID-19 could be diagnosed without ruling out varicella zoster virus and herpes simplex virus with Tzanck smear or polymerase chain reaction (PCR) for the Herpesviridae family in the vesicle fluid or on the skin [36, 37]. In our opinion, even if seeking DNA of Herpesviridae family members is ideally advisable, clinical diagnosis may be reliable in most cases, and the role of herpes viruses as mere superinfection in patients with dysfunctional immune response associated with COVID-19 needs to be considered . To our knowledge, SARS-CoV-2 has not been hitherto isolated by means of reverse transcriptase PCR in the vesicle fluid of papulovesicular rash [33, 31].
No standardized treatments for COVID-19-related papulovesicular exanthem are available, also given that it is self-healing within a short time frame. Thus, a “wait-and-see” strategy may be recommended.Go to:
Chilblain-Like Acral Pattern
Clinical Features and Association with COVID-19 Severity
COVID-19-related chilblain-like acral lesions have been first described in a 13-year-old boy by Italian authors in early March . Since then, several “outbreaks” of chilblain-like acral lesions chiefly involving young adults and children from different countries worldwide have been posted on social media and published in the scientific literature [40, 41, 42, 43, 44, 45, 46]. Caucasians seem to be significantly more affected than other ethnic groups [47, 48]. Chilblain-like acral lesions were the second most frequent cutaneous manifestation (n = 46/159; 28.9%) in the multicenter Italian study shown in Table Table1.1. Different pathogenetic hypotheses, including increased interferon release induced by COVID-19 and consequent cytokine-mediated inflammatory response, have been suggested . Furthermore, virus-induced endothelial damage as well as an obliterative microangiopathy and coagulation abnormalities could be mechanisms involved in the pathogenesis of these lesions . Chilblain-like acral lesions associated with COVID-19 were depicted as erythematous-violaceous patches or plaques predominantly involving the feet and, to a lesser extent, hands [40, 51]. Rare cases of chilblain-like lesions involving other acral sites, such as the auricular region, were also reported . The occurrence of blistering lesions varied according to the case series analyzed; Piccolo et al. , indeed, reported the presence of blistering lesions in 23 out of 54 patients, while other authors did not describe bullous lesions in their series [40, 47]. Dermoscopy of these lesions revealed the presence of an indicative pattern represented by a red background area with purpuric globules . Pain/burning sensation as well as pruritus were commonly reported symptoms, even if a small proportion of patients presented with asymptomatic lesions [40, 44, 47]. Unlike other COVID-19-related cutaneous findings, chilblain-like acral lesions tended to mostly involve patients without systemic symptoms.
The frequent occurrence of chilblain-like lesions in the absence of cold exposure and the involvement of patients without evident COVID-19-related symptoms raised the question whether these manifestations were actually associated with SARS-CoV-2 infection.
Histopathological and Pathophysiological Findings
Chilblain-like lesions share many histopathological features with idiopathic and autoimmunity-related chilblains, including epidermal necrotic keratinocytes, dermal edema, perivascular and perieccrine sweat gland lymphocytic inflammation. Vascular changes such as endotheliitis and microthrombi may be found [40, 45, 54, 55] (Fig. (Fig.22).
Data on the real association between chilblain-like acral lesions and COVID-19 are controversial.
The first case series failed to perform SARS-CoV-2 testing in all patients, also due to logistic problems and economic restrictions, and diagnosed COVID-19 only in a minority of patients with chilblain-like acral lesions [40, 44, 47]. Subsequently, some authors systematically sought SARS-CoV-2 with serology and/or nasopharyngeal swab in patients with chilblain-like acral lesions. In their cohort of 38 children with pseudo-chilblain, Caselli et al.  showed no evidence of SARS-CoV-2 infection by PCR or serology. Chilblain-like acral lesions appeared not to be directly associated with COVID-19 also in the case series by Herman et al. . These authors failed to detect SARS-CoV-2 in nasopharyngeal swabs and skin biopsies and demonstrated no specific anti-SARS-CoV-2 immunoglobulin IgM or IgG antibodies in blood samples. Therefore, they concluded that lifestyle changes associated with lockdown measures might be a possible explanation for these lesions . Similar results were obtained also by other authors [58, 59, 60, 61, 62, 63] weakening the hypothesis of a direct etiological link between SARS-CoV-2 and chilblain-like acral lesions.
Opposite conclusions have been drawn by Colmenero et al. , who demonstrated by immunohistochemistry and electron microscopy the presence of SARS-CoV-2 in endothelial cells of skin biopsies of 7 children with chilblain-like acral lesions, suggesting that virus-induced vascular damage and secondary ischemia could explain the pathophysiology of these lesions.
In the absence of definitive data on chilblain-like acral lesions’ pathogenesis, the occurrence of such lesions should prompt self-isolation and confirmatory testing for SARS-CoV-2 infection .
In the absence of significant therapeutic options for chilblain-like acral lesions associated with COVID-19 and given their tendency to spontaneously heal, a “wait-and-see” strategy may be suggested.Go to:
Livedo Reticularis/Racemosa-Like Pattern
Clinical Features and Association with COVID-19 Severity
Livedo describes a reticulate pattern of slow blood flow, with consequent desaturation of blood and bluish cutaneous discoloration. It has been divided into: (i) livedo reticularis, which develops as tight, symmetrical, lace-like, dusky patches forming complete rings surrounding a pale center, generally associated with cold-induced cutaneous vasoconstriction or vascular flow disturbances such as seen in polycythemia and (ii) livedo racemosa, characterized by larger, irregular and asymmetrical rings than seen in livedo reticularis, more frequently associated with focal impairment of blood flow, as it can be seen in Sneddon’s syndrome .
In our classification, the livedo reticularis/racemosa-like pattern has been distinguished by the purpuric “vasculitic” pattern because the former likely recognizes a occlusive/microthrombotic vasculopathic etiology, while the latter can be more likely considered the expression of a “true” vasculitic process . Instead, the classification by Galván Casas et al.  merged these two patterns into the category “livedo/necrosis”.
In a French study on vascular lesions associated with COVID-19, livedo was observed in 1 out of 7 patients . In the large cases series of 716 patients by Freeman et al. , livedo reticularis-like lesions, retiform purpura and livedo racemosa-like lesions accounted for 3.5, 2.6 and 0.6% of all cutaneous manifestations, respectively. In the multicentric Italian study, livedo reticularis/racemosa-like lesions accounted for 2.5% of cutaneous manifestations (Table (Table11).
The pathogenic mechanisms at the basis of small blood vessel occlusion are yet unknown, even if neurogenic, microthrombotic or immune complex-mediated etiologies have been postulated .
Livedo reticularis-like lesions are frequently mild, transient and not associated with thromboembolic complications [68, 69]. On the contrary, livedo racemosa-like lesions and retiform purpura have often been described in patients with severe coagulopathy [60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72].
Histopathological and Pathophysiological Findings
The histopathology of livedoid lesions associated with COVID-19 has been described by Magro et al. , who observed in 3 patients pauci-inflammatory microthrombotic vasculopathy. The same group demonstrated that in the thrombotic retiform purpura of patients with severe COVID-19, the vascular thrombosis in the skin and internal organs is associated with a minimal interferon response permitting increased viral replication with release of viral proteins that localize to the endothelium inducing widespread complement activation , which is frequent in COVID-19 patients and probably involved in the pathophysiology of its clinical complications .
In view of the absence of significant therapeutic options for livedo reticularis/racemosa-like lesions associated with COVID-19, a “wait-and-see” strategy may be suggested.Go to:
Purpuric “Vasculitic” Pattern
Clinical Features and Association with COVID-19 Severity
The first COVID-19-associated cutaneous manifestation with purpuric features was reported by Joob et al. , who described a petechial rash misdiagnosed as dengue in a COVID-19 patient. Purpuric lesions have been suggested to occur more frequently in elderly patients with severe COVID-19, likely representing the cutaneous manifestations associated with the highest rate of COVID-19-related mortality . This hypothesis is corroborated by the unfavorable prognosis observed in several cases reported in the literature [77, 78].
The purpuric pattern reflects the presence of vasculitic changes probably due to the direct damage of endothelial cells by the virus or dysregulated host inflammatory responses induced by COVID-19.
These lesions are likely to be very rare, representing 8.2% of skin manifestations included in the Italian multicentric study shown in Table Table1.1. In their case series of 7 patients with vascular skin lesions related to COVID-19, Bouaziz et al.  reported 2 patients with purpuric lesions with (n = 1) and without (n = 1) necrosis. In the series by Freeman et al. , 12/716 (1.8%) and 11/716 (1.6%) cases of patients with palpable purpura and dengue-like eruption, respectively, have been reported. Galván Casas et al.  reported 21 patients with “livedo/necrosis,” most of whom presenting cutaneous signs in concomitance with systemic symptoms’ onset.
Purpuric lesions may be generalized , localized in the intertriginous regions  or arranged in an acral distribution . Vasculitic lesions may evolve into hemorrhagic blisters . In most severe cases, extensive acute necrosis and association with severe coagulopathy may be seen . Dermoscopy of purpuric lesions revealed the presence of papules with incomplete violaceous rim and a central yellow globule .
When performed, histopathology of skin lesions showed leukocytoclastic vasculitis [77, 79], severe neutrophilic infiltrate within the small vessel walls and in their proximity , intense lymphocytic perivascular infiltrates , presence of fibrin [79, 81] and endothelial swelling  (Fig. (Fig.22).
Topical corticosteroids have been successfully used for treating mild cases of purpuric lesions . Cases with necrotic-ulcerative lesions and widespread presentation may be treated with systemic corticosteroids.Go to:
Other COVID-19-Associated Cutaneous Manifestations
Other peculiar rare COVID-19-related cutaneous manifestations that cannot be pigeonholed in the classification proposed by our group  include, among others, the erythema multiforme-like eruption , pityriasis rosea-like rash , multi-system inflammatory syndrome in children , anagen effluvium  and a pseudoherpetic variant of Grover disease . However, the spectrum of possible COVID-19-associated skin manifestations is likely to be still incomplete, and it is expected that new entities associated with this infection will be described.Go to:
COVID-19-associated cutaneous manifestations have been increasingly reported in the last few months, garnering attention both from the international scientific community and from the media. A few months after the outbreak of the pandemic, many narrative and systematic reviews concerning the dermatological manifestations of COVID-19 have been published [2, 3, 6, 88, 89, 90, 91]. A summary of clinical features, histopathological findings, severity of COVID-19 systemic symptoms and therapeutic options of COVID-19-related skin manifestations has been provided in Table Table22.
Summary of clinical features, histopathological findings, severity of COVID-19 systemic symptoms and therapeutic options of COVID-19-related skin manifestations
Itching urticarial rash predominantly involving the trunk and limbs; angioedema may also rarely occur
Vacuolar interface dermatitis associated with superficial perivascular lymphocytic infiltrate
Low-dose systemic corticosteroids combined with nonsedating antihistamines
Topical corticosteroids for mild cases; systemic corticosteroids for severe cases
(i) Widespread polymorphic pattern consisting of small papules, vesicles and pustules of different sizes; (ii) localized pattern consisting of papulovesicular lesions, usually involving the mid chest/upper abdominal region or the back
Prominent acantholysis and dyskeratosis associated with unilocular intraepidermal vesicles in a suprabasal location
Wait and see
Chilblain-like acral pattern
Erythematous-violaceous patches or plaques predominantly involving the feet or, to a lesser extent, hands. Pain/burning sensation as well as pruritus were commonly reported symptoms
Perivascular and periadnexal dermal lymphocytic infiltrates
Wait and see
Livedo reticularis/racemosa-like pattern
Livedo reticularis-like lesions: mild, transient, symmetrical, lace-like, dusky patches forming complete rings surrounding a pale center. Livedo racemosa-like lesions: large, irregular and asymmetrical violaceous annular lesions frequently described in patients with severe coagulopathy
Livedo reticularis-like lesions: intermediate severity; livedo racemosa-like lesions: high severity
Pauci-inflammatory microthrombotic vasculopathy
Wait and see
Purpuric “vasculitic” pattern
Purpuric lesions may be generalized, arranged in an acral distribution or localized in the intertriginous regions. Purpuric elements may evolve into hemorrhagic blisters, possibly leading to necrotic-ulcerative lesions
Leukocytoclastic vasculitis, severe perivascular neutrophilic and lymphocytic infiltrate, presence of fibrin and endothelial swelling
Topical corticosteroids for mild cases; systemic corticosteroids for severe cases
The correlation between severity of COVID-19 systemic symptoms and skin manifestations has been inferred mainly from the study by Freeman et al. .
Albeit several hypotheses on pathophysiological mechanisms at the basis of these skin findings are present in the literature [50, 92, 93], none of them is substantiated by strong evidence, and this field needs to be largely elucidated. Moreover, cutaneous eruptions due to viruses other than SARS-CoV-2 [35, 37] or drugs prescribed for the management of this infection [94, 95] always need to be ruled out.
Experimental pathophysiological studies and clinical data derived from large case series are still needed for shedding light onto this novel, underexplored and fascinating topic.
Although COVID-19-associated cutaneous manifestations have been increasingly reported, their pathophysiological mechanisms need to be extensively explored. The conditions may be distinguished in six clinical phenotypes, each showing different histopathological patterns.
Conflict of Interest Statement
The authors have no conflicts of interest to declare.:
This paper did not receive any funding.
Giovanni Genovese wrote the paper with the contribution of Chiara Moltrasio. Angelo Valerio Marzano and Emilio Berti supervised the work and revised the paper for critical revision of important intellectual content.Go to:
We would like to thank Dr. Cosimo Misciali, Dr. Paolo Sena and Prof. Pietro Quaglino for kindly providing us with histopathological pictures.Go to:
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• Immunogenicity might be elicited, while sterile immunity was not established.
A previously symptomless 86-year-old man received the first dose of the BNT162b2 mRNA COVID-19 vaccine. He died 4 weeks later from acute renal and respiratory failure. Although he did not present with any COVID-19-specific symptoms, he tested positive for SARS-CoV-2 before he died. Spike protein (S1) antigen-binding showed significant levels for immunoglobulin (Ig) G, while nucleocapsid IgG/IgM was not elicited. Acute bronchopneumonia and tubular failure were assigned as the cause of death at autopsy; however, we did not observe any characteristic morphological features of COVID-19. Postmortem molecular mapping by real-time polymerase chain reaction revealed relevant SARS-CoV-2 cycle threshold values in all organs examined (oropharynx, olfactory mucosa, trachea, lungs, heart, kidney and cerebrum) except for the liver and olfactory bulb. These results might suggest that the first vaccination induces immunogenicity but not sterile immunity.
Stevens-Johnson syndrome/toxic epidermal necrolysis (SJS/TEN) is a spectrum of acute, delayed-type hypersensitivity reactions that affect the skin and the mucous membranes. Medications are the culprit cause of these disorders in addition to infections and in very rare instances vaccinations. We report a case of TEN in a 49-year-old woman with no previous medical history. The disorder developed one week after receiving the first dose of COVID-19 vaccine with no other identifiable causes. The patient received two doses of tumor necrosis factor-alpha inhibitor (etanercept) and she stopped developing new lesions after two days of the initial dose; complete healing was observed after 22 days and no side effects were observed in our patient. This case demonstrates an extremely rare complication to the COVID-19 vaccine. The benefits of receiving the COVID-19 outweigh the potential risk.
Toxic epidermal necrolysis (TEN) is a rare immune-mediated, life-threatening skin reaction characterized by blistering and extensive epidermal detachment of more than 30% of body surface area. The incidence is estimated to be 0.4 to 1.9 cases per million population per year worldwide and an estimated mortality rate of 25% to 35% [1, 2]. Medication is usually the cause of TEN (e.g., certain antibiotics and antiepileptics) . Vaccination-induced Stevens-Johnson syndrome (SJS)/TEN is rare, with less than twenty reported cases in the published literature, with the measles vaccine being reported to cause both SJS and TEN, varicella, smallpox, anthrax, tetanus, and influenza vaccines were reported to cause SJS alone, and MMR (measles, mumps, rubella), hantavirus and meningococcal B vaccines were reported to cause TEN [4, 5, 6]. The patient usually develops a fever and other flu-like symptoms one to three weeks after being exposed to medication followed by painful erythematous to purpuric skin lesions that tend to coalescence. Next erosions and vesiculobullous lesions and epidermal detachment over wide body surface area develop. Mucous membranes are also involved, and the patient develops oral ulcers, vaginal ulcers, and possible acute conjunctivitis . In this paper, we report a case of TEN following the administration of the Pfizer COVID-19 vaccine (Pfizer, Inc., New York, USA).
Background: Coronavirus disease (COVID-19) is the pandemic caused by SARS-CoV-2 that has caused more than 2.2 million deaths worldwide. We summarize the reported pathologic findings on biopsy and autopsy in patients with severe/fatal COVID-19 and documented the presence and/or effect of SARS-CoV-2 in all organs.
Methods and findings: A systematic search of the PubMed, Embase, MedRxiv, Lilacs and Epistemonikos databases from January to August 2020 for all case reports and case series that reported histopathologic findings of COVID-19 infection at autopsy or tissue biopsy was performed. 603 COVID-19 cases from 75 of 451 screened studies met inclusion criteria. The most common pathologic findings were lungs: diffuse alveolar damage (DAD) (92%) and superimposed acute bronchopneumonia (27%); liver: hepatitis (21%), heart: myocarditis (11.4%). Vasculitis was common only in skin biopsies (25%). Microthrombi were described in the placenta (57.9%), lung (38%), kidney (20%), Central Nervous System (CNS) (18%), and gastrointestinal (GI) tract (2%). Injury of endothelial cells was common in the lung (18%) and heart (4%). Hemodynamic changes such as necrosis due to hypoxia/hypoperfusion, edema and congestion were common in kidney (53%), liver (48%), CNS (31%) and GI tract (18%). SARS-CoV-2 viral particles were demonstrated within organ-specific cells in the trachea, lung, liver, large intestine, kidney, CNS either by electron microscopy, immunofluorescence, or immunohistochemistry. Additional tissues were positive by Polymerase Chain Reaction (PCR) tests only. The included studies were from numerous countries, some were not peer reviewed, and some studies were performed by subspecialists, resulting in variable and inconsistent reporting or over statement of the reported findings.
Conclusions: The main pathologic findings of severe/fatal COVID-19 infection are DAD, changes related to coagulopathy and/or hemodynamic compromise. In addition, according to the observed organ damage myocarditis may be associated with sequelae.
Mechanisms Underlying Disease Severity and Progression
Authors: Mary Kathryn Bohn,1,2, Alexandra Hall,1 Lusia Sepiashvili,1,2, Benjamin Jung,1,2 Shannon, Steele,1 and Khosrow Adeli1,2,3
The global epidemiology of coronavirus disease 2019 (COVID-19) suggests a wide spectrum of clinical severity, ranging from asymptomatic to fatal. Although the clinical and laboratory characteristics of COVID-19 patients have been well characterized, the pathophysiological mechanisms underlying disease severity and progression remain unclear. This review highlights key mechanisms that have been proposed to contribute to COVID-19 progression from viral entry to multisystem organ failure, as well as the central role of the immune response in successful viralclearance or progression to death.
Coronavirus disease 2019 (COVID-19) is caused by a novel beta-coronavirus known as Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). As of June 15, 2020, the number of global confirmed cases has surpassed 8 million, with over 400,000 reported mortalities. The unparalleled pathogenicity and global impact of this pandemic has rapidly engaged the scientific community in urgently needed research. Preliminary reports from the Chinese Center for Disease Control and Prevention have estimated that the large majority of confirmed SARS-CoV-2 cases are mild (81%), with ~14% progressing to severe pneumonia and 5% developing acute respiratory distress syndrome (ARDS), sepsis, and/or multisystem organ failure (MOF) (144). Although more data is urgently needed to elucidate the global epidemiology of COVID-19 (80), a wide spectrum of clinical severity is evident, with most patients able to mount a sufficient and appropriate immune response, ultimately leading to viral clearance and case resolution. However, a significant subset of patients present with severe clinical manifestations, requiring life-supporting treatment (51). The pathophysiological mechanisms behind key events in the progression from mild to severe disease remain unclear, warranting further investigation to inform therapeutic decisions. Here, we review the current literature and summarize key proposed mechanisms of COVID-19 pathophysiological progression (FIGURE 1). Key Pathophysiological Mechanisms: Our Current Understanding Viral Invasion The first step in COVID-19 pathogenesis is viral invasion via its target host cell receptors. SARSCoV-2 viral entry has been described in detail elsewhere (138). In brief, SARS-CoV-2 consists of four main structural glycoproteins: spike (S), membrane (M), envelope (E), and nucleocapsid (N). The M, E, and N proteins are critical for viral particle assembly and release, whereas the S protein is responsible for viral binding and entry into host cells (33, 76, 89, 143, 148). Similar to SARS-CoV, several researchers have identified human angiotensin converting enzyme 2 (ACE2) as an entry receptor for SARS-CoV-2 (75, 99, 148, 156). SARSCoV-2 is mostly transmissible through large respiratory droplets, directly infecting cells of the upper and lower respiratory tract, especially nasal ciliated and alveolar epithelial cells (161). In addition to the lungs, ACE2 is also expressed in various other human tissues, such as the small intestine, kidneys, heart, thyroid, testis, and adipose tissue, indicating the virus may directly infect cells of other organ systems when viremia is present (77). Interestingly, although the S proteins of SARS-CoV-2 and SARSCoV share 72% homology in amino acid sequences, SARS-CoV-2 has been reported to have a higher affinity for the ACE2 receptor (18, 21, 143). Following host cell binding, viral and cell membranes fuse, enabling the virus to enter into the cell (89). For many coronaviruses, including SARS-CoV, host cell binding alone is insufficient to facilitate membrane fusion, requiring S-protein priming or cleavage by host cell proteases or transmembrane serine proteases (9, 10, 90, 94, 108). Indeed, Hoffman and colleagues demonstrated that S-protein priming by transmembrane serine protease 2 (TMPRSS2), which may be substituted by cathepsin B/L, is required to facilitate SARS-CoV-2 entry into host cells (58). In addition, unlike other coronaviruses, SARS-CoV-2 has been reported to possess a furin-like cleavage site in the S-protein domain, located between the S1 and S2 subunits (31, 138). Furin-like proteases are ubiquitously expressed, albeit at low levels, indicating that S-protein priming at this cleavage site may contribute to the widened cell tropism and enhanced transmissibility of SARS-CoV-2 (123). However, whether furin-like protease-mediated cleavage is required for SARS-CoV-2 host entry has yet to be determined. Blocking or inhibiting these processing enzymes may serve as a potential antiviral target (130). Interestingly, SARS-CoV-2 has developed a unique S1/S2 cleavage site in its S protein, characterized by a four-amino acid insertion, which seems to be absent in all other coronaviruses (4). This molecular mimicry has been identified as an efficient evolutionary adaptation that some viruses have acquired for exploiting the host cellular machinery. Once the nucleocapsid is deposited into the cytoplasm of the host cell, the RNA genome is replicated and translated into structural and accessory proteins. Vesicles containing the newly formed viral particles are then transported to and fuse with the plasma membrane, releasing them to infect other host cells in the same fashion (33, 89, 105). Although much progress has been made in our understanding of the mechanisms underlying SARS-CoV-2 invasion, additional research is needed to delineate exactly how cleavage of the S proteins by TMPRSS2 confers viral particle entry as well as how S-protein cleavage by membrane proteases contributes to viral penetration.
The aim of this study was to evaluate the relationship between infection with SARS-CoV-2 and autoimmunity.
Coronavirus disease 2019 (COVID-19) is an infectious disease caused by severe acute respiratory syndrome (SARS) associated coronavirus 2 (SARS-CoV-2). Although most of the infected individuals are asymptomatic, a proportion of patients with COVID-19 develop severe disease with multiple organ injuries. Evidence suggests that some medications used to treat autoimmune rheumatologic diseases might have therapeutic effect in patients with severe COVID-19 infections, drawing attention to the relationship between COVID-19 and autoimmune diseases. COVID-19 shares similarities with autoimmune diseases in clinical manifestations, immune responses and pathogenic mechanisms. Robust immune reactions participate in the pathogenesis of both disease conditions. Autoantibodies as a hallmark of autoimmune diseases can also be detected in COVID-19 patients. Moreover, some patients have been reported to develop autoimmune diseases, such as Guillain–Barré syndrome or systemic lupus erythematosus, after COVID-19 infection. It is speculated that SARS-CoV-2 can disturb self-tolerance and trigger autoimmune responses through cross-reactivity with host cells. The infection risk and prognosis of COVID-19 in patients with autoimmune diseases remains controversial, but patient adherence to medication regimens to prevent autoimmune disease flares is strongly recommended.
We present a review of the association between COVID-19 and autoimmune diseases, focusing on similarities in immune responses, cross-reactivity of SARS-CoV-2, the development of autoimmune diseases in COVID-19 patients and the risk of COVID-19 infection in patients with preexisting autoimmune conditions.
Since December 2019, a novel infection named coronavirus disease 2019 (COVID-19) broke out in Wuhan, China, and has been sweeping across the globe. COVID-19 was officially declared a pandemic by WHO on 11 March 2020 . The disease is caused by a newly identified strain of severe acute respiratory syndrome (SARS) associated coronavirus, which was named SARS-CoV-2 after SARS-CoV that caused the epidemic of SARS in 2002 .
SARS-CoV-2 belongs to the coronavirus family, which are enveloped viruses with a spherical morphology and a single-stranded RNA (ssRNA) genome . The spike glycoproteins (S protein) cross through the peplos of the virus and form a crown-like surface . Through the receptor binding domain (RBD) located in the S1 subunit of the S protein, the virus can ligate to the host cell receptor angiotensin-converting enzyme 2 (ACE2) and invade into the cell [5–7].
In many cases, hosts infected by SARS-CoV-2 present with flu-like symptoms, such as fever, fatigue and dry cough. Headache, myalgia, sore throat, nausea and diarrhoea can also be seen in patients with COVID-19 [8,9]. Shortness of breath and hypoxemia occur in severe cases. In critical cases, the disease progresses rapidly and patients can develop septic shock and multiorgan dysfunction . As such, COVID-19 can be a systemic disease affecting multiple organ systems, including the skin, kidneys, respiratory system, cardiovascular system, digestive system, nervous system and haematological system . The dysregulated immune response and increased pro-inflammatory cytokines induced by SARS-CoV-2 contribute to the disease pathogenesis and organ damage, which brought attention to immune-regulatory therapy in the treatment of COVID-19 . Medications used to treat autoimmune diseases are widely used in critical cases of COVID-19 . Further, some autoantibodies can be detected in patients with COVID-19 . These observations suggest that examining pathways known to contribute to the pathogenesis of autoimmunity might provide clues to better understand and treat COVID-19.
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is the virus that causes coronavirus disease 2019 (COVID-19), is associated with high mortality and hospitalization rates. However, many patients who are infected with SARS-CoV-2 remain asymptomatic or develop mild symptoms.
In a recent study published on the preprint server bioRxiv*, researchers focus on the relationship between the pre-existing airway neutrophils and SARS-CoV-2 infection to determine the impact that neutrophils have on COVID-19.
An overview of neutrophils
Neutrophils are the first and predominant immune cells that are recruited to the respiratory tract in response to viral infection. Upon their arrival, neutrophils release various inflammatory mediators in an effort to rapidly eliminate the pathogen from the infected area.
Neutrophils are capable of recognizing infectious sites as well as act as sites of infections which, together, leads to an acute inflammatory response. An uncontrolled massive inflammatory response, which is also known as the cytokine storm, has been documented in patients with severe COVID-19.
“Despite their importance in anti-viral immunity and response to viral pathogens, neutrophils have been somewhat overlooked for their role in the pathogenesis of SARS-CoV-2 infection.”
Coronaviruses (CoV) have recently emerged as potentially serious pathogens that can cause significant human morbidity and death. The severe acute respiratory syndrome (SARS)-CoV was identified as the etiologic agent of the 2002-2003 international SARS outbreak. Yet, how SARS evades innate immune responses to cause human disease remains poorly understood. In this study, we show that a protein encoded by SARS-CoV designated as open reading frame-9b (ORF-9b) localizes to mitochondria and causes mitochondrial elongation by triggering ubiquitination and proteasomal degradation of dynamin-like protein 1, a host protein involved in mitochondrial fission. Also, acting on mitochondria, ORF-9b targets the mitochondrial-associated adaptor molecule MAVS signalosome by usurping PCBP2 and the HECT domain E3 ligase AIP4 to trigger the degradation of MAVS, TRAF3, and TRAF 6. This severely limits host cell IFN responses. Reducing either PCBP2 or AIP4 expression substantially reversed the ORF-9b-mediated reduction of MAVS and the suppression of antiviral transcriptional responses. Finally, transient ORF-9b expression led to a strong induction of autophagy in cells. The induction of autophagy depended upon ATG5, a critical autophagy regulator, but the inhibition of MAVS signaling did not. These results indicate that SARS-CoV ORF-9b manipulates host cell mitochondria and mitochondrial function to help evade host innate immunity. This study has uncovered an important clue to the pathogenesis of SARS-CoV infection and illustrates the havoc that a small ORF can cause in cells.