Probes for INS

The recent coronavirus disease 2019 (COVID-19) pandemic is caused by severe acute respiratory syndrome coronavirus 2. COVID-19 was first reported in China (December 2019) and is now prevalent across the globe. Entry of severe acute respiratory syndrome coronavirus 2 into mammalian cells requires the binding of viral Spike (S) proteins to the angiotensin-converting enzyme 2 receptor. Once entered, the S protein is primed by a specialized serine protease, transmembrane serine protease 2 in the host cell. Importantly, besides the respiratory symptoms that are consistent with other common respiratory virus infections when patients become viremic, a significant number of COVID-19 patients also develop liver comorbidities. We explored whether a specific target cell-type in the mammalian liver could be implicated in disease pathophysiology other than the general deleterious response to cytokine storms. Here, we used single-cell RNA-seq to survey the human liver and identified potentially implicated liver cell-type for viral ingress. We analyzed ~300,000 single cells across five different (i.e., human fetal, healthy, cirrhotic, tumor, and adjacent normal) liver tissue types. This study reports on the co-expression of angiotensin-converting enzyme 2 and transmembrane serine protease 2 in a TROP2+ liver progenitor population. Importantly, we detected enrichment of this cell population in the cirrhotic liver when compared with tumor tissue. These results indicated that in COVID-19-associated liver dysfunction and cell death, a viral infection of TROP2+ progenitors in the liver might significantly impair liver regeneration in patients with liver cirrhosis.

Infection by SARS-CoV-2 may be associated with testicular dysfunction that could affect male fertility.Testicles of fatal COVID-19 cases were investigated to detect virus in tissue and to evaluate histopathological and transcriptomic changes.Three groups were compared: a. uninfected controls (subjects dying of trauma or sudden cardiac death; n = 10); b. subjects dying of COVID-19 (virus-negative in testes; n = 15); c. subjects dying of COVID-19 (virus-positive in testes; n = 9). SARS-CoV-2 genome and nucleocapsid antigen were probed using RT-PCR, in situ hybridization, immunohistochemistry (IHC). Infiltrating leukocytes were typed by IHC. mRNA transcripts of immune-related and testis-specific genes were quantified using the nCounter method.SARS-CoV-2 was detected in testis tissue of 9/24 (37%) COVID-19 cases accompanied by scattered T-cell and macrophage infiltrates. Size of testicles and counts of spermatogenic cells were not significantly different among groups. Analysis of mRNA transcripts showed that in virus-positive testes immune processes were activated (interferon-alpha and -gamma pathways). By contrast, transcription of 12 testis-specific genes was downregulated, independently of virus positivity in tissue. By IHC, expression of the luteinizing hormone/choriogonadotropin receptor was enhanced in virus-positive compared to virus-negative testicles, while expression of receptors for androgens and the follicle-stimulating hormone were not significantly different among groups.In lethal COVID-19 cases, infection of testicular cells is not uncommon. Viral infection associates with activation of interferon pathways and downregulation of testis-specific genes involved in spermatogenesis. Due to the exceedingly high numbers of infected people in the pandemic, the impact of virus on fertility should be further investigated.

: The outbreak of COVID-19 disease, caused by SARS-CoV-2 beta-coronovirus, urges a focused search for the underlying mechanisms and treatment options. The lung is the major target organ of COVID-19, wherein the primary cause of mortality is hypoxic respiratory failure, resulting from acute respiratory disease syndrome (ARDS), with severe hypoxemia, often requiring assisted ventilation. While similar in some ways to ARDS secondary to other causes, lungs of some patients dying with COVID-19 exhibit distinct features of vascular involvement, including severe endothelial injury and cell death via apoptosis and/or pyroptosis, widespread capillary inflammation and thrombosis. Furthermore, the pulmonary pathology of COVID-19 is characterized by focal inflammatory cell infiltration, impeding alveolar gas exchange resulting in areas of local tissue hypoxia, consistent with potential amplification of COVID-19 pathogenicity by hypoxia. Vascular endothelial cells play essential roles in both innate and adaptive immune responses, and are considered to be “conditional innate immune cells” centrally participating in various inflammatory, immune pathologies. Activated endothelial cells produce cytokines/chemokines, dynamically recruit and activate inflammatory cells and platelets, and centrally participate in pro-thrombotic processes (thrombotic microangiopathies). Initial reports presented pathological findings of localized direct infection of vascular endothelial cells with SARS-CoV-2, yet emerging evidence does not support direct infection of endothelial or other vascular wall cell and thus widespread endothelial cell dysfunction and inflammation may be better explained as secondary responses to epithelial cell infection and inflammation. Endothelial cells are also actively engaged in a cross-talk with the complement system, the essential arm of innate immunity. Recent reports present evidence for complement deposition in SARS-CoV-2-damaged lung microcirculation, further strengthening the idea that, in severe cases of COVID-19, complement activation is an essential player, generating destructive hemorrhagic, capillariitis-like tissue damage, clotting, and hyper-inflammation. Thus, complement-targeted therapies are actively in development. This review is intended to explore in detail these ideas.

In the 2002 outbreak of severe acute respiratory syndrome (SARS) a number of patients presented abnormalities in the thyroid functioning, neuroendocrine and calcium homeostasis. It was detected in autopsies from SARS Coronavirus (SARS-CoV) patients that the thyroid gland was significantly affected by the disease, with extensive injury and death of follicular and parafollicular cells. In the present SARS-CoV-2 pandemic some studies start to report acute thyroiditis and alterations in the levels of thyroid hormones [(triiodothyronine (T3), thyroxine (T4), thyroid stimulating hormone (TSH)]. Thyroid cells present high levels of mRNA expression of angiotensin-converting enzyme 2 (ACE2), the host receptor for SARS-CoV-2. It remains poorly studied the thyroid expression of proteins that predispose to SARS-CoV-2 infection and if thyroid cells can be a direct or indirect target of SARS-CoV-2 infection.

The widespread coronavirus disease 2019 (COVID-19) is caused by infection with the novel coronavirus SARS-CoV-2. Currently, we have limited understanding of which cells become infected with SARS-CoV-2 in human tissues and where viral RNA localizes on the subcellular level. Here, we present a platform for preparing autopsy tissue for visualizing SARS-CoV-2 RNA using RNA fluorescence in situ hybridization (FISH) with amplification by hybridization chain reaction. We developed probe sets that target different regions of SARS-CoV-2 (including ORF1a and N), as well as probe sets that specifically target SARS-CoV-2 subgenomic mRNAs. We validated these probe sets in cell culture and tissues (lung, lymph node, and placenta) from infected patients. Using this technology, we observe distinct subcellular localization patterns of the ORF1a and N regions. In human lung tissue, we performed multiplexed RNA FISH HCR for SARS-CoV-2 and cell-type-specific marker genes. We found viral RNA in cells containing the alveolar type 2 (AT2) cell marker gene (SFTPC) and the alveolar macrophage marker gene (MARCO) but did not identify viral RNA in cells containing the alveolar type 1 (AT1) cell marker gene (AGER). Moreover, we observed distinct subcellular localization patterns of viral RNA in AT2 cells and alveolar macrophages. In sum, we demonstrate the use of RNA FISH HCR for visualizing different RNA species from SARS-CoV-2 in cell lines and FFPE (formalin fixation and paraffin embedding) autopsy specimens. We anticipate that this platform could be broadly useful for studying SARS-CoV-2 pathology in tissues, as well as extended for other applications, including investigating the viral life cycle, viral diagnostics, and drug screening. IMPORTANCE Here, we developed an in situ RNA detection assay for RNA generated by the SARS-CoV-2 virus. We found viral RNA in lung, lymph node, and placenta samples from pathology specimens from COVID patients. Using high-magnification microscopy, we can visualize the subcellular distribution of these RNA in single cells.

The SARS-CoV-2 pandemic has had a significant impact worldwide, particularly in middle- and low-income countries. While this impact has been well-recognized in certain age groups, the effects, both direct and indirect, on the neonatal population remain largely unknown. There are placental changes associated, though the contributions to maternal and fetal illness have not been fully determined. The rate of premature delivery has increased and SARS-CoV-2 infection is proportionately higher in premature neonates, which appears to be related to premature delivery for maternal reasons rather than an increase in spontaneous preterm labor. There is much room for expansion, including long-term data on outcomes for affected babies. Though uncommon, there has been evidence of adverse events in neonates, including Multisystem Inflammatory Syndrome in Children, associated with COVID-19 (MIS-C). There are recommendations for reduction of viral transmission to neonates, though more research is required to determine the role of passive immunization of the fetus via maternal vaccination. There is now considerable evidence suggesting that the severe visitation restrictions implemented early in the pandemic have negatively impacted the care of the neonate and the experiences of both parents and healthcare professionals alike. Ongoing collaboration is required to determine the full impact, and guidelines for future management. IMPACT: Comprehensive review of current available evidence related to impact of the COVID-19 pandemic on neonates, effects on their health, impact on their quality of care and indirect influences on their clinical course, including comparisons with other age groups. Reference to current evidence for maternal experience of infection and how it impacts the fetus and then neonate. Outline of the need for ongoing research, including specific areas in which there are significant gaps in knowledge.

RATIONALE: Patients with idiopathic pulmonary fibrosis (IPF) have worse outcomes following COVID-19. SARSCoV-2 (2019-nCoV) spike protein (S1) harbors an RGD motif in its receptor-binding domain (RBD). Although SARS-CoV-2 is to exploit human Angiotensin Converting Enzyme-2 (ACE2) receptors for cell entry. Single Cell RNA-seq showed that normal lung expresses low levels of ACE2 with very low expression (1.5%) in Alveolar type 2 epithelial cells. It is possible that SARS-CoV-2 needs a cellular co-receptor, which could include integrins, to promote alveolar cell internalization and pneumonitis.METHODS: Solid-phase binding assays were used to investigate S1 binding to ACE2 or αv containing integrins. Pseudovirus entry assays were used to measure the internalization of SARS-CoV-2 into Human embryonic kidney 293T cells expressing different combinations of potential receptors. RNAscope was used to visualize the co-localization of SARS-CoV-2, ACE2, and integrin mRNAs. Immunohistochemistry was used to evaluate the expression of αvβ6 integrins and ACE2 in lung tissue.RESULTS: Binding assays demonstrated that the RGD containing αvβ3 and αvβ6 integrins bound robustly to the SARS-CoV-2 S1 subunit of Spike protein and overexpression of the αvβ6 integrin modestly augments ACE2 mediated SARS-CoV-2 pseudoviral entry into epithelial cells. In COVID-19 damaged lung ACE2 levels are low but the αvβ6 integrin levels are increased in alveolar epithelium whereas both ACE2 and αvβ6 integrin are increased in lung sections from idiopathic pulmonary fibrosis compared with normal lung samples. CONCLUSION: The SARS-CoV-2 S1 subunit can bind αvβ6 integrins augmenting ACE2-dependent internalization of pseudovirus. In IPF patients, ACE2 levels and αvβ6 integrin levels are increased. Increased binding of the SARS-CoV-2 to ACE2 and the αvβ6 integrin within fibrotic lung may explain the increased risk of severe COVID in patients with IPF.

The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has resulted in a global health emergency. In addition to common respiratory symptoms, some patients with COVID-19 infections may experience a range of extra-pulmonary manifestations, such as digestive system involvement. Patients with COVID-19 have been reported to suffer from acute mesenteric ischemia (AMI) that is associated with disease-related severity and mortality. However, in the context of COVID-19, the exact cause of AMI has yet to be clearly defined. This review provides a comprehensive overview of the available data and elucidates the possible underlying mechanisms linking COVID-19 to AMI, in addition to highlighting therapeutic approaches for clinicians. Finally, given the severe global impact of COVID-19, we emphasize the importance of coordinated vaccination programs.

SARS-CoV-2, the etiological agent of COVID-19, causes life-threatening disease. This novel coronavirus enters host cells via the respiratory tract, promoting the formation of severe pulmonary lesions and systemic disease. Few animal models can simulate the clinical signs and pathology of COVID-19 patients. Diverse preclinical studies using K18-hACE2 mice and Syrian golden hamsters, which are highly permissive to SARS-CoV-2 in the respiratory tract, are emerging; however, the systemic pathogenesis and cellular tropism of these models remain obscure. We intranasally infected K18-hACE2 mice and Syrian golden hamsters with SARS-CoV-2, and compared the clinical features, pathogenesis, cellular tropism, and infiltrated immune-cell subsets. In K18-hACE2 mice, SARS-CoV-2 persistently replicated in alveolar cells and caused pulmonary and extra-pulmonary disease, resulting in fatal outcomes. Conversely, in Syrian golden hamsters, transient SARS-CoV-2 infection in bronchial cells caused reversible pulmonary disease, without mortality. Our findings provide comprehensive insights into the pathogenic spectrum of COVID-19 using pre-clinical models.

Severe cases of SARS-CoV-2 infection are characterized by hypercoagulopathies and systemic endotheliitis of the lung microvasculature. The dynamics of vascular damage, and whether it is a direct consequence of endothelial infection or an indirect consequence of an immune cell-mediated cytokine storm remain unknown. Using a vascularized lung-on-chip model, we find that infection of alveolar epithelial cells leads to limited apical release of virions, consistent with reports of monoculture infection. However, viral RNA and proteins are rapidly detected in underlying endothelial cells, which are themselves refractory to apical infection in monocultures. Although endothelial infection is unproductive, it leads to the formation of cell clusters with low CD31 expression, a progressive loss of barrier integrity and a pro-coagulatory microenvironment. Viral RNA persists in individual cells generating an inflammatory response, which is transient in epithelial cells but persistent in endothelial cells and typified by IL-6 secretion even in the absence of immune cells. Inhibition of IL-6 signalling with tocilizumab reduces but does not prevent loss of barrier integrity. SARS-CoV-2-mediated endothelial cell damage thus occurs independently of cytokine storm.

Several models were developed to study the pathogenicity of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as well as the in vivo efficacy of vaccines and therapeutics. Since wild-type mice are naturally resistant to infection by ancestral SARS-CoV-2 strains, several transgenic mouse models expressing human angiotensin-converting enzyme 2 (hACE2) were developed. An alternative approach has been to develop mouse-adapted SARS-CoV-2 strains. Here, we compared the clinical progression, viral replication kinetics and dissemination, pulmonary tropism, and host innate immune response dynamics between the mouse-adapted MA10 strain and its parental strain (USA-WA1/2020) following intranasal inoculation of K18-hACE2 mice, a widely used model. Compared to its parental counterpart, the MA10 strain induced earlier clinical decline with significantly higher viral replication and earlier neurodissemination. Importantly, the MA10 strain also showed a wider tropism, with infection of bronchiolar epithelia. While both SARS-CoV-2 strains induced comparable pulmonary cytokine/chemokine responses, many proinflammatory and monocyte-recruitment chemokines, such as interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α), IP-10/CXCL10, and MCP-1/CCL2, showed an earlier peak in MA10-infected mice. Furthermore, both strains induced a similar downregulation of murine Ace2, with only a transient downregulation of Tmprss2 and no alterations in hACE2 expression. Overall, these data demonstrate that in K18-hACE2 mice, the MA10 strain has a pulmonary tropism that more closely resembles SARS-CoV-2 tropism in humans (airways and pneumocytes) than its parental strain. Its rapid replication and neurodissemination and early host pulmonary responses can have a significant impact on the clinical outcomes of infection and are, therefore, critical features to consider for study designs using these strains and mouse model. IMPORTANCE The COVID-19 pandemic, caused by SARS-CoV-2, is still significantly impacting health care systems around the globe. Refined animal models are needed to study SARS-CoV-2 pathogenicity as well as efficacy of vaccines and therapeutics. In line with this, thorough evaluation of animal models and virus strains/variants are paramount for standardization and meaningful comparisons. Here, we demonstrated differences in replication dynamics between the Wuhan-like USA-WA1/2020 strain and the derivative mouse-adapted MA10 strain in K18-hACE2 mice. The MA10 strain showed accelerated viral replication and neurodissemination, differential pulmonary tropism, and earlier pulmonary innate immune responses. The observed differences allow us to better refine experimental designs when considering the use of the MA10 strain in the widely utilized K18-hACE2 murine model.

Vaccination has been a game changer in our efforts to address the coronavirus disease 2019 (COVID-19) pandemic. However, the disease might still represent a clinical crisis for several more years, in part because of the inevitable emergence of variants capable of evading the pre-existing immunity. Drugs affecting viral spread will help curtail transmission, but therapeutics are needed to treat the more severe cases requiring hospitalization. A deep analysis of the evolving immune landscape of COVID-19 suggests that understanding the molecular bases of the distinct clinical stages is paramount if we are to limit the burden of inflammation, which can lead to death in frail individuals, according to age, sex and comorbidities. Different phases can be defined using immune biomarkers and need specific therapeutic approaches, tailored to the underlying immune contexture.