Choroid plexus defects in Down syndrome brain organoids enhance neurotropism of SARS-CoV-2.

Why individuals with Down syndrome (DS) are more susceptible to SARS-CoV-2-induced neuropathology remains elusive. Choroid plexus (ChP) plays critical roles in barrier function and immune response modulation and expresses the ACE2 receptor and the chromosome 21-encoded TMPRSS2 protease, suggesting its substantial role in establishing SARS-CoV-2 infection in the brain. To explore this, we established brain organoids from DS and isogenic euploid iPSC that consist of a core of functional cortical neurons surrounded by a functional ChP-like epithelium (ChPCOs). DS-ChPCOs recapitulated abnormal DS cortical development and revealed defects in ciliogenesis and epithelial cell polarity in ChP-like epithelium. We then demonstrated that the ChP-like epithelium facilitates infection and replication of SARS-CoV-2 in cortical neurons and that this is increased in DS. Inhibiting TMPRSS2 and furin activity reduced viral replication in DS-ChPCOs to euploid levels. This model enables dissection of the role of ChP in neurotropic virus infection and euploid forebrain development and permits screening of therapeutics for SARS-CoV-2-induced neuropathogenesis.

Association of specific ACE2 and TMPRSS2 variants with circulatory cytokines of COVID-19 Emirati patients.

Introduction: The COVID-19 pandemic represented one of the most significant challenges to researchers and healthcare providers. Several factors determine the disease severity, whereas none alone can explain the tremendous variability. The Single nucleotide variants (SNVs) in angiotensin-converting enzyme-2 (ACE2) and transmembrane serine protease type-2 (TMPRSS2) genes affect the virus entry and are considered possible risk factors for COVID-19. Methods: We compiled a panel of gene variants from both genes and used in-silico analysis to predict their significance. We performed biological validation to assess their capacity to alter the ACE2 interaction with the virus spike protein. Subsequently, we conducted a retrospective comparative genome analysis on those variants in the Emirati patients with different disease severity (total of 96) along with 69 healthy control subjects. Results: Our results showed that the Emirati population lacks the variants that were previously reported as associated with disease severity, whereas a new variant in ACE2 "Chr X:g.15584534" was associated with disease severity specifically among female patients. In-silico analysis revealed that the new variant can determine the ACE2 gene transcription. Several cytokines (GM-CSF and IL-6) and chemokines (MCP-1/CCL2, IL-8/CXCL8, and IP-10/CXCL10) were markedly increased in COVID-19 patients with a significant correlation with disease severity. The newly reported genetic variant of ACE2 showed a positive correlation with CD40L, IL-1ß, IL-2, IL-15, and IL-17A in COVID-19 patients. Conclusion: Whereas COVID-19 represents now a past pandemic, our study underscores the importance of genetic factors specific to a population, which can influence both the susceptibility to viral infections and the level of severity; subsequently expected required preparedness in different areas of the world.

Pleiotropy of positive selection in ancient ACE2 suggests an alternative hypothesis for bat-specific adaptations to host coronaviruses.

Angiotensin-convertingenzyme 2 (ACE2) has dual functions, regulating cardiovascular physiology and serving as the receptor for coronaviruses. Bats, the only true flying mammals and natural viral reservoirs, have evolved positive alterations in traits related to both functions of ACE2. This suggests significant evolutionary changes in ACE2 during bat evolution. To test this hypothesis, we examine the selection pressure in ACE2 along the ancestral branch of all bats (AncBat-ACE2), where powered flight and bat-coronavirus coevolution occurred, and detect a positive selection signature. To assess the functional effects of positive selection, we resurrect AncBat-ACE2 and its mutant (AncBat-ACE2-mut) created by replacing the positively selected sites. Compared to AncBat-ACE2-mut, AncBat-ACE2 exhibits stronger enzymatic activity, enhances mice's performance in exercise fatigue, and shows lower affinity to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Our findings indicate the functional pleiotropy of positive selection in the ancient ACE2 of bats, providing an alternative hypothesis for the evolutionary origin of bats' defense against coronaviruses.

In vitro mRNA-S maternal vaccination induced altered immune regulation at the maternal-fetal interface.

BACKGROUND: Maternal-fetal immunology is intricate, and the effects of mRNA-S maternal vaccination on immune regulation at the maternal-fetal interface require further investigation. Our study endeavors to elucidate these immunological changes, enhancing our comprehension of maternal and fetal health outcomes. By analyzing immune profiles and cytokine responses, we aim to provide valuable insights into the impact of mRNA-S vaccination on the delicate balance of immune regulation during pregnancy, addressing critical questions in the field of reproductive pharmacology. OBJECTIVES: This investigation sought to examine the prospective influence of mRNA-S-based vaccines and extracellular vesicles (EVs) containing the Spike (S) protein at the maternal-fetal interface. Our primary emphasis was on evaluating their effects on maternal decidua cells and fetal chorion trophoblast cells (hFM-CTCs). METHODS: We validated the generation of EVs containing the S protein from small human airway epithelial cell lines (HSAECs) following mRNA-S vaccine exposure. We assessed the expression of angiotensin-converting enzyme 2 (ACE2) gene and protein in fetal membranes and the placenta, with specific attention to decidual cells and fetal membrane chorion cells. To assess cellular functionality, these cells were exposed to both recombinant S protein and EVs loaded with S proteins (eSPs). RESULTS: Our findings revealed that cells and EVs subjected to mRNA-S-based vaccination exhibited altered protein expression levels of S proteins. At the feto-maternal interface, both placental and fetal membrane tissues demonstrated similar ACE-2 expression levels. Among individual cellular layers, syncytiotrophoblast cells in the placenta and chorion cells in the fetal membrane exhibited elevated ACE-2 expression. Notably, EVs derived from HSAECs activated the MAPK pathway in decidual cells. Additionally, decidual cells displayed a substantial increase in gene expression of chemokines like CXCL-10 and CXCL-11, as well as proinflammatory cytokines such as IL-6 in response to eSPs. However, the levels of Ccl-2 and IL-1ß remained unchanged in decidual cells under the same conditions. Conversely, hFM-CTCs demonstrated significant alterations in the proinflammatory cytokines and chemokines with respect to eSPs. CONCLUSION: In conclusion, our study indicates that mRNA-S-based maternal vaccination during pregnancy may influence the maternal-fetal interface's COVID-19 interaction and immune regulation. Further investigation is warranted to assess safety and implications.

Combination of spironolactone and DPP-4 inhibitors for treatment of SARS-CoV-2 infection: a literature review.

Coronavirus disease 2019 (COVID-19) is still causing hospitalization and death, and vaccination appears to become less effective with each emerging variant. Spike, non-spike, and other possible unrecognized mutations have reduced the efficacy of recommended therapeutic approaches, including monoclonal antibodies, plasma transfusion, and antivirals. SARS-CoV-2 binds to angiotensin-converting enzyme 2 (ACE2) and probably dipeptidyl peptidase 4 (DPP-4) to initiate the process of endocytosis by employing host proteases such as transmembrane serine protease-2 (TMPRSS-2) and ADAM metallopeptidase domain 17 (ADAM17). Spironolactone reduces the amount of soluble ACE2 and antagonizes TMPRSS-2 and ADAM17. DPP-4 inhibitors play immunomodulatory roles and may block viral entry. The efficacy of treatment with a combination of spironolactone and DPP-4 inhibitors does not appear to be affected by viral mutations. Therefore, the combination of spironolactone and DPP-4 inhibitors might improve the clinical outcome for COVID-19 patients by decreasing the efficiency of SARS-CoV-2 entry into cells and providing better anti-inflammatory, antiproliferative, and antifibrotic effects than those achieved using current therapeutic approaches such as antivirals and monoclonal antibodies.

SARS-CoV-2 strains bearing Omicron BA.1 spike replicate in C57BL/6 mice.

Introduction: SARS-CoV-2, the cause of the COVID pandemic, is an RNA virus with a high propensity to mutate. Successive virus variants, including variants of concern (VOC), have emerged with increased transmission or immune escape. The original pandemic virus and early variants replicated poorly, if at all, in mice at least partly due to a mismatch between the receptor binding domain on the viral spike protein and the murine angiotensin converting enzyme 2 (ACE2). Omicron VOC emerged in late 2021 harboring > 50 new mutations, 35 of them in the spike protein. This variant resulted in a very large wave of infections, even in the face of prior immunity, albeit being inherently less severe than earlier variants. Reflecting the lower severity reported in humans, Omicron displayed attenuated infection in hamsters and also in the K18-hACE2 mouse model. K18-hACE2 mice express both the human ACE2 as well as the endogenous mouse ACE2. Methods: Here we infected hACE2 knock-in mice that express only human ACE2 and no murine ACE2, or C57BL/6 wildtype mice with SARS-CoV-2 D614G (first-wave isolate), Delta or Omicron BA.1 variants and assessed infectivity and downstream innate immune responses. Results: While replication of SARS-CoV-2 Omicron was lower in the lungs of hACE2 knock-in mice compared with SARS-CoV-2 D614G and VOC Delta, it replicated more efficiently than the earlier variants in C57BL/6 wildtype mice. This opens the opportunity to test the effect of host genetics on SARS-CoV-2 infections in wildtype mice. As a proof of principle, we tested Omicron infection in mice lacking expression of the interferon-alpha receptor-1 (IFNAR1). In these mice we found that loss of type I IFN receptor signaling resulted in higher viral loads in the lungs were detected. Finally, using a chimeric virus of first wave SARS-CoV-2 harboring the Omicron spike protein, we show that Omicron spike increase infection of C57BL/6 wildtype mice, but non-spike genes of Omicron confer attenuation of viral replication. Discussion: Since this chimeric virus efficiently infected C57BL/6 wildtype mice, and replicated in their lungs, our findings illustrate a pathway for genetic mapping of virushost interactions during SARS-CoV-2 infection.

Susceptibility to recombinant SARS-CoV-2 spike protein entry in the lungs of high-fat diet-induced obese mice.

Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Obesity is a major risk factor for the development of COVID-19. Angiotensin-converting enzyme 2 (ACE2) is an essential receptor for cell entry of SARS-CoV-2. The receptor-binding domain of the S1 subunit (S1-RBD protein) in the SARS-CoV-2 spike glycoprotein binds to ACE2 on host cells, through which the virus enters several organs, including the lungs. Considering these findings, recombinant ACE2 might be utilized as a decoy protein to attenuate SARS-CoV-2 infection. Here, we examined whether obesity increases ACE2 expression in the lungs and whether recombinant ACE2 administration diminishes the entry of S1-RBD protein into lung cells. We observed that high-fat diet-induced obesity promoted ACE2 expression in the lungs by increasing serum levels of LPS derived from the intestine. S1-RBD protein entered the lungs specifically through ACE2 expressed in host lungs and that the administration of recombinant ACE2 attenuated this entry. We conclude that obesity makes hosts susceptible to recombinant SARS-CoV-2 spike proteins due to elevated ACE2 expression in lungs, and this model of administering S1-RBD protein can be applied to new COVID-19 treatments.

AlphaFold2 Predictions of Conformational Ensembles and Atomistic Simulations of the SARS-CoV-2 Spike XBB Lineages Reveal Epistatic Couplings between Convergent Mutational Hotspots that Control ACE2 Affinity.

In this study, we combined AlphaFold-based atomistic structural modeling, microsecond molecular simulations, mutational profiling, and network analysis to characterize binding mechanisms of the SARS-CoV-2 spike protein with the host receptor ACE2 for a series of Omicron XBB variants including XBB.1.5, XBB.1.5+L455F, XBB.1.5+F456L, and XBB.1.5+L455F+F456L. AlphaFold-based structural and dynamic modeling of SARS-CoV-2 Spike XBB lineages can accurately predict the experimental structures and characterize conformational ensembles of the spike protein complexes with the ACE2. Microsecond molecular dynamics simulations identified important differences in the conformational landscapes and equilibrium ensembles of the XBB variants, suggesting that combining AlphaFold predictions of multiple conformations with molecular dynamics simulations can provide a complementary approach for the characterization of functional protein states and binding mechanisms. Using the ensemble-based mutational profiling of protein residues and physics-based rigorous calculations of binding affinities, we identified binding energy hotspots and characterized the molecular basis underlying epistatic couplings between convergent mutational hotspots. Consistent with the experiments, the results revealed the mediating role of the Q493 hotspot in the synchronization of epistatic couplings between L455F and F456L mutations, providing a quantitative insight into the energetic determinants underlying binding differences between XBB lineages. We also proposed a network-based perturbation approach for mutational profiling of allosteric communications and uncovered the important relationships between allosteric centers mediating long-range communication and binding hotspots of epistatic couplings. The results of this study support a mechanism in which the binding mechanisms of the XBB variants may be determined by epistatic effects between convergent evolutionary hotspots that control ACE2 binding.

Analysis the molecular similarity of least common amino acid sites in ACE2 receptor to predict the potential susceptible species for SARS-CoV-2.

SARS-CoV-2 infections in animals have been reported globally. However, the understanding of the complete spectrum of animals susceptible to SARS-CoV-2 remains limited. The virus's dynamic nature and its potential to infect a wide range of animals are crucial considerations for a One Health approach that integrates both human and animal health. This study introduces a bioinformatic approach to predict potential susceptibility to SARS-CoV-2 in both domestic and wild animals. By examining genomic sequencing, we establish phylogenetic relationships between the virus and its potential hosts. We focus on the interaction between the SARS-CoV-2 genome sequence and specific regions of the host species' ACE2 receptor. We analyzed and compared ACE2 receptor sequences from 29 species known to be infected, selecting 10 least common amino acid sites (LCAS) from key binding domains based on similarity patterns. Our analysis included 49 species across primates, carnivores, rodents, and artiodactyls, revealing complete consistency in the LCAS and identifying them as potentially susceptible. We employed the LCAS similarity pattern to predict the likelihood of SARS-CoV-2 infection in unexamined species. This method serves as a valuable screening tool for assessing infection risks in domestic and wild animals, aiding in the prevention of disease outbreaks.

Effects of Chronic Inflammatory Activation of Murine and Human Arterial Endothelial Cells at Normal Lipoprotein and Cholesterol Levels In Vivo and In Vitro.

The activation of endothelial cells is crucial for immune defense mechanisms but also plays a role in the development of atherosclerosis. We have previously shown that inflammatory stimulation of endothelial cells on top of elevated lipoprotein/cholesterol levels accelerates atherogenesis. The aim of the current study was to investigate how chronic endothelial inflammation changes the aortic transcriptome of mice at normal lipoprotein levels and to compare this to the inflammatory response of isolated endothelial cells in vitro. We applied a mouse model expressing constitutive active IκB kinase 2 (caIKK2)-the key activator of the inflammatory NF-κB pathway-specifically in arterial endothelial cells and analyzed transcriptomic changes in whole aortas, followed by pathway and network analyses. We found an upregulation of cell death and mitochondrial beta-oxidation pathways with a predicted increase in endothelial apoptosis and necrosis and a simultaneous reduction in protein synthesis genes. The highest upregulated gene was ACE2, the SARS-CoV-2 receptor, which is also an important regulator of blood pressure. Analysis of isolated human arterial and venous endothelial cells supported these findings and also revealed a reduction in DNA replication, as well as repair mechanisms, in line with the notion that chronic inflammation contributes to endothelial dysfunction.

Two new and effective food-extracted immunomodulatory agents exhibit anti-inflammatory response activity in the hACE2 acute lung injury murine model of COVID-19.

Objective: The coronavirus disease 2019 (COVID-19) spread rapidly and claimed millions of lives worldwide. Acute respiratory distress syndrome (ARDS) is the major cause of COVID-19-associated deaths. Due to the limitations of current drugs, developing effective therapeutic options that can be used rapidly and safely in clinics for treating severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infections is necessary. This study aims to investigate the effects of two food-extracted immunomodulatory agents, ajoene-enriched garlic extract (AGE) and cruciferous vegetables-extracted sulforaphane (SFN), on anti-inflammatory and immune responses in a SARS-CoV-2 acute lung injury mouse model. Methods: In this study, we established a mouse model to mimic the SARS-CoV-2 infection acute lung injury model via intratracheal injection of polyinosinic:polycytidylic acid (poly[I:C]) and SARS-CoV-2 recombinant spike protein (SP). After the different agents treatment, lung sections, bronchoalveolar lavage fluid (BALF) and fresh faeces were harvested. Then, H&E staining was used to examine symptoms of interstitial pneumonia. Flow cytometry was used to examine the change of immune cell populations. Multiplex cytokines assay was used to examine the inflammatory cytokines.16S rDNA high-throughput sequencing was used to examine the change of gut microbiome. Results: Our results showed that AGE and SFN significantly suppressed the symptoms of interstitial pneumonia, effectively inhibited the production of inflammatory cytokines, decreased the percentage of inflammatory cell populations, and elevated T cell populations in the mouse model. Furthermore, we also observed that the gut microbiome of genus Paramuribaculum were enriched in the AGE-treated group. Conclusion: Here, for the first time, we observed that these two novel, safe, and relatively inexpensive immunomodulatory agents exhibited the same effects on anti-inflammatory and immune responses as neutralizing monoclonal antibodies (mAbs) against interleukin 6 receptor (IL-6R), which have been suggested for treating COVID-19 patients. Our results revealed the therapeutic ability of these two immunomodulatory agents in a mouse model of SARS-CoV-2 acute lung injury by promoting anti-inflammatory and immune responses. These results suggest that AGE and SFN are promising candidates for the COVID-19 treatment.

TMPRSS2-mediated SARS-CoV-2 uptake boosts innate immune activation, enhances cytopathology, and drives convergent virus evolution.

The accessory protease transmembrane protease serine 2 (TMPRSS2) enhances severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) uptake into ACE2-expressing cells, although how increased entry impacts downstream viral and host processes remains unclear. To investigate this in more detail, we performed infection assays in engineered cells promoting ACE2-mediated entry with and without TMPRSS2 coexpression. Electron microscopy and inhibitor experiments indicated TMPRSS2-mediated cell entry was associated with increased virion internalization into endosomes, and partially dependent upon clathrin-mediated endocytosis. TMPRSS2 increased panvariant uptake efficiency and enhanced early rates of virus replication, transcription, and secretion, with variant-specific profiles observed. On the host side, transcriptional profiling confirmed the magnitude of infection-induced antiviral and proinflammatory responses were linked to uptake efficiency, with TMPRSS2-assisted entry boosting early antiviral responses. In addition, TMPRSS2-enhanced infections increased rates of cytopathology, apoptosis, and necrosis and modulated virus secretion kinetics in a variant-specific manner. On the virus side, convergent signatures of cell-uptake-dependent innate immune induction were recorded in viral genomes, manifesting as switches in dominant coupled Nsp3 residues whose frequencies were correlated to the magnitude of the cellular response to infection. Experimentally, we demonstrated that selected Nsp3 mutations conferred enhanced interferon antagonism. More broadly, we show that TMPRSS2 orthologues from evolutionarily diverse mammals facilitate panvariant enhancement of cell uptake. In summary, our study uncovers previously unreported associations, linking cell entry efficiency to innate immune activation kinetics, cell death rates, virus secretion dynamics, and convergent selection of viral mutations. These data expand our understanding of TMPRSS2's role in the SARS-CoV-2 life cycle and confirm its broader significance in zoonotic reservoirs and animal models.

Biophysical principles predict fitness of SARS-CoV-2 variants.

SARS-CoV-2 employs its spike protein's receptor binding domain (RBD) to enter host cells. The RBD is constantly subjected to immune responses, while requiring efficient binding to host cell receptors for successful infection. However, our understanding of how RBD's biophysical properties contribute to SARS-CoV-2's epidemiological fitness remains largely incomplete. Through a comprehensive approach, comprising large-scale sequence analysis of SARS-CoV-2 variants and the identification of a fitness function based on binding thermodynamics, we unravel the relationship between the biophysical properties of RBD variants and their contribution to viral fitness. We developed a biophysical model that uses statistical mechanics to map the molecular phenotype space, characterized by dissociation constants of RBD to ACE2, LY-CoV016, LY-CoV555, REGN10987, and S309, onto an epistatic fitness landscape. We validate our findings through experimentally measured and machine learning (ML) estimated binding affinities, coupled with infectivity data derived from population-level sequencing. Our analysis reveals that this model effectively predicts the fitness of novel RBD variants and can account for the epistatic interactions among mutations, including explaining the later reversal of Q493R. Our study sheds light on the impact of specific mutations on viral fitness and delivers a tool for predicting the future epidemiological trajectory of previously unseen or emerging low-frequency variants. These insights offer not only greater understanding of viral evolution but also potentially aid in guiding public health decisions in the battle against COVID-19 and future pandemics.

Gene variants rs5182, rs2074192, and rs4343 in the renin-angiotensin-aldosterone system are associated with symptom severity, higher odds of hospitalization, and death in COVID-19.

OBJECTIVES: To analyze the gene variants of the renin-angiotensin-aldosterone system and determine their association with the severity and outcome of COVID-19. METHODS: A total of 104 patients were included in the study: 34 asymptomatic patients with COVID-19 as controls and 70 symptomatic patients as cases. The genetic variants ACE rs4343, ACE2 rs2074192, AGTR1 rs5182, and AGT rs4762 were identified using TaqMan genotyping tests. RESULTS: Patients with the T/T genotype of AGTR1 rs5182 have a higher probability of developing symptomatic COVID-19 (odds ratio [OR] 12.25, 95% confidence interval [CI] 1.34-111.9, P ≤0.001) and a higher risk of hospitalization because of disease (OR 14.00, 95% CI 1.53-128.49, P = 0.012). The haplotype CTG (AGTR1 rs5182, ACE2 rs2074192, ACE rs4343) decreased the odds of death related to COVID-19 in the study population (OR 0.03, 95% CI 0.0-0.06, P = 0.026). CONCLUSIONS: The T/T genotype of the AGTR1 rs5182 variant increased the probability of symptomatic COVID-19 and hospitalization, whereas the haplotype CTG (consisting of AGTR1 rs5182, ACE2 rs2074192, and ACE rs4343) decreased the odds of death related to COVID-19 by 97% in the hospitalized patients with COVID-19. These results support the participation of renin-angiotensin-aldosterone system gene variants as modifiers of the severity of symptoms associated with SARS-CoV-2 infection and the outcome of COVID-19.

A systematic mutation analysis of 13 major SARS-CoV-2 variants.

SARS-CoV-2 evolves constantly with various novel mutations. Due to their enhanced infectivity, transmissibility and immune evasion, a comprehensive understanding of the association between these mutations and the respective functional changes is crucial. However, previous mutation studies of major SARS-CoV-2 variants remain limited. Here, we performed systematic analyses of full-length amino acids mutation, phylogenetic features, protein physicochemical properties, molecular dynamics and immune escape as well as pseudotype virus infection assays among thirteen major SARS-CoV-2 variants. We found that Omicron exhibited the most abundant and complex mutation sites, higher indices of hydrophobicity and flexibility than other variants. The results of molecular dynamics simulation suggest that Omicron has the highest number of hydrogen bonds and strongest binding free energy between the S protein and ACE2 receptor. Furthermore, we revealed 10 immune escape sites in 13 major variants, some of them were reported previously, but four of which (i.e. 339/373/477/496) are first reported to be specific to Omicron, whereas 462 is specific to Epslion. The infectivity of these variants was confirmed by the pseudotype virus infection assays. Our findings may help us understand the functional consequences of the mutations within various variants and the underlying mechanisms of the immune escapes conferred by the S proteins.

A murine model of post-acute neurological sequelae following SARS-CoV-2 variant infection.

Viral variant is one known risk factor associated with post-acute sequelae of COVID-19 (PASC), yet the pathogenesis is largely unknown. Here, we studied SARS-CoV-2 Delta variant-induced PASC in K18-hACE2 mice. The virus replicated productively, induced robust inflammatory responses in lung and brain tissues, and caused weight loss and mortality during the acute infection. Longitudinal behavior studies in surviving mice up to 4 months post-acute infection revealed persistent abnormalities in neuropsychiatric state and motor behaviors, while reflex and sensory functions recovered over time. In the brain, no detectable viral RNA and minimal residential immune cell activation was observed in the surviving mice post-acute infection. Transcriptome analysis revealed persistent activation of immune pathways, including humoral responses, complement, and phagocytosis, and gene expression levels associated with ataxia telangiectasia, impaired cognitive function and memory recall, and neuronal dysfunction and degeneration. Furthermore, surviving mice maintained potent systemic T helper 1 prone cellular immune responses and strong sera neutralizing antibodies against Delta and Omicron variants months post-acute infection. Overall, our findings suggest that infection in K18-hACE2 mice recapitulates the persistent clinical symptoms reported in long-COVID patients and provides new insights into the role of systemic and brain residential immune factors in PASC pathogenesis.

Genetic variations in ACE2 gene associated with metabolic syndrome in southern China: a case-control study.

Metabolic syndrome (MetS) is closely related to cardiovascular and cerebrovascular diseases, and genetic predisposition is one of the main triggers for its development. To identify the susceptibility genes for MetS, we investigated the relationship between angiotensin-converting enzyme 2 (ACE2) single nucleotide polymorphisms (SNPs) and MetS in southern China. In total, 339 MetS patients and 398 non-MetS hospitalized patients were recruited. Four ACE2 polymorphisms (rs2074192, rs2106809, rs879922, and rs4646155) were genotyped using the polymerase chain reaction-ligase detection method and tested for their potential association with MetS and its related components. ACE2 rs2074192 and rs2106809 minor alleles conferred 2.485-fold and 3.313-fold greater risks of MetS in women. ACE2 rs2074192 and rs2106809 variants were risk factors for obesity, diabetes, and low-high-density lipoprotein cholesterolemia. However, in men, the ACE2 rs2074192 minor allele was associated with an approximately 0.525-fold reduction in MetS prevalence. Further comparing the components of MetS, ACE2 rs2074192 and rs2106809 variants reduced the risk of obesity and high triglyceride levels. In conclusion, ACE2 rs2074192 and rs2106809 SNPs were independently associated with MetS in a southern Chinese population and showed gender heterogeneity, which can be partially explained by obesity. Thus, these SNPs may be utilized as predictive biomarkers and molecular targets for MetS. A limitation of this study is that environmental and lifestyle differences, as well as genetic heterogeneity among different populations, were not considered in the analysis.

The lethal K18-hACE2 knock-in mouse model mimicking the severe pneumonia of COVID-19 is practicable for antiviral development.

Animal models of COVID-19 facilitate the development of vaccines and antivirals against SARS-CoV-2. The efficacy of antivirals or vaccines may differ in different animal models with varied degrees of disease. Here, we introduce a mouse model expressing human angiotensin-converting enzyme 2 (ACE2). In this model, ACE2 with the human cytokeratin 18 promoter was knocked into the Hipp11 locus of C57BL/6J mouse by CRISPR - Cas9 (K18-hACE2 KI). Upon intranasal inoculation with high (3 × 105 PFU) or low (2.5 × 102 PFU) dose of SARS-CoV-2 wildtype (WT), Delta, Omicron BA.1, or Omicron BA.2 variants, all mice showed obvious infection symptoms, including weight loss, high viral loads in the lung, and interstitial pneumonia. 100% lethality was observed in K18-hACE2 KI mice infected by variants with a delay of endpoint for Delta and BA.1, and a significantly attenuated pathogenicity was observed for BA.2. The pneumonia of infected mice was accompanied by the infiltration of neutrophils and pulmonary fibrosis in the lung. Compared with K18-hACE2 Tg mice and HFH4-hACE2 Tg mice, K18-hACE2 KI mice are more susceptible to SARS-CoV-2. In the antivirals test, REGN10933 and Remdesivir had limited antiviral efficacies in K18-hACE2 KI mice upon the challenge of SARS-CoV-2 infections, while Nirmatrelvir, monoclonal antibody 4G4, and mRNA vaccines potently protected the mice from death. Our results suggest that the K18-hACE2 KI mouse model is lethal and stable for SARS-CoV-2 infection, and is practicable and stringent to antiviral development.

Some mechanistic underpinnings of molecular adaptations of SARS-COV-2 spike protein by integrating candidate adaptive polymorphisms with protein dynamics.

We integrate evolutionary predictions based on the neutral theory of molecular evolution with protein dynamics to generate mechanistic insight into the molecular adaptations of the SARS-COV-2 spike (S) protein. With this approach, we first identified candidate adaptive polymorphisms (CAPs) of the SARS-CoV-2 S protein and assessed the impact of these CAPs through dynamics analysis. Not only have we found that CAPs frequently overlap with well-known functional sites, but also, using several different dynamics-based metrics, we reveal the critical allosteric interplay between SARS-CoV-2 CAPs and the S protein binding sites with the human ACE2 (hACE2) protein. CAPs interact far differently with the hACE2 binding site residues in the open conformation of the S protein compared to the closed form. In particular, the CAP sites control the dynamics of binding residues in the open state, suggesting an allosteric control of hACE2 binding. We also explored the characteristic mutations of different SARS-CoV-2 strains to find dynamic hallmarks and potential effects of future mutations. Our analyses reveal that Delta strain-specific variants have non-additive (i.e., epistatic) interactions with CAP sites, whereas the less pathogenic Omicron strains have mostly additive mutations. Finally, our dynamics-based analysis suggests that the novel mutations observed in the Omicron strain epistatically interact with the CAP sites to help escape antibody binding.

Mutations in the SARS-CoV-2 spike proteins affected the ACE2-binding affinity during the development of Omicron pandemic variants.

Mutations in SARS-CoV-2 caused multiple waves of pandemics. To identify the function of such mutations, we investigated the binding affinity of the S protein with its receptor, ACE2. Omicron BA.1 showed significantly lower binding affinity with human ACE2 than prototype SARS-CoV-2 and Alpha strain, indicating that pre-Omicron to Omicron transition was not mediated by increasing the ACE2-binding affinity. Meanwhile, the later Omicron variants, BA.5 and XBB.1.5, showed significantly higher ACE2-binding affinity, suggesting that the increased ACE2-binding could be involved in the variant transition within Omicron strains. Furthermore, Alpha and Omicron variants, but not prototype SARS-CoV-2, bound mouse ACE2, which lead to a hypothesis that early Omicron strains evolved from Alpha strain by acquiring multiple mutations in mice.