SARS-CoV-2 BA.2.86 enters lung cells and evades neutralizing antibodies with high efficiency.

BA.2.86, a recently identified descendant of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron BA.2 sublineage, contains ∼35 mutations in the spike (S) protein and spreads in multiple countries. Here, we investigated whether the virus exhibits altered biological traits, focusing on S protein-driven viral entry. Employing pseudotyped particles, we show that BA.2.86, unlike other Omicron sublineages, enters Calu-3 lung cells with high efficiency and in a serine- but not cysteine-protease-dependent manner. Robust lung cell infection was confirmed with authentic BA.2.86, but the virus exhibited low specific infectivity. Further, BA.2.86 was highly resistant against all therapeutic antibodies tested, efficiently evading neutralization by antibodies induced by non-adapted vaccines. In contrast, BA.2.86 and the currently circulating EG.5.1 sublineage were appreciably neutralized by antibodies induced by the XBB.1.5-adapted vaccine. Collectively, BA.2.86 has regained a trait characteristic of early SARS-CoV-2 lineages, robust lung cell entry, and evades neutralizing antibodies. However, BA.2.86 exhibits low specific infectivity, which might limit transmissibility.

A multi-epitope/CXCL11 prime/pull coronavirus mucosal vaccine boosts the frequency and the function of lung-resident memory CD4+ and CD8+ T cells and enhanced protection against COVID-19-like symptoms and death caused by SARS-CoV-2 infection.

IMPORTANCE: Although the current rate of SARS-CoV-2 infections has decreased significantly, COVID-19 still ranks very high as a cause of death worldwide. As of October 2023, the weekly mortality rate is still at 600 deaths in the United States alone, which surpasses even the worst mortality rates recorded for influenza. Thus, the long-term outlook of COVID-19 is still a serious concern outlining the need for the next-generation vaccine. This study found that a prime/pull coronavirus vaccine strategy increased the frequency of functional SARS-CoV-2-specific CD4+ and CD8+ memory T cells in the lungs of SARS-CoV-2-infected triple transgenic HLA-DR*0101/HLA-A*0201/hACE2 mouse model, thereby resulting in low viral titer and reduced COVID-19-like symptoms.

Lung dendritic-cell metabolism underlies susceptibility to viral infection in diabetes.

People with diabetes feature a life-risking susceptibility to respiratory viral infection, including influenza and SARS-CoV-2 (ref. 1), whose mechanism remains unknown. In acquired and genetic mouse models of diabetes, induced with an acute pulmonary viral infection, we demonstrate that hyperglycaemia leads to impaired costimulatory molecule expression, antigen transport and T cell priming in distinct lung dendritic cell (DC) subsets, driving a defective antiviral adaptive immune response, delayed viral clearance and enhanced mortality. Mechanistically, hyperglycaemia induces an altered metabolic DC circuitry characterized by increased glucose-to-acetyl-CoA shunting and downstream histone acetylation, leading to global chromatin alterations. These, in turn, drive impaired expression of key DC effectors including central antigen presentation-related genes. Either glucose-lowering treatment or pharmacological modulation of histone acetylation rescues DC function and antiviral immunity. Collectively, we highlight a hyperglycaemia-driven metabolic-immune axis orchestrating DC dysfunction during pulmonary viral infection and identify metabolic checkpoints that may be therapeutically exploited in mitigating exacerbated disease in infected diabetics.

Type I interferon signaling induces a delayed antiproliferative response in respiratory epithelial cells during SARS-CoV-2 infection.

ABSTRACT: Disease progression during SARS-CoV-2 infection is tightly linked to the fate of lung epithelial cells, with severe cases of COVID-19 characterized by direct injury of the alveolar epithelium and an impairment in its regeneration from progenitor cells. The molecular pathways that govern respiratory epithelial cell death and proliferation during SARS-CoV-2 infection, however, remain unclear. We now report a high-throughput CRISPR screen for host genetic modifiers of the survival and proliferation of SARS-CoV-2-infected Calu-3 respiratory epithelial cells. The top four genes identified in our screen encode components of the same type I interferon (IFN-I) signaling complex­IFNAR1, IFNAR2, JAK1, and TYK2. The fifth gene, ACE2, was an expected control encoding the SARS-CoV-2 viral receptor. Surprisingly, despite the antiviral properties of IFN-I signaling, its disruption in our screen was associated with an increase in Calu-3 cell fitness. We validated this effect and found that IFN-I signaling did not sensitize SARS-CoV-2-infected cultures to cell death but rather inhibited the proliferation of surviving cells after the early peak of viral replication and cytopathic effect. We also found that IFN-I signaling alone, in the absence of viral infection, was sufficient to induce this delayed antiproliferative response in both Calu-3 cells and iPSC-derived type 2 alveolar epithelial cells. Together, these findings highlight a cell autonomous antiproliferative response by respiratory epithelial cells to persistent IFN-I signaling during SARS-CoV-2 infection. This response may contribute to the deficient alveolar regeneration that has been associated with COVID-19 lung injury and represents a promising area for host-targeted therapeutic development.

Avian influenza A viruses exhibit plasticity in sialylglycoconjugate receptor usage in human lung cells.

IMPORTANCE: It is well known that influenza A viruses (IAV) initiate host cell infection by binding to sialic acid, a sugar molecule present at the ends of various sugar chains called glycoconjugates. These sugar chains can vary in chain length, structure, and composition. However, it remains unknown if IAV strains preferentially bind to sialic acid on specific glycoconjugate type(s) for host cell infection. Here, we utilized CRISPR gene editing to abolish sialic acid on different glycoconjugate types in human lung cells, and evaluated human versus avian IAV infections. Our studies show that both human and avian IAV strains can infect human lung cells by utilizing any of the three major sialic acid-containing glycoconjugate types, specifically N-glycans, O-glycans, and glycolipids. Interestingly, simultaneous elimination of sialic acid on all three major glycoconjugate types in human lung cells dramatically decreased human IAV infection, yet had little effect on avian IAV infection. These studies show that avian IAV strains effectively utilize other less prevalent glycoconjugates for infection, whereas human IAV strains rely on a limited repertoire of glycoconjugate types. The remarkable ability of avian IAV strains to utilize diverse glycoconjugate types may allow for easy transmission into new host species.

Characterization of the SARS-CoV-2 BA.5.5 and BQ.1.1 Omicron variants in mice and hamsters.

The continued evolution and emergence of novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants have resulted in challenges to vaccine and antibody efficacy. The emergence of each new variant necessitates the need to re-evaluate and refine animal models used for countermeasure testing. Here, we tested a recently circulating SARS-CoV-2 Omicron lineage variant, BQ.1.1, in multiple rodent models including K18-human ACE2 (hACE2) transgenic, C57BL/6J, and 129S2 mice, and Syrian golden hamsters. In contrast to a previously dominant BA.5.5 Omicron variant, inoculation of K18-hACE2 mice with BQ.1.1 resulted in substantial weight loss, a characteristic seen in pre-Omicron variants. BQ.1.1 also replicated to higher levels in the lungs of K18-hACE2 mice and caused greater lung pathology than the BA.5.5 variant. However, in C57BL/6J mice, 129S2 mice, and Syrian hamsters, BQ.1.1 did not cause increased respiratory tract infection or disease compared to animals administered BA.5.5. Moreover, the rates of direct contact or airborne transmission in hamsters were not significantly different after BQ.1.1 and BA.5.5 infections. Taken together, these data suggest that the BQ.1.1 Omicron variant has increased virulence in rodent species that express hACE2, possibly due to the acquisition of unique spike mutations relative to earlier Omicron variants. IMPORTANCE As severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to evolve, there is a need to rapidly assess the efficacy of vaccines and antiviral therapeutics against newly emergent variants. To do so, the commonly used animal models must also be re-evaluated. Here, we determined the pathogenicity of the BQ.1.1 SARS-CoV-2 variant in multiple SARS-CoV-2 animal models including transgenic mice expressing human ACE2 (hACE2), two strains of conventional laboratory mice, and Syrian hamsters. While BQ.1.1 and BA.5.5 infection resulted in similar levels of viral burden and clinical disease in hamsters and the conventional strains of laboratory mice tested, increases in lung infection were detected in hACE2-expressing transgenic mice, which corresponded with greater levels of pro-inflammatory cytokines and lung pathology. Taken together, our data highlight important differences in two closely related Omicron SARS-CoV-2 variant strains and provide a foundation for evaluating countermeasures.

In-vivo and human evidence for potential efficacy of therapeutic polyclonal RSV neutralizing antibodies for palivizumab-resistant RSV infections.

BACKGROUND: Monoclonal antibody (palivizumab), intravenous immune globulin (IGIV), or respiratory syncytial virus (RSV)-polyclonal-hyperimmune-globulin (RSV-IG as Respigam®, RI-001, RI-002) are used with ribavirin in RSV-infected immunocompromised patients, with debated efficacy. Palivizumab-resistance (PR) can arise during treatment of persistent infections in this population. RSV-IG may confer benefit in PR-RSV infection. METHODS: RSV-IG [RI-001] was provided for an immunocompromised infant with RSV-pneumonitis refractory to ribavirin and palivizumab. RSV-neutralizing antibody, respiratory RSV load (qPCR), and F-gene-sequence-detection of PR was determined. Prophylactic RSV-IG [RI-002] or palivizumab was administered in a cotton-rat model infected with wild-type and PR-RSV. Lung RSV load and neutralizing antibody were measured. RESULTS: As protective RI-001-neutralizing antibody titers waned in the infant, a subpopulation of PR-escape mutants were detected with a fatal RSV-burden in the lungs. In PR-RSV-infected cotton rats, prophylactic RI-002 reduced RSV-load in the lungs (2.45 vs 0.28 log10 PFU/g lung-tissue reduction, respectively, p < 0.05) and provided protective RSV-neutralizing antibody. CONCLUSIONS: RSV-IG and ribavirin use in immunocompromised patients requires further study.

SARS-CoV-2 spike protein induces lung endothelial cell dysfunction and thrombo-inflammation depending on the C3a/C3a receptor signalling.

The spike protein of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) can interact with endothelial cells. However, no studies demonstrated the direct effect of the spike protein subunit 1 (S1) in inducing lung vascular damage and the potential mechanisms contributing to lung injury. Here, we found that S1 injection in mice transgenic for human angiotensin converting enzyme 2 (ACE2) induced early loss of lung endothelial thromboresistance at 3 days, as revealed by thrombomodulin loss and von Willebrand factor (vWF) increase. In parallel, vascular and epithelial C3 deposits and enhanced C3a receptor (C3aR) expression were observed. These changes preceded diffuse alveolar damage and lung vascular fibrin(ogen)/platelets aggregates at 7 days, as well as inflammatory cell recruitment and fibrosis. Treatment with C3aR antagonist (C3aRa) inhibited lung C3 accumulation and C3a/C3aR activation, limiting vascular thrombo-inflammation and fibrosis. Our study demonstrates that S1 triggers vascular dysfunction and activates complement system, instrumental to lung thrombo-inflammatory injury. By extension, our data indicate C3aRa as a valuable therapeutic strategy to limit S1-dependent lung pathology.

Evaluation of the Interactions between Mumps Virus and Guinea Pig.

Mumps is a highly contagious viral disease that can be prevented by vaccination. In the last decade, we have encountered repeated outbreaks of mumps in highly vaccinated populations, which call into question the effectiveness of available vaccines. Animal models are crucial for understanding virus-host interactions, and viruses such as mumps virus (MuV), whose only natural host is the human, pose a particular challenge. In our study, we examined the interaction between MuV and the guinea pig. Our results present the first evidence that guinea pigs of the Hartley strain can be infected in vivo after intranasal and intratesticular inoculation. We observed a significant viral replication in infected tissues up to 5 days following infection and induction of cellular and humoral immune responses as well as histopathological changes in infected lungs and testicles, without clinical signs of disease. Transmission of the infection through direct contact between animals was not possible. Our results demonstrate that guinea pigs and guinea pig primary cell cultures represent a promising model for immunological and pathogenetic studies of the complex MuV infection. IMPORTANCE Understanding of mumps virus (MuV) pathogenesis and the immune responses against MuV infection is limited. One of the reasons is the lack of relevant animal models. This study explores the interaction between MuV and the guinea pig. We demonstrated that all tested guinea pig tissue homogenates and primary cell cultures are highly susceptible to MuV infection and that α2,3-sialylated glycans (MuV cellular receptors) are being abundantly expressed at their surface. The virus remains in the guinea pig lungs and trachea for up to 4 days following intranasal infection. Although asymptomatic, MuV infection strongly activates both humoral and cellular immune response in infected animals and provides protection against virus challenge. Infection of the lungs and testicles after intranasal and intratesticular inoculation, respectively, is also supported by histopathological changes in these organs. Our findings give perspective for application of guinea pigs in research on MuV pathogenesis, antiviral response, and vaccine development and testing.

A replicon RNA vaccine can induce durable protective immunity from SARS-CoV-2 in nonhuman primates after neutralizing antibodies have waned.

The global SARS-CoV-2 pandemic prompted rapid development of COVID-19 vaccines. Although several vaccines have received emergency approval through various public health agencies, the SARS-CoV-2 pandemic continues. Emergent variants of concern, waning immunity in the vaccinated, evidence that vaccines may not prevent transmission and inequity in vaccine distribution have driven continued development of vaccines against SARS-CoV-2 to address these public health needs. In this report, we evaluated a novel self-amplifying replicon RNA vaccine against SARS-CoV-2 in a pigtail macaque model of COVID-19 disease. We found that this vaccine elicited strong binding and neutralizing antibody responses against homologous virus. We also observed broad binding antibody against heterologous contemporary and ancestral strains, but neutralizing antibody responses were primarily targeted to the vaccine-homologous strain. While binding antibody responses were sustained, neutralizing antibody waned to undetectable levels in some animals after six months but were rapidly recalled and conferred protection from disease when the animals were challenged 7 months after vaccination as evident by reduced viral replication and pathology in the lower respiratory tract, reduced viral shedding in the nasal cavity and lower concentrations of pro-inflammatory cytokines in the lung. Cumulatively, our data demonstrate in pigtail macaques that a self-amplifying replicon RNA vaccine can elicit durable and protective immunity to SARS-CoV-2 infection. Furthermore, these data provide evidence that this vaccine can provide durable protective efficacy and reduce viral shedding even after neutralizing antibody responses have waned to undetectable levels.

Generation of Human Embryonic Stem Cell-Derived Lung Organoids for Modeling Infection and Replication Differences between Human Adenovirus Types 3 and 55 and Evaluating Potential Antiviral Drugs.

Human adenoviruses type 3 (HAdV-3) and type 55 (HAdV-55) are frequently encountered, highly contagious respiratory pathogens with high morbidity rate. In contrast to HAdV-3, one of the most predominant types in children, HAdV-55 is a reemergent pathogen associated with more severe community-acquired pneumonia (CAP) in adults, especially in military camps. However, the infectivity and pathogenicity differences between these viruses remain unknown as in vivo models are not available. Here, we report a novel system utilizing human embryonic stem cells-derived 3-dimensional airway organoids (hAWOs) and alveolar organoids (hALOs) to investigate these two viruses. Firstly, HAdV-55 replicated more robustly than HAdV-3. Secondly, cell tropism analysis in hAWOs and hALOs by immunofluorescence staining revealed that HAdV-55 infected more airway and alveolar stem cells (basal and AT2 cells) than HAdV-3, which may lead to impairment of self-renewal functions post-injury and the loss of cell differentiation in lungs. Additionally, the viral life cycles of HAdV-3 and -55 in organoids were also observed using Transmission Electron Microscopy. This study presents a useful pair of lung organoids for modeling infection and replication differences between respiratory pathogens, illustrating that HAdV-55 has relatively higher replication efficiency and more specific cell tropism in human lung organoids than HAdV-3, which may result in relatively higher pathogenicity and virulence of HAdV-55 in human lungs. The model system is also suitable for evaluating potential antiviral drugs, as demonstrated with cidofovir. IMPORTANCE Human adenovirus (HAdV) infections are a major threat worldwide. HAdV-3 is one of the most predominant respiratory pathogen types found in children. Many clinical studies have reported that HAdV-3 causes less severe disease. In contrast, HAdV-55, a reemergent acute respiratory disease pathogen, is associated with severe community-acquired pneumonia in adults. Currently, no ideal in vivo models are available for studying HAdVs. Therefore, the mechanism of infectivity and pathogenicity differences between human adenoviruses remain unknown. In this study, a useful pair of 3-dimensional (3D) airway organoids (hAWOs) and alveolar organoids (hALOs) were developed to serve as a model. The life cycles of HAdV-3 and HAdV-55 in these human lung organoids were documented for the first time. These 3D organoids harbor different cell types, which are similar to the ones found in humans. This allows for the study of the natural target cells for infection. The finding of differences in replication efficiency and cell tropism between HAdV-55 and -3 may provide insights into the mechanism of clinical pathogenicity differences between these two important HAdV types. Additionally, this study provides a viable and effective in vitro tool for evaluating potential anti-adenoviral treatments.

SARS-CoV-2 Mutations Lead to a Decrease in the Number of Tissue-Specific MicroRNA-Binding Regions in the Lung.

RNA interference in vertebrates acts as an antiviral mechanism only in undifferentiated embryonic stem cells and is mediated by microRNAs. In somatic cells, host microRNAs also bind to the genomes of RNA viruses, regulating their translation and replication. It has been shown that viral (+)RNA can evolve under the influence of host cell miRNAs. In more than two years of the pandemic, the SARS-CoV-2 virus has mutated significantly. It is quite possible that some mutations could be retained in the virus genome under the influence of miRNAs produced by alveolar cells. We demonstrated that microRNAs in human lung tissue exert evolutionary pressure on the SARS-CoV-2 genome. Moreover, a significant number of sites of host microRNA binding with the virus genome are located in the NSP3-NSP5 region responsible for autoproteolysis of viral polypeptides.

A SARS-CoV-2-Related Virus from Malayan Pangolin Causes Lung Infection without Severe Disease in Human ACE2-Transgenic Mice.

Coronavirus disease 2019 (COVID-19), which is caused by the novel coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is the most severe emerging infectious disease in the current century. The discovery of SARS-CoV-2-related coronaviruses (SARSr-CoV-2) in bats and pangolins in South Asian countries indicates that SARS-CoV-2 likely originated from wildlife. To date, two SARSr-CoV-2 strains have been isolated from pangolins seized in Guangxi and Guangdong by the customs agency of China, respectively. However, it remains unclear whether these viruses cause disease in animal models and whether they pose a transmission risk to humans. In this study, we investigated the biological features of a SARSr-CoV-2 strain isolated from a smuggled Malayan pangolin (Manis javanica) captured by the Guangxi customs agency, termed MpCoV-GX, in terms of receptor usage, cell tropism, and pathogenicity in wild-type BALB/c mice, human angiotensin-converting enzyme 2 (ACE2)-transgenic mice, and human ACE2 knock-in mice. We found that MpCoV-GX can utilize ACE2 from humans, pangolins, civets, bats, pigs, and mice for cell entry and infect cell lines derived from humans, monkeys, bats, minks, and pigs. The virus could infect three mouse models but showed limited pathogenicity, with mild peribronchial and perivascular inflammatory cell infiltration observed in lungs. Our results suggest that this SARSr-CoV-2 virus from pangolins has the potential for interspecies infection, but its pathogenicity is mild in mice. Future surveillance among these wildlife hosts of SARSr-CoV-2 is needed to monitor variants that may have higher pathogenicity and higher spillover risk. IMPORTANCE SARS-CoV-2, which likely spilled over from wildlife, is the third highly pathogenic human coronavirus. Being highly transmissible, it is perpetuating a pandemic and continuously posing a severe threat to global public health. Several SARS-CoV-2-related coronaviruses (SARSr-CoV-2) in bats and pangolins have been identified since the SARS-CoV-2 outbreak. It is therefore important to assess their potential of crossing species barriers for better understanding of their risk of future emergence. In this work, we investigated the biological features and pathogenicity of a SARSr-CoV-2 strain isolated from a smuggled Malayan pangolin, named MpCoV-GX. We found that MpCoV-GX can utilize ACE2 from 7 species for cell entry and infect cell lines derived from a variety of mammalian species. MpCoV-GX can infect mice expressing human ACE2 without causing severe disease. These findings suggest the potential of cross-species transmission of MpCoV-GX, and highlight the need of further surveillance of SARSr-CoV-2 in pangolins and other potential animal hosts.

The Activation of the RIG-I/MDA5 Signaling Pathway upon Influenza D Virus Infection Impairs the Pulmonary Proinflammatory Response Triggered by Mycoplasma bovis Superinfection.

Concurrent infections with multiple pathogens are often described in cattle with respiratory illness. However, how the host-pathogen interactions influence the clinical outcome has been only partially explored in this species. Influenza D virus (IDV) was discovered in 2011. Since then, IDV has been detected worldwide in different hosts. A significant association between IDV and bacterial pathogens in sick cattle was shown in epidemiological studies, especially with Mycoplasma bovis. In an experimental challenge, IDV aggravated M. bovis-induced pneumonia. However, the mechanisms through which IDV drives an increased susceptibility to bacterial superinfections remain unknown. Here, we used the organotypic lung model precision-cut lung slices to study the interplay between IDV and M. bovis coinfection. Our results show that a primary IDV infection promotes M. bovis superinfection by increasing the bacterial replication and the ultrastructural damages in lung pneumocytes. In our model, IDV impaired the innate immune response triggered by M. bovis by decreasing the expression of several proinflammatory cytokines and chemokines that are important for immune cell recruitment and the bacterial clearance. Stimulations with agonists of cytosolic helicases and Toll-like receptors (TLRs) revealed that a primary activation of RIG-I/MDA5 desensitizes the TLR2 activation, similar to what was observed with IDV infection. The cross talk between these two pattern recognition receptors leads to a nonadditive response, which alters the TLR2-mediated cascade that controls the bacterial infection. These results highlight innate immune mechanisms that were not described for cattle so far and improve our understanding of the bovine host-microbe interactions and IDV pathogenesis. IMPORTANCE Since the spread of the respiratory influenza D virus (IDV) infection to the cattle population, the question about the impact of this virus on bovine respiratory disease (BRD) remains still unanswered. Animals affected by BRD are often coinfected with multiple pathogens, especially viruses and bacteria. In particular, viruses are suspected to enhance secondary bacterial superinfections. Here, we use an ex vivo model of lung tissue to study the effects of IDV infection on bacterial superinfections. Our results show that IDV increases the susceptibility to the respiratory pathogen Mycoplasma bovis. In particular, IDV seems to activate immune pathways that inhibit the innate immune response against the bacteria. This may allow M. bovis to increase its proliferation and to delay its clearance from lung tissue. These results suggest that IDV could have a negative impact on the respiratory pathology of cattle.

Neonatal imprinting of alveolar macrophages via neutrophil-derived 12-HETE.

Resident-tissue macrophages (RTMs) arise from embryonic precursors1,2, yet the developmental signals that shape their longevity remain largely unknown. Here we demonstrate in mice genetically deficient in 12-lipoxygenase and 15-lipoxygenase (Alox15-/- mice) that neonatal neutrophil-derived 12-HETE is required for self-renewal and maintenance of alveolar macrophages (AMs) during lung development. Although the seeding and differentiation of AM progenitors remained intact, the absence of 12-HETE led to a significant reduction in AMs in adult lungs and enhanced senescence owing to increased prostaglandin E2 production. A compromised AM compartment resulted in increased susceptibility to acute lung injury induced by lipopolysaccharide and to pulmonary infections with influenza A virus or SARS-CoV-2. Our results highlight the complexity of prenatal RTM programming and reveal their dependency on in trans eicosanoid production by neutrophils for lifelong self-renewal.

Spike and nsp6 are key determinants of SARS-CoV-2 Omicron BA.1 attenuation.

The SARS-CoV-2 Omicron variant is more immune evasive and less virulent than other major viral variants that have so far been recognized1-12. The Omicron spike (S) protein, which has an unusually large number of mutations, is considered to be the main driver of these phenotypes. Here we generated chimeric recombinant SARS-CoV-2 encoding the S gene of Omicron (BA.1 lineage) in the backbone of an ancestral SARS-CoV-2 isolate, and compared this virus with the naturally circulating Omicron variant. The Omicron S-bearing virus robustly escaped vaccine-induced humoral immunity, mainly owing to mutations in the receptor-binding motif; however, unlike naturally occurring Omicron, it efficiently replicated in cell lines and primary-like distal lung cells. Similarly, in K18-hACE2 mice, although virus bearing Omicron S caused less severe disease than the ancestral virus, its virulence was not attenuated to the level of Omicron. Further investigation showed that mutating non-structural protein 6 (nsp6) in addition to the S protein was sufficient to recapitulate the attenuated phenotype of Omicron. This indicates that although the vaccine escape of Omicron is driven by mutations in S, the pathogenicity of Omicron is determined by mutations both in and outside of the S protein.

HIGH HEPARANASE LEVEL IN SURVIVORS OF COVID-19 - INDICATOR OF VASCULAR AND PULMONARY RECOVERY?

ABSTRACT: Background: Severe progression of coronavirus disease 2019 (COVID-19) causes respiratory failure and critical illness. Recently, COVID-19 has been associated with heparanase (HPSE)-induced endothelial barrier dysfunction and inflammation, so called endothelitis, and therapeutic treatment with heparin or low-molecular-weight heparin (LMWH) targeting HPSE has been postulated. Because, up to this date, clinicians are unable to measure the severity of endothelitis, which can lead to multiorgan failure and concomitant death, we investigated plasma levels of HPSE and heparin-binding protein (HBP) in COVID-19 intensive care patients to render a possible link between endothelitis and these plasma parameters. Therefore, a prospective prolonged cohort study was conducted, including 47 COVID-19 patients from the intensive care unit. Plasma levels of HPSE, and HBP were measured daily by enzyme-linked immunosorbent assay in survivors (n = 35) and nonsurvivors (n = 12) of COVID-19 from admission until discharge or death. All patients were either treated with heparin or LMWH, aiming for an activated partial thromboplastin time of ≥60 seconds or an anti-Xa level of >0.8 IU/mL using enoxaparin, depending on the clinical status of the patient (patients with extracorporeal membrane oxygenation or >0.1 µg/kg/min noradrenaline received heparin, all others enoxaparin). Results: We found significantly higher plasma levels of HPSE and HBP in survivors and nonsurvivors of COVID-19, compared with healthy controls. Still, interestingly, plasma HPSE levels were significantly higher ( P < 0.001) in survivors compared with nonsurvivors of COVID-19. In contrast, plasma HBP levels were significantly reduced ( P < 0.001) in survivors compared with nonsurvivors of COVID-19. Furthermore, when patients received heparin, they had significantly lower HPSE ( P = 2.22 e - 16) and significantly higher HBP ( P = 0.00013) plasma levels as when they received LMWH. Conclusion: Our results demonstrated that patients, who recover from COVID-19-induced vascular and pulmonary damage and were discharged from the intensive care unit, have significantly higher plasma HPSE level than patients who succumb to COVID-19. Therefore, HPSE is not suitable as marker for disease severity in COVID-19 but maybe as marker for patient's recovery. In addition, patients receiving therapeutic heparin treatment displayed significantly lower heparanse plasma level than upon therapeutic treatment with LMWH.

Lung Organoids as Model to Study SARS-CoV-2 Infection.

Coronavirus disease-2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has become a global pandemic and has severely affected socio-economic conditions and people's life. The lung is the major target organ infected and (seriously) damaged by SARS-CoV-2, so a comprehensive understanding of the virus and the mechanism of infection are the first choices to overcome COVID-19. Recent studies have demonstrated the enormous value of human organoids as platforms for virological research, making them an ideal tool for researching host-pathogen interactions. In this study, the various existing lung organoids and their identification biomarkers and applications are summarized. At the same time, the seven coronaviruses currently capable of infecting humans are outlined. Finally, a detailed summary of existing studies on SARS-CoV-2 using lung organoids is provided and includes pathogenesis, drug development, and precision treatment. This review highlights the value of lung organoids in studying SARS-CoV-2 infection, bringing hope that research will alleviate COVID-19-associated lung infections.

3D Ex vivo tissue platforms to investigate the early phases of influenza a virus- and SARS-CoV-2-induced respiratory diseases.

Pandemic outbreaks of viruses such as influenza virus or SARS-CoV-2 are associated with high morbidity and mortality and thus pose a massive threat to global health and economics. Physiologically relevant models are needed to study the viral life cycle, describe the pathophysiological consequences of viral infection, and explore possible drug targets and treatment options. While simple cell culture-based models do not reflect the tissue environment and systemic responses, animal models are linked with huge direct and indirect costs and ethical questions. Ex vivo platforms based on tissue explants have been introduced as suitable platforms to bridge the gap between cell culture and animal models. We established a murine lung tissue explant platform for two respiratory viruses, influenza A virus (IAV) and SARS-CoV-2. We observed efficient viral replication, associated with the release of inflammatory cytokines and the induction of an antiviral interferon response, comparable to ex vivo infection in human lung explants. Endolysosomal entry could be confirmed as a potential host target for pharmacological intervention, and the potential repurposing potentials of fluoxetine and interferons for host-directed therapy previously seen in vitro could be recapitulated in the ex vivo model.

In Vivo Profiling of Individual Multiciliated Cells during Acute Influenza A Virus Infection.

Influenza virus infections are thought to be initiated in a small number of cells; however, the heterogeneity across the cellular responses of the epithelial cells during establishment of disease is incompletely understood. Here, we used an H1N1 influenza virus encoding a fluorescent reporter gene, a cell lineage-labeling transgenic mouse line, and single-cell RNA sequencing to explore the range of responses in a susceptible epithelial cell population during an acute influenza A virus (IAV) infection. Focusing on multiciliated cells, we identified a subpopulation that basally expresses interferon-stimulated genes (ISGs), which we hypothesize may be important for the early response to infection. We subsequently found that a population of infected ciliated cells produce most of the ciliated cell-derived inflammatory cytokines, and nearly all bystander ciliated cells induce a broadly antiviral state. From these data together, we propose that variable preexisting gene expression patterns in the initial cells targeted by the virus may ultimately affect the establishment of viral disease. IMPORTANCE Influenza A virus poses a significant threat to public health, and each year, millions of people in the United States alone are exposed to the virus. We do not currently, however, fully understand why some individuals clear the infection asymptomatically and others become severely ill. Understanding how these divergent phenotypes arise could eventually be leveraged to design therapeutics that prevent severe disease. As a first step toward understanding these different infection states, we used a technology that allowed us to determine how thousands of individual murine lung epithelial cells behaved before and during IAV infection. We found that small subsets of epithelial cells exhibited an antiviral state prior to infection, and similarly, some cells made high levels of inflammatory cytokines during infection. We propose that different ratios of these individual cellular responses may contribute to the broader antiviral state of the lung and may ultimately affect disease severity.