Distribution of importin-α isoforms in poultry species and their tissue- and age-related differences.

While importin-α is well studied in mammals, the knowledge in avian species is still limited. In this study, we compared the mRNA expression patterns of five importin-α isoforms in the respiratory tract, liver, and spleen of chickens, turkeys, and pekin ducks in two different age-groups. In addition, we determined the distribution of importin-α in selected tissue of conchae, trachea, and lung of post-hatch chickens at all cellular levels by immunohistochemical staining. Our results indicate that importin-α3 is the most abundant isoform in the respiratory tract of chickens, turkeys, and pekin ducks. Moreover, importin-α is expressed as a gradient with lowest mRNA levels in the conchae and highest levels in the lung. The mRNA expression levels of most isoforms were higher in tissues from post-hatch chickens and turkeys in comparison to the corresponding embryos. In contrast to that, duck embryos mostly show higher mRNA expression levels of importin-α than post-hatch ducks.

Hamster model for post-COVID-19 alveolar regeneration offers an opportunity to understand post-acute sequelae of SARS-CoV-2.

COVID-19 survivors often suffer from post-acute sequelae of SARS-CoV-2 infection (PASC). Current evidence suggests dysregulated alveolar regeneration as a possible explanation for respiratory PASC, which deserves further investigation in a suitable animal model. This study investigates morphological, phenotypical and transcriptomic features of alveolar regeneration in SARS-CoV-2 infected Syrian golden hamsters. We demonstrate that CK8+ alveolar differentiation intermediate (ADI) cells occur following SARS-CoV-2-induced diffuse alveolar damage. A subset of ADI cells shows nuclear accumulation of TP53 at 6- and 14-days post infection (dpi), indicating a prolonged arrest in the ADI state. Transcriptome data show high module scores for pathways involved in cell senescence, epithelial-mesenchymal transition, and angiogenesis in cell clusters with high ADI gene expression. Moreover, we show that multipotent CK14+ airway basal cell progenitors migrate out of terminal bronchioles, aiding alveolar regeneration. At 14 dpi, ADI cells, peribronchiolar proliferates, M2-macrophages, and sub-pleural fibrosis are observed, indicating incomplete alveolar restoration. The results demonstrate that the hamster model reliably phenocopies indicators of a dysregulated alveolar regeneration of COVID-19 patients. The results provide important information on a translational COVID-19 model, which is crucial for its application in future research addressing pathomechanisms of PASC and in testing of prophylactic and therapeutic approaches for this syndrome.

T-705-Derived Prodrugs Show High Antiviral Efficacies against a Broad Range of Influenza A Viruses with Synergistic Effects When Combined with Oseltamivir.

Emerging influenza A viruses (IAV) bear the potential to cause pandemics with unpredictable consequences for global human health. In particular, the WHO has declared avian H5 and H7 subtypes as high-risk candidates, and continuous surveillance of these viruses as well as the development of novel, broadly acting antivirals, are key for pandemic preparedness. In this study, we sought to design T-705 (Favipiravir) related inhibitors that target the RNA-dependent RNA polymerase and evaluate their antiviral efficacies against a broad range of IAVs. Therefore, we synthesized a library of derivatives of T-705 ribonucleoside analogues (called T-1106 pronucleotides) and tested their ability to inhibit both seasonal and highly pathogenic avian influenza viruses in vitro. We further showed that diphosphate (DP) prodrugs of T-1106 are potent inhibitors of H1N1, H3N2, H5N1, and H7N9 IAV replication. Importantly, in comparison to T-705, these DP derivatives achieved 5- to 10-fold higher antiviral activity and were non-cytotoxic at the therapeutically active concentrations. Moreover, our lead DP prodrug candidate showed drug synergy with the neuraminidase inhibitor oseltamivir, thus opening up another avenue for combinational antiviral therapy against IAV infections. Our findings may serve as a basis for further pre-clinical development of T-1106 prodrugs as an effective countermeasure against emerging IAVs with pandemic potential.

In Vitro Investigation of the Interaction of Avian Metapneumovirus and Newcastle Disease Virus with Turkey Respiratory and Reproductive Tissue.

In poultry, several respiratory viral infections lead to a drop in egg production associated with high economic losses. While the virus-host interactions at the respiratory epithelium are well studied, less is known about these interactions in the oviduct. To investigate possible differences between virus infections at these epithelial structures, we compared the interactions of two important poultry viruses on turkey organ cultures. Two members of the order Mononegavirales, the Avian Metapneumovirus (AMPV) and the Newcastle disease virus (NDV), were selected to conduct the in vitro experiments since these viruses can infect both the trachea and oviduct. In addition, we used different strains of these viruses, a subtype A and a subtype B strain for AMPV and the NDV strains Komarow and Herts'33, to detect possible differences not only between the tissues but also between different viral strains. Turkey tracheal and oviduct organ cultures (TOC and OOC) were prepared to investigate viral replication, antigen localisation, lesion development, and the expression pattern of interferon-λ and importin-α isoforms. All viruses replicated more efficiently in the oviduct than in the tracheal epithelium (p < 0.05). In addition, we observed higher expression levels of both, IFN-λ and importin-α in OOCs compared to TOCs. Our results indicated strain-dependent differences, with the AMPV-B- and Herts'33 strains being more virulent in organ cultures than the AMPV-A- and Komarow strains, based on the higher viral genome loads, more severe histological lesions, and higher upregulation of IFN-λ. Overall, our findings reveal tissue- and virus strain-dependent differences, which may have consequences for disease development in the host tissue and, subsequently, possible treatment strategies.

Characterization of young and aged ferrets as animal models for SARS-CoV-2 infection with focus on neutrophil extracellular traps.

Neutrophil extracellular traps (NETs) are net-like structures released by activated neutrophils upon infection [e.g., severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)] as part of the innate immune response that have protective effects by pathogen entrapment and immobilization or result in detrimental consequences for the host due to the massive release of NETs and their impaired degradation by nucleases like DNase-1. Higher amounts of NETs are associated with coronavirus disease 2019 (COVID-19) severity and are a risk factor for severe disease outcome. The objective of our study was to investigate NET formation in young versus aged ferrets to evaluate their value as translational model for SARS-CoV-2-infection and to correlate different NET markers and virological parameters. In each of the two groups (young and aged), nine female ferrets were intratracheally infected with 1 mL of 106 TCID50/mL SARS-CoV-2 (BavPat1/2020) and euthanized at 4, 7, or 21 days post-infection. Three animals per group served as negative controls. Significantly more infectious virus and viral RNA was found in the upper respiratory tract of aged ferrets. Interestingly, cell-free DNA and DNase-1 activity was generally higher in bronchoalveolar lavage fluid (BALF) but significantly lower in serum of aged compared to young ferrets. In accordance with these data, immunofluorescence microscopy revealed significantly more NETs in lungs of aged compared to young infected ferrets. The association of SARS-CoV-2-antigen in the respiratory mucosa and NET markers in the nasal conchae, but the absence of virus antigen in the lungs, confirms the nasal epithelium as the major location for virus replication as described for young ferrets. Furthermore, a strong positive correlation was found between virus shedding and cell-free DNA or the level of DNAse-1 activity in aged ferrets. Despite the increased NET formation in infected lungs of aged ferrets, the animals did not show a strong NET phenotype and correlation among tested NET markers. Therefore, ferrets are of limited use to study SARS-CoV-2 pathogenesis associated with NET formation. Nevertheless, the mild to moderate clinical signs, virus shedding pattern, and the lung pathology of aged ferrets confirm those animals as a relevant model to study age-dependent COVID-19 pathogenesis.

H7N9 avian influenza virus infection in men is associated with testosterone depletion.

Human infections with H7N9 avian influenza A virus that emerged in East China in 2013 and caused high morbidity rates were more frequently detected in men than in women over the last five epidemic waves. However, molecular markers associated with poor disease outcomes in men are still unknown. In this study, we systematically analysed sex hormone and cytokine levels in males and females with laboratory-confirmed H7N9 influenza in comparison to H7N9-negative control groups as well as laboratory-confirmed seasonal H1N1/H3N2 influenza cases (n = 369). Multivariable analyses reveal that H7N9-infected men present with considerably reduced testosterone levels associated with a poor outcome compared to non-infected controls. Regression analyses reveal that testosterone levels in H7N9-infected men are negatively associated with the levels of several pro-inflammatory cytokines, such as IL-6 and IL-15. To assess whether there is a causal relationship between low testosterone levels and avian H7N9 influenza infection, we used a mouse model. In male mice, we show that respiratory H7N9 infection leads to a high viral load and inflammatory cytokine response in the testes as well as a reduction in pre-infection plasma testosterone levels. Collectively, these findings suggest that monitoring sex hormone levels may support individualized management for patients with avian influenza infections.

Assessing and improving the validity of COVID-19 autopsy studies - A multicentre approach to establish essential standards for immunohistochemical and ultrastructural analyses.

BACKGROUND: Autopsy studies have provided valuable insights into the pathophysiology of COVID-19. Controversies remain about whether the clinical presentation is due to direct organ damage by SARS-CoV-2 or secondary effects, such as overshooting immune response. SARS-CoV-2 detection in tissues by RT-qPCR and immunohistochemistry (IHC) or electron microscopy (EM) can help answer these questions, but a comprehensive evaluation of these applications is missing. METHODS: We assessed publications using IHC and EM for SARS-CoV-2 detection in autopsy tissues. We systematically evaluated commercially available antibodies against the SARS-CoV-2 proteins in cultured cell lines and COVID-19 autopsy tissues. In a multicentre study, we evaluated specificity, reproducibility, and inter-observer variability of SARS-CoV-2 IHC. We correlated RT-qPCR viral tissue loads with semiquantitative IHC scoring. We used qualitative and quantitative EM analyses to refine criteria for ultrastructural identification of SARS-CoV-2. FINDINGS: Publications show high variability in detection and interpretation of SARS-CoV-2 abundance in autopsy tissues by IHC or EM. We show that IHC using antibodies against SARS-CoV-2 nucleocapsid yields the highest sensitivity and specificity. We found a positive correlation between presence of viral proteins by IHC and RT-qPCR-determined SARS-CoV-2 viral RNA load (N= 35; r=-0.83, p-value <0.0001). For EM, we refined criteria for virus identification and provide recommendations for optimized sampling and analysis. 135 of 144 publications misinterpret cellular structures as virus using EM or show only insufficient data. We provide publicly accessible digitized EM sections as a reference and for training purposes. INTERPRETATION: Since detection of SARS-CoV-2 in human autopsy tissues by IHC and EM is difficult and frequently incorrect, we propose criteria for a re-evaluation of available data and guidance for further investigations of direct organ effects by SARS-CoV-2. FUNDING: German Federal Ministry of Health, German Federal Ministry of Education and Research, Berlin University Alliance, German Research Foundation, German Center for Infectious Research.

SARS-CoV-2 Infection Dysregulates Cilia and Basal Cell Homeostasis in the Respiratory Epithelium of Hamsters.

Similar to many other respiratory viruses, SARS-CoV-2 targets the ciliated cells of the respiratory epithelium and compromises mucociliary clearance, thereby facilitating spread to the lungs and paving the way for secondary infections. A detailed understanding of mechanism involved in ciliary loss and subsequent regeneration is crucial to assess the possible long-term consequences of COVID-19. The aim of this study was to characterize the sequence of histological and ultrastructural changes observed in the ciliated epithelium during and after SARS-CoV-2 infection in the golden Syrian hamster model. We show that acute infection induces a severe, transient loss of cilia, which is, at least in part, caused by cilia internalization. Internalized cilia colocalize with membrane invaginations, facilitating virus entry into the cell. Infection also results in a progressive decline in cells expressing the regulator of ciliogenesis FOXJ1, which persists beyond virus clearance and the termination of inflammatory changes. Ciliary loss triggers the mobilization of p73+ and CK14+ basal cells, which ceases after regeneration of the cilia. Although ciliation is restored after two weeks despite the lack of FOXJ1, an increased frequency of cilia with ultrastructural alterations indicative of secondary ciliary dyskinesia is observed. In summary, the work provides new insights into SARS-CoV-2 pathogenesis and expands our understanding of virally induced damage to defense mechanisms in the conducting airways.

Microgliosis and neuronal proteinopathy in brain persist beyond viral clearance in SARS-CoV-2 hamster model.

BACKGROUND: Neurological symptoms such as cognitive decline and depression contribute substantially to post-COVID-19 syndrome, defined as lasting symptoms several weeks after initial SARS-CoV-2 infection. The pathogenesis is still elusive, which hampers appropriate treatment. Neuroinflammatory responses and neurodegenerative processes may occur in absence of overt neuroinvasion. METHODS: Here we determined whether intranasal SARS-CoV-2 infection in male and female syrian golden hamsters results in persistent brain pathology. Brains 3 (symptomatic) or 14 days (viral clearance) post infection versus mock (n = 10 each) were immunohistochemically analyzed for viral protein, neuroinflammatory response and accumulation of tau, hyperphosphorylated tau and alpha-synuclein protein. FINDINGS: Viral protein in the nasal cavity led to pronounced microglia activation in the olfactory bulb beyond viral clearance. Cortical but not hippocampal neurons accumulated hyperphosphorylated tau and alpha-synuclein, in the absence of overt inflammation and neurodegeneration. Importantly, not all brain regions were affected, which is in line with selective vulnerability. INTERPRETATION: Thus, despite the absence of virus in brain, neurons develop signatures of proteinopathies that may contribute to progressive neuronal dysfunction. Further in depth analysis of this important mechanism is required. FUNDING: Federal Ministry of Health (BMG; ZMV I 1-2520COR501), Federal Ministry of Education and Research (BMBF 01KI1723G), Ministry of Science and Culture of Lower Saxony in Germany (14 - 76103-184 CORONA-15/20), German Research Foundation (DFG; 398066876/GRK 2485/1), Luxemburgish National Research Fund (FNR, Project Reference: 15686728, EU SC1-PHE-CORONAVIRUS-2020 MANCO, no > 101003651).

Pre-existing helminth infection impairs the efficacy of adjuvanted influenza vaccination in mice.

The world health organization estimates that more than a quarter of the human population is infected with parasitic worms that are called helminths. Many helminths suppress the immune system of their hosts to prolong their survival. This helminth-induced immunosuppression "spills over" to unrelated antigens and can suppress the immune response to vaccination against other pathogens. Indeed, several human studies have reported a negative correlation between helminth infections and responses to vaccinations. Using mice that are infected with the parasitic nematode Litomosoides sigmodontis as a model for chronic human filarial infections, we reported previously that concurrent helminth infection impaired the vaccination-induced protection against the human pathogenic 2009 pandemic H1N1 influenza A virus (2009 pH1N1). Vaccinated, helminth-infected mice produced less neutralizing, influenza-specific antibodies than vaccinated naïve control mice. Consequently helminth-infected and vaccinated mice were not protected against a challenge infection with influenza virus but displayed high virus burden in the lung and a transient weight loss. In the current study we tried to improve the vaccination efficacy using vaccines that are licensed for humans. We either introduced a prime-boost vaccination regimen using the non-adjuvanted anti-influenza vaccine Begripal or employed the adjuvanted influenza vaccine Fluad. Although both strategies elevated the production of influenza-specific antibodies and protected mice from the transient weight loss that is caused by an influenza challenge infection, sterile immunity was not achieved. Helminth-infected vaccinated mice still had high virus burden in the lung while non-helminth-infected vaccinated mice rapidly cleared the virus. In summary we demonstrate that basic improvements of influenza vaccination regimen are not sufficient to confer sterile immunity on the background of helminth-induced immunosuppression, despite amelioration of pathology i.e. weight loss. Our findings highlight the risk of failed vaccinations in helminth-endemic areas, especially in light of the ongoing vaccination campaign to control the COVID-19 pandemic.

Impaired Degradation of Neutrophil Extracellular Traps: A Possible Severity Factor of Elderly Male COVID-19 Patients.

Neutrophil extracellular traps (NETs) have been described as a potential trigger of severe COVID-19. NETs are known as extracellular DNA fibers released by neutrophils in response to infection. If the host is unable to balance efficient clearance of NETs by dornases (DNases), detrimental consequences occur. Elevated levels of NETs in COVID-19 patients are associated with higher risk of morbid thrombotic complications. Here, we studied the level of NET markers and DNase activity in a cohort of COVID-19 patients compared to healthy controls. Our data confirmed an increased level of NET markers in the plasma of COVID-19 patients, with a higher level in male compared to female patients. At the same time, there was an increased DNase activity detectable in COVID-19 patients compared to healthy controls. Importantly, there was a negative correlation of DNase activity with the age of male patients. The antimicrobial peptide LL-37, which is known to stabilize NETs against DNase degradation, is embedded in NETs upon severe acute respiratory syndrome coronavirus-2-infection. The LL-37 plasma level correlates with the NET-marker level in male COVID-19 patients, indicating a potential role of LL-37 in the risk of NET-associated thrombosis in male COVID-19 patients by stabilizing NETs against DNase degradation. In conclusion, our data identify two potential risk factors of elderly male patients which may lead to inefficient NET degradation and a subsequently higher risk of NET-associated thrombosis during COVID-19: reduced DNase activity and an increased LL-37 level.

Ferrets are valuable models for SARS-CoV-2 research.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), resulted in an ongoing pandemic with millions of deaths worldwide. Infection of humans can be asymptomatic or result in fever, fatigue, dry cough, dyspnea, and acute respiratory distress syndrome with multiorgan failure in severe cases. The pathogenesis of COVID-19 is not fully understood, and various models employing different species are currently applied. Ferrets can be infected with SARS-CoV-2 and efficiently transmit the virus to contact animals. In contrast to hamsters, ferrets usually show mild disease and viral replication restricted to the upper airways. Most reports have used the intranasal inoculation route, while the intratracheal infection model is not well characterized. Herein, we present clinical, virological, and pathological data from young ferrets intratracheally inoculated with SARS-CoV-2. Infected animals showed no significant clinical signs, and had transient infection with peak viral RNA loads at 4 days postinfection, mild to moderate rhinitis, and pulmonary endothelialitis/vasculitis. Viral antigen was exclusively found in the respiratory epithelium of the nasal cavity, indicating a particular tropism for cells in this location. Viral antigen was associated with epithelial damage and influx of inflammatory cells, including activated neutrophils releasing neutrophil extracellular traps. Scanning electron microscopy of the nasal respiratory mucosa revealed loss of cilia, shedding, and rupture of epithelial cells. The currently established ferret SARS-CoV-2 infection models are comparatively discussed with SARS-CoV-2 pathogenesis in mink, and the advantages and disadvantages of both species as research models for zoonotic betacoronaviruses are highlighted.

Infant immunity against viral infections is advanced by the placenta-dependent vertical transfer of maternal antibodies.

Neonatal passive immunity, derived from transplacental transfer of IgG antibodies from mother to fetus during pregnancy, can mitigate the risk for severe infections in the early postnatal period. Understanding the placenta as the gateway organ in this process, we aimed to evaluate the influence of specific factors modulating the transplacental IgG transfer rate (TPTR) in 141 mother/neonate pairs. We further evaluated the potential health advantage elicited by maternal IgG with regard to respiratory tract infections during infancy and early childhood. Data and biological samples collected within the prospective longitudinal pregnancy cohort study PRINCE (Prenatal Identification of Children's Health) were used for these analyses. We tested IgG antibody levels against seven pathogens (measles, mumps, rubella, tetanus, diphtheria, pertussis and influenza A) by ELISA and detected seropositivity in 72.6-100% of pregnant women and in 76.3-100% of their neonates, respectively. Cord blood IgG levels reached 137-160% of levels detected in maternal blood. Strikingly, assessment of TPTR for all seven antigens highlighted that TPTR strongly depends on individual placental function. Subsequent in-depth analysis of anti-influenza A IgG revealed a link between cord blood levels and uterine perfusion, measured by uterine artery pulsatility index. Moreover, higher cord blood anti-influenza A IgG levels were associated with a significantly reduced risk for respiratory tract infections during the first six months of life, indicating a high degree of cross-reactivity and possible pathogen-agnostic effects of anti-influenza A antibodies. Taken together, our data suggest that early life immunity is modulated by maternal IgG levels and individual placental features such as perfusion. Vaccination of pregnant women, i.e. against influenza, can increase neonatal antibody levels and hereby protect against early life respiratory infections. Consequently, specific guidelines should evolve in order to safeguard neonates born from pregnancies with poorer placental capacity for vertical transfer of protective antibodies.

Oral SARS-CoV-2 reduction by local treatment: A plasma technology application?

The SARS-CoV-2 pandemic reemphasized the importance of and need for efficient hygiene and disinfection measures. The coronavirus' efficient spread capitalizes on its airborne transmission routes via virus aerosol release from human oral and nasopharyngeal cavities. Besides the upper respiratory tract, efficient viral replication has been described in the epithelium of these two body cavities. To this end, the idea emerged to employ plasma technology to locally reduce mucosal viral loads as an additional measure to reduce patient infectivity. We here outline conceptual ideas of such treatment concepts within what is known in the antiviral actions of plasma treatment so far.

Offspring born to influenza A virus infected pregnant mice have increased susceptibility to viral and bacterial infections in early life.

Influenza during pregnancy can affect the health of offspring in later life, among which neurocognitive disorders are among the best described. Here, we investigate whether maternal influenza infection has adverse effects on immune responses in offspring. We establish a two-hit mouse model to study the effect of maternal influenza A virus infection (first hit) on vulnerability of offspring to heterologous infections (second hit) in later life. Offspring born to influenza A virus infected mothers are stunted in growth and more vulnerable to heterologous infections (influenza B virus and MRSA) than those born to PBS- or poly(I:C)-treated mothers. Enhanced vulnerability to infection in neonates is associated with reduced haematopoetic development and immune responses. In particular, alveolar macrophages of offspring exposed to maternal influenza have reduced capacity to clear second hit pathogens. This impaired pathogen clearance is partially reversed by adoptive transfer of alveolar macrophages from healthy offspring born to uninfected dams. These findings suggest that maternal influenza infection may impair immune ontogeny and increase susceptibility to early life infections of offspring.

Inefficient Placental Virus Replication and Absence of Neonatal Cell-Specific Immunity Upon Sars-CoV-2 Infection During Pregnancy.

Pregnant women have been carefully observed during the COVID-19 pandemic, as the pregnancy-specific immune adaptation is known to increase the risk for infections. Recent evidence indicates that even though most pregnant have a mild or asymptomatic course, a severe course of COVID-19 and a higher risk of progression to diseases have also been described, along with a heightened risk for pregnancy complications. Yet, vertical transmission of the virus is rare and the possibility of placental SARS-CoV-2 infection as a prerequisite for vertical transmission requires further studies. We here assessed the severity of COVID-19 and onset of neonatal infections in an observational study of women infected with SARS-CoV-2 during pregnancy. Our placental analyses showed a paucity of SARS-CoV-2 viral expression ex vivo in term placentae under acute infection. No viral placental expression was detectable in convalescent pregnant women. Inoculation of placental explants generated from placentas of non-infected women at birth with SARS-CoV-2 in vitro revealed inefficient SARS-CoV-2 replication in different types of placental tissues, which provides a rationale for the low ex vivo viral expression. We further detected specific SARS-CoV-2 T cell responses in pregnant women within a few days upon infection, which was undetectable in cord blood. Our present findings confirm that vertical transmission of SARS-CoV-2 is rare, likely due to the inefficient virus replication in placental tissues. Despite the predominantly benign course of infection in most mothers and negligible risk of vertical transmission, continuous vigilance on the consequences of COVID-19 during pregnancy is required, since the maternal immune activation in response to the SARS-CoV2 infection may have long-term consequences for children's health.

Vascular Inflammation Is Associated with Loss of Aquaporin 1 Expression on Endothelial Cells and Increased Fluid Leakage in SARS-CoV-2 Infected Golden Syrian Hamsters.

Vascular changes represent a characteristic feature of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection leading to a breakdown of the vascular barrier and subsequent edema formation. The aim of this study was to provide a detailed characterization of the vascular alterations during SARS-CoV-2 infection and to evaluate the impaired vascular integrity. Groups of ten golden Syrian hamsters were infected intranasally with SARS-CoV-2 or phosphate-buffered saline (mock infection). Necropsies were performed at 1, 3, 6, and 14 days post-infection (dpi). Lung samples were investigated using hematoxylin and eosin, alcian blue, immunohistochemistry targeting aquaporin 1, CD3, CD204, CD31, laminin, myeloperoxidase, SARS-CoV-2 nucleoprotein, and transmission electron microscopy. SARS-CoV-2 infected animals showed endothelial hypertrophy, endothelialitis, and vasculitis. Inflammation mainly consisted of macrophages and lower numbers of T-lymphocytes and neutrophils/heterophils infiltrating the vascular walls as well as the perivascular region at 3 and 6 dpi. Affected vessels showed edema formation in association with loss of aquaporin 1 on endothelial cells. In addition, an ultrastructural investigation revealed disruption of the endothelium. Summarized, the presented findings indicate that loss of aquaporin 1 entails the loss of intercellular junctions resulting in paracellular leakage of edema as a key pathogenic mechanism in SARS-CoV-2 triggered pulmonary lesions.

Vasculitis and Neutrophil Extracellular Traps in Lungs of Golden Syrian Hamsters With SARS-CoV-2.

Neutrophil extracellular traps (NETs) have been identified as one pathogenetic trigger in severe COVID-19 cases and therefore well-described animal models to understand the influence of NETs in COVID-19 pathogenesis are needed. SARS-CoV-2 infection causes infection and interstitial pneumonia of varying severity in humans and COVID-19 models. Pulmonary as well as peripheral vascular lesions represent a severe, sometimes fatal, disease complication of unknown pathogenesis in COVID-19 patients. Furthermore, neutrophil extracellular traps (NETs), which are known to contribute to vessel inflammation or endothelial damage, have also been shown as potential driver of COVID-19 in humans. Though most studies in animal models describe the pulmonary lesions characterized by interstitial inflammation, type II pneumocyte hyperplasia, edema, fibrin formation and infiltration of macrophages and neutrophils, detailed pathological description of vascular lesions or NETs in COVID-19 animal models are lacking so far. Here we report different types of pulmonary vascular lesions in the golden Syrian hamster model of COVID-19. Vascular lesions included endothelialitis and vasculitis at 3 and 6 days post infection (dpi), and were almost nearly resolved at 14 dpi. Importantly, virus antigen was present in pulmonary lesions, but lacking in vascular alterations. In good correlation to these data, NETs were detected in the lungs of infected animals at 3 and 6 dpi. Hence, the Syrian hamster seems to represent a useful model to further investigate the role of vascular lesions and NETs in COVID-19 pathogenesis.

Antiviral drug screen identifies DNA-damage response inhibitor as potent blocker of SARS-CoV-2 replication.

SARS-CoV-2 has currently precipitated the COVID-19 global health crisis. We developed a medium-throughput drug-screening system and identified a small-molecule library of 34 of 430 protein kinase inhibitors that were capable of inhibiting the SARS-CoV-2 cytopathic effect in human epithelial cells. These drug inhibitors are in various stages of clinical trials. We detected key proteins involved in cellular signaling pathways mTOR-PI3K-AKT, ABL-BCR/MAPK, and DNA-damage response that are critical for SARS-CoV-2 infection. A drug-protein interaction-based secondary screen confirmed compounds, such as the ATR kinase inhibitor berzosertib and torin2 with anti-SARS-CoV-2 activity. Berzosertib exhibited potent antiviral activity against SARS-CoV-2 in multiple cell types and blocked replication at the post-entry step. Berzosertib inhibited replication of SARS-CoV-1 and the Middle East respiratory syndrome coronavirus (MERS-CoV) as well. Our study highlights key promising kinase inhibitors to constrain coronavirus replication as a host-directed therapy in the treatment of COVID-19 and beyond as well as provides an important mechanism of host-pathogen interactions.