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Abstract 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.

Influenza infection during pregnancy can affect health of offspring but it is not clear how this affects immune responses. Here the authors use a mouse model to show that influenza infection during pregnancy can increase susceptibility to secondary infection and alter immune cell function in offspring.

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.

Male sex was repeatedly identified as a risk factor for death and intensive care admission. However, it is yet unclear whether sex hormones are associated with disease severity in COVID-19 patients. In this study, we analysed sex hormone levels (estradiol and testosterone) of male and female COVID-19 patients (n = 50) admitted to an intensive care unit (ICU) in comparison to control non-COVID-19 patients at the ICU (n = 42), non-COVID-19 patients with the most prevalent comorbidity (coronary heart diseases) present within the COVID-19 cohort (n = 39) and healthy individuals (n = 50). We detected significantly elevated estradiol levels in critically ill male COVID-19 patients compared to all control cohorts. Testosterone levels were significantly reduced in critically ill male COVID-19 patients compared to control cohorts. No statistically significant differences in sex hormone levels were detected in critically ill female COVID-19 patients, albeit similar trends towards elevated estradiol levels were observed. Linear regression analysis revealed that among a broad range of cytokines and chemokines analysed, IFN-γ levels are positively associated with estradiol levels in male and female COVID-19 patients. Furthermore, male COVID-19 patients with elevated estradiol levels were more likely to receive ECMO treatment. Thus, we herein identified that disturbance of sex hormone metabolism might present a hallmark in critically ill male COVID-19 patients.

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.

Summary: Importin-α adaptor proteins orchestrate dynamic nuclear transport processes involved in cellular homeostasis. Here, we show that importin-α3, one of the main NF-κB transporters, is the most abundantly expressed classical nuclear transport factor in the mammalian respiratory tract. Importin-α3 promoter activity is regulated by TNF-α-induced NF-κB in a concentration-dependent manner. High-level TNF-α-inducing highly pathogenic avian influenza A viruses (HPAIVs) isolated from fatal human cases harboring human-type polymerase signatures (PB2 627K, 701N) significantly downregulate importin-α3 mRNA expression in primary lung cells. Importin-α3 depletion is restored upon back-mutating the HPAIV polymerase into an avian-type signature (PB2 627E, 701D) that can no longer induce high TNF-α levels. Importin-α3-deficient mice show reduced NF-κB-activated antiviral gene expression and increased influenza lethality. Thus, importin-α3 plays a key role in antiviral immunity against influenza. Lifting the bottleneck in importin-α3 availability in the lung might provide a new strategy to combat respiratory virus infections. : Thiele et al. show that importin-α3 is one of the major nuclear transporters of NF-κB in the mammalian lung. High-level TNF-α-inducing HPAIVs inhibit importin-α3 mRNA transcription by interfering with its promoter activity. Thus, HPAIVs may evade antiviral immunity in the respiratory tract by generating a bottleneck in importin-α3 availability. Keywords: lung, influenza, pneumonia, immune sensor, cytokine storm

Deciphering complex virus-host interactions is crucial for pandemic preparedness. In this study, we assessed the impact of recently postulated cellular factors ANP32A and ANP32B of influenza A virus (IAV) species specificity on viral pathogenesis in a genetically modified mouse model. Infection of ANP32A−/− and ANP32A+/+ mice with a seasonal H3N2 IAV or a highly pathogenic H5N1 human isolate did not result in any significant differences in virus tropism, innate immune response or disease outcome. However, infection of ANP32B−/− mice with H3N2 or H5N1 IAV revealed significantly reduced virus loads, inflammatory cytokine response and reduced pathogenicity compared to ANP32B+/+ mice. Genome-wide transcriptome analyses in ANP32B+/+ and ANP32B−/− mice further uncovered novel immune-regulatory pathways that correlate with reduced pathogenicity in the absence of ANP32B. These data show that ANP32B but not ANP32A promotes IAV pathogenesis in mice. Moreover, ANP32B might possess a yet unknown immune-modulatory function during IAV infection. Targeting ANP32B or its regulated pathways might therefore pose a new strategy to combat severe influenza.

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.