In brain tissue of three harbor seals of the German North Sea coast, high virus loads of highly pathogenic avian influenza virus (HPAIV) H5N8 were detected. Identification of different virus variants indicates high exposure to HPAIV circulating in wild birds, but there is no evidence for H5 specific antibodies in healthy seals. Replication of avian viruses in seals may allow HPAIV to acquire mutations needed to adapt to mammalian hosts as shown by PB2 627K variants detected in these cases.

The ongoing threat of COVID-19 has highlighted the need for effective prophylaxis and convenient therapies, especially for outpatient settings. We have previously developed highly potent single-domain (VHH) antibodies, also known as nanobodies, that target the Receptor Binding Domain (RBD) of the SARS-CoV-2 Spike protein and neutralize the Wuhan strain of the virus. In this study, we present a new generation of anti-RBD nanobodies with superior properties. The primary representative of this group, Re32D03, neutralizes Alpha to Delta as well as Omicron BA.2.75; other members neutralize, in addition, Omicron BA.1, BA.2, BA.4/5, and XBB.1. Crystal structures of RBD-nanobody complexes reveal how ACE2-binding is blocked and also explain the nanobodies' tolerance to immune escape mutations. Through the cryo-EM structure of the Ma16B06-BA.1 Spike complex, we demonstrated how a single nanobody molecule can neutralize a trimeric spike. We also describe a method for large-scale production of these nanobodies in Pichia pastoris, and for formulating them into aerosols. Exposing hamsters to these aerosols, before or even 24 h after infection with SARS-CoV-2, significantly reduced virus load, weight loss and pathogenicity. These results show the potential of aerosolized nanobodies for prophylaxis and therapy of coronavirus infections.

RNA viruses present a constant threat to human health, often with limited options for vaccination or therapy. Notable examples include influenza viruses and coronaviruses, which have pandemic potential. Filo- and henipaviruses cause more limited outbreaks, but with high case fatality rates. All RNA viruses rely on the activity of a virus-encoded RNA-dependent RNA polymerase (RdRp). An antiviral nucleoside analogue, 4'-Fluorouridine (4'-FlU), targets RdRp and diminishes the replication of several RNA viruses, including influenza A virus and SARS-CoV-2, through incorporation into nascent viral RNA and delayed chain termination. However, the effective concentration of 4'-FlU varied among different viruses, raising the need to fortify its efficacy. Here we show that inhibitors of dihydroorotate dehydrogenase (DHODH), an enzyme essential for pyrimidine biosynthesis, can synergistically enhance the antiviral effect of 4'-FlU against influenza A viruses, SARS-CoV-2, henipaviruses, and Ebola virus. Even 4'-FlU-resistant mutant influenza A virus was re-sensitized towards 4'-FlU by DHODH inhibition. The addition of uridine rescued influenza A virus replication, strongly suggesting uridine depletion as a mechanism of this synergy. 4'-FlU was also highly effective against SARS-CoV-2 in a hamster model of COVID. We propose that the impairment of endogenous uridine synthesis by DHODH inhibition enhances the incorporation of 4'-FlU into viral RNAs. This strategy may be broadly applicable to enhance the efficacy of pyrimidine nucleoside analogues for antiviral therapy.

Long-term consequences of SARS-CoV-2 infection affect millions of people and strain public health systems. The underlying pathomechanisms remain unclear, necessitating further research in appropriate animal models. This study aimed to characterize the trajectory of lung regeneration over 112 days in the male hamster model by combining morphological, transcriptomic and functional readouts. We demonstrate that in the acute phase, SARS-CoV-2 Delta-infected, male, aged hamsters show a severe impairment of lung function at rest. In the chronic phase, similar impairments persisted up to 7 weeks post-infection but were only evident after exercise on a rodent treadmill. The male hamster model recapitulates chronic pulmonary fibrotic changes observed in many patients with respiratory long COVID, but lacks extra-pulmonary long-term lesions. We show that sub-pleural and interstitial pulmonary fibrosis as well as alveolar bronchiolization persist until 112 dpi. Interestingly, CK8⁺ alveolar differentiation intermediate (ADI) cells are becoming less prominent in the alveolar proliferation areas from 28 dpi on. Instead, CK14⁺ airway basal cells and SCGB1A1⁺ club cells, expressing cell proliferation markers, mainly populate alveolar bronchiolization areas at later time-points. We postulate that pulmonary fibrosis and SCGB1A1⁺ club cell-rich areas of alveolar bronchiolization represent potential risk factors for other diseases in long-COVID survivors.

Self-renewal of the intestinal epithelium originates from stem cells located at the crypt base. Upregulation of various stem cell markers in intestinal epithelial neoplasms indicates a potential role of stem cells in tumorigenesis. In this study, the immunoreactivity of potential intestinal stem cell markers ( Sry box transcription factor 9 [Sox9], homeodomain-only protein [Hopx], survivin) and tuft cell marker doublecortin-like kinase 1 (DCLK1) in normal canine intestine and intestinal epithelial neoplasms was investigated. Formalin-fixed paraffin-embedded (FFPE) small and large intestine as well as intestinal neoplasms (55 colorectal adenomas [CRAs], 17 small intestinal adenocarcinomas [SICs], and 12 colorectal adenocarcinomas [CRCs]) were analyzed immunohistologically. Potential stem cell markers Sox9, Hopx, and survivin were detected in the crypts of normal canine small and large intestine. DCLK1⁺ tuft cells were present in decreasing numbers along the crypt-villus axis of the jejunum and rarely detectable in large intestine. In canine intestinal epithelial tumors, nuclear Sox9 immunoreactivity was detectable in 84.9% (CRA), 80% (CRC), and 77% of epithelial neoplastic cells (SIC). Hopx and survivin were expressed within cytoplasm and nuclei of neoplastic cells in benign and malignant tumors. DCLK1 showed a cytoplasmic reaction within neoplastic cells. The combined score of Hopx, DCLK1, and survivin varied among the examined cases. Overall, malignant tumors showed lower DCLK1 scores but higher Hopx scores in comparison with benign tumors. For survivin, no differences were detectable. In conclusion, stem cell markers Sox9, Hopx, and survivin were detectable at the crypt base and the immunoreactivity of Sox9, DCLK1, survivin, and Hopx was increased in canine intestinal adenomas and adenocarcinomas compared with normal mucosa.

Since the onset of the COVID-19 pandemic, SARS-CoV-2 neutralising monoclonal antibodies (mAbs) are being developed for clinical use. With the appearance of new virus variants, most mAbs lost their virus-neutralising activity, highlighting the complexity of mAb development under conditions of continuous SARS-CoV-2 evolution. Hamsters were treated with SARS-CoV-2 neutralising mAbs and then challenged with SARS-CoV-2. Recombinant VSV expressing the spike protein of SARS-CoV-2 was utilised in an in vitro system to select for antibody escape variants. Surface plasmon resonance measurements were performed to characterise the binding affinity and epitope of various mAbs. Fc-mediated effector functions of neutralising and non-neutralising mAb combinations were determined via multiple in vitro assays. Few of the mAb treated and infected hamsters experienced breakthrough infections, which derived from mutated virus that emerged in vivo. We developed an in vitro antibody escape assay that recapitulated the in vivo situation and we found that somatic hypermutations (SHM) affected the profile of viral escape hotspots that mAbs selected for. Pairwise combination of mAbs binding non-overlapping epitopes suppressed the emergence of viral mutants. The formulation with a third, non-neutralising mAb enhanced the Fc-mediated effector functions of the mAb treatment in an additive manner. We conclude that treatment with single mAbs rapidly leads to the formation of novel virus variants. An important function of SHM is to suppress the emergence of viral antibody escape variants. Our data suggest that the anticipatory B cell memory can be harnessed to design combinations of SARS-CoV-2 neutralising mAbs that have a reduced risk to induce viral escape. This study was supported by public funding from the German Research Foundation (DFG), the Federal Ministry of Education and Research (BMBF), the COVID-19-Research Network of the State of Lower Saxony (COFONI), the German Centre for Infection Research (DZIF), and the Helmholtz Association of German Research Centres.