Background During the initial COVID-19 response, Germany's Federal Government implemented several nonpharmaceutical interventions (NPIs) that were instrumental in suppressing early exponential spread of SARS-CoV-2. NPI effect on the transmission of other respiratory viruses has not been examined at the national level thus far. Methods Upper respiratory tract specimens from 3580 patients with acute respiratory infection (ARI), collected within the nationwide German ARI Sentinel, underwent RT-PCR diagnostics for multiple respiratory viruses. The observation period (weeks 1-38 of 2020) included the time before, during and after a far-reaching contact ban. Detection rates for different viruses were compared to 2017-2019 sentinel data (15350 samples; week 1-38, 11823 samples). Findings The March 2020 contact ban, which was followed by a mask mandate, was associated with an unprecedented and sustained decline of multiple respiratory viruses. Among these, rhinovirus was the single agent that resurged to levels equalling those of previous years. Rhinovirus rebound was first observed in children, after schools and daycares had reopened. By contrast, other nonenveloped viruses (i.e. gastroenteritis viruses reported at the national level) suppressed after the shutdown did not rebound. Interpretation Contact restrictions with a subsequent mask mandate in spring may substantially reduce respiratory virus circulation. This reduction appears sustained for most viruses, indicating that the activity of influenza and other respiratory viruses during the subsequent winter season might be low,whereas rhinovirus resurgence, potentially driven by transmission in educational institutions in a setting of waning population immunity, might signal predominance of rhinovirus-related ARIs. Funding Robert Koch-Institute and German Ministry of Health.

We investigated the inhibitory effects of the antifungal protein (AFP) from Aspergillus giganteus on the growth of several filamentous fungi. For this purpose, the MICs of AFP were determined and ranged from 0.1 g/ml for Fusarium oxysporum to 200 g/ml for Aspergillus nidulans. The antifungal activity of AFP was diminished in the presence of cations. We were able to show that incubation of AFP-sensitive fungi with the protein resulted in membrane permeabilization using an assay based on the uptake of the fluorescent dye SYTOX Green. No permeabilization by AFP could be detected at concentrations below the species-specific MIC. Furthermore, AFP-induced permeabilization could readily be detected after 5 min of incubation. Localization experiments with fluorescein isothiocyanate-labeled AFP and immunofluorescence staining with an AFP-specific antibody supported the observation that the protein interacts with membranes. After treatment of AFP-sensitive fungi with AFP, the protein was localized at the plasma membrane, whereas it was mainly detected inside the cells of AFP-resistant fungi. We conclude from these data that the growth-inhibitory effect of AFP is caused by permeabilization of the fungal membranes.The imperfect filamentous fungus Aspergillus giganteus was found to secrete two small basic proteins, namely, the antifungal protein (AFP) and ␣-sarcin (22). ␣-Sarcin is a cytotoxic protein belonging to the ribotoxins, a family of ribosome-inactivating proteins (2,6,7,13,17,24,30). AFP is a highly basic polypeptide of 51 amino acids with a high content of cysteine, tyrosine, and lysine residues. The isoelectric point was estimated to be 8.8; thus, the protein is positively charged under neutral conditions. AFP is folded into five highly twisted antiparallel strands, defining a small and compact structure with four stabilizing disulfide bridges (3,20,29).Only three small, basic antifungal proteins have been isolated from filamentous fungi to date: AFP from A. giganteus (22), PAF from Penicillium chrysogenum (18), and Anafp from Aspergillus niger (14). Recently, the naf gene was isolated from Penicillium nalgiovense and is identical to the paf gene from P. chrysogenum and encodes a protein with transient antifungal activity (8). All these antifungal proteins are secreted and inhibit the growth of numerous filamentous fungi without affecting bacteria and yeast. Most notably, they are significantly similar in regard to their structures, sizes, and basic characters. The PAF protein exhibits 42.6% sequence similarity with the AFP sequence (18).Not much is known about the modes of action of the antifungal proteins secreted by fungi. It has, however, been shown that defensins, a class of antimicrobial proteins which are quite similar to antifungal proteins in terms of their structures, sizes, and disulfide bridges, interact with anionic phospholipids of bacterial membranes (15). In addition, it has been found that some plant defensins have a receptor-mediated interaction with the fungal plasma membrane (27). However, noth...

In this paper, we report the detection, purification and characterization of the first metalloprotease inhibitor (IMPI) from invertebrates. IMPI was purified from the hemolymph of last-instar larvae of Galleria mellonella by precipitation with trichloroacetic acid and heat followed by affinity chromatography on a thermolysin-Sepharose column and gel filtration or reverse-phase high-performance liquid chromatography. For the detection of inhibitor activity, a new azocoll assay was established. IMPI was only detectable in larvae that had been injected with bacterial or fungal provocators, suggesting that it is induced nonspecifically during the humoral immune response. Injection of larvae with IMPI rendered them resistant to thermolysin, in quantities that normally would be lethal for them. IMPI was shown to be specific for metalloproteases. The molecular mass of IMPI was determined by mass spectrometry to be 8360 Da. Purified IMPI was heterogeneous, owing to different degrees of glycosylation with hexose/hexosamine and deoxyhexose residues. Ten cysteine residues were found in the molecule, and these are presumed to form five disulfide bridges. The amino terminus was blocked, but a partial amino-acid sequence starting from the thermolysin cleavage site was determined; this sequence exhibited no similarity with other known proteins, suggesting that the IMPI represents a new type of protease inhibitor. Keywords : metalloproteases; metalloprotease inhibitor ; insect immunity ; Galleria mellonella.Insects are particularly resistant to micro-organisms. The endogenous defense of insects is based on cellular and humoral immune responses. The latter includes the synthesis of a broad spectrum of potent antimicrobial proteins that act directly against invading micro-organisms [1,2]. The response of insects to wounding or infection includes hemolymph coagulation and an activation of the prophenoloxidase cascade. The latter process is activated by serine proteases and regulated by serine-protease inhibitors in order to avoid excessive activation of and damage to host tissues [3,4]. In addition, protease inhibitors are thought to inhibit undesired proteolytic enzymes released by damaged cells or invading pathogens. Entomopathogenic fungi are the only group of micro-organisms that can infect insect hosts directly through their chitinous exoskeleton. Fungal extracellular proteases contribute to both integument penetration and digestion of host proteins. They are reported to determine the host specificity and the virulence of the producing fungal species [5,6]. Protease inhibitors detected in the cuticle or hemolymph of several insect species have been proposed to regulate not only endogenous proteases but also act against toxic fungal proteases hibitors and one cysteine protease inhibitor, but no metalloprotease inhibitor [12].Recently, it was reported that injection of zymosan or heatinactivated yeast cells resulted in reduced or delayed mortality of G. mellonella larvae after injection of living pathogenic fungal cells [13]. T...

Respiratory viruses are a cause of upper respiratory tract infections (URTI), but can be associated with severe lower respiratory tract infections (LRTI) in immunocompromised patients. The objective of this study was to investigate the genetic variability of influenza virus, parainfluenza virus and respiratory syncytial virus (RSV) and the duration of viral shedding in hematological patients. Nasopharyngeal swabs from hematological patients were screened for influenza, parainfluenza and RSV on admission as well as on development of respiratory symptoms. Consecutive swabs were collected until viral clearance. Out of 672 tested patients, a total of 111 patients (17%) were infected with one of the investigated viral agents: 40 with influenza, 13 with parainfluenza and 64 with RSV; six patients had influenza/RSV or parainfluenza/RSV co-infections. The majority of infected patients (n = 75/111) underwent stem cell transplantation (42 autologous, 48 allogeneic, 15 autologous and allogeneic). LRTI was observed in 48 patients, of whom 15 patients developed severe LRTI, and 13 patients with respiratory tract infection died. Phylogenetic analysis revealed a variety of influenza A(H1N1)pdm09, A(H3N2), influenza B, parainfluenza 3 and RSV A, B viruses. RSV A was detected in 54 patients, RSV B in ten patients. The newly emerging RSV A genotype ON1 predominated in the study cohort and was found in 48 (75%) of 64 RSV-infected patients. Furthermore, two distinct clusters were detected for RSV A genotype ON1, identical RSV G gene sequences in these patients are consistent with nosocomial transmission. Long-term viral shedding for more than 30 days was significantly associated with prior allogeneic transplantation (p = 0.01) and was most pronounced in patients with RSV infection (n = 16) with a median duration of viral shedding for 80 days (range 35–334 days). Long-term shedding of respiratory viruses might be a catalyzer of nosocomial transmission and must be considered for efficient infection control in immunocompromised patients.

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