Abstract
The origin of SARS-CoV-2 variants of concern remains unclear. Here, we test whether intra-host virus evolution during persistent infections could be a contributing factor by characterizing ...the long-term SARS-CoV-2 infection dynamics in an immunosuppressed kidney transplant recipient. Applying RT-qPCR and next-generation sequencing (NGS) of sequential respiratory specimens, we identify several mutations in the viral genome late in infection. We demonstrate that a late viral isolate exhibiting genome mutations similar to those found in variants of concern first identified in UK, South Africa, and Brazil, can escape neutralization by COVID-19 antisera. Moreover, infection of susceptible mice with this patient’s escape variant elicits protective immunity against re-infection with either the parental virus and the escape variant, as well as high neutralization titers against the alpha and beta SARS-CoV-2 variants, B.1.1.7 and B.1.351, demonstrating a considerable immune control against such variants of concern. Upon lowering immunosuppressive treatment, the patient generated spike-specific neutralizing antibodies and resolved the infection. Our results suggest that immunocompromised patients could be a source for the emergence of potentially harmful SARS-CoV-2 variants.
Infections with emerging and re-emerging arboviruses are of increasing concern for global health. Tick-transmitted RNA viruses of the genus Thogotovirus in the Orthomyxoviridae family have ...considerable zoonotic potential, as indicated by the recent emergence of Bourbon virus in the USA. To successfully infect humans, arboviruses have to escape the restrictive power of the interferon defense system. This is exemplified by the high sensitivity of thogotoviruses to the antiviral action of the interferon-induced myxovirus resistance protein A (MxA) that inhibits the polymerase activity of incoming viral ribonucleoprotein complexes. Acquiring resistance to human MxA would be expected to enhance the zoonotic potential of these pathogens. Therefore, we screened a panel of 10 different thogotovirus isolates obtained from various parts of the world for their sensitivity to MxA. A single isolate from Nigeria, Jos virus, showed resistance to the antiviral action of MxA in cell culture and in MxA-transgenic mice, whereas the prototypic Sicilian isolate SiAr126 was fully MxA-sensitive. Further analysis identified two amino acid substitutions (G327R and R328V) in the viral nucleoprotein as determinants for MxA resistance. Importantly, when introduced into SiAr126, the R328V mutation resulted in complete MxA escape of the recombinant virus, without causing any viral fitness loss. The escape mutation abolished viral nucleoprotein recognition by MxA and allowed unhindered viral growth in MxA-expressing cells and in MxA-transgenic mice. These findings demonstrate that thogotoviruses can overcome the species barrier by escaping MxA restriction and reveal that these tick-transmitted viruses may have a greater zoonotic potential than previously suspected.
This paper introduces heart sound detection by radar systems, which enables touch-free and continuous monitoring of heart sounds. The proposed measurement principle entails two enhancements in modern ...vital sign monitoring. First, common touch-based auscultation with a phonocardiograph can be simplified by using biomedical radar systems. Second, detecting heart sounds offers a further feasibility in radar-based heartbeat monitoring. To analyse the performance of the proposed measurement principle, 9930 seconds of eleven persons-under-tests' vital signs were acquired and stored in a database using multiple, synchronised sensors: a continuous wave radar system, a phonocardiograph (PCG), an electrocardiograph (ECG), and a temperature-based respiration sensor. A hidden semi-Markov model is utilised to detect the heart sounds in the phonocardiograph and radar data and additionally, an advanced template matching (ATM) algorithm is used for state-of-the-art radar-based heartbeat detection. The feasibility of the proposed measurement principle is shown by a morphology analysis between the data acquired by radar and PCG for the dominant heart sounds S1 and S2: The correlation is 82.97 ± 11.15% for 5274 used occurrences of S1 and 80.72 ± 12.16% for 5277 used occurrences of S2. The performance of the proposed detection method is evaluated by comparing the F-scores for radar and PCG-based heart sound detection with ECG as reference: Achieving an F1 value of 92.22 ± 2.07%, the radar system approximates the score of 94.15 ± 1.61% for the PCG. The accuracy regarding the detection timing of heartbeat occurrences is analysed by means of the root-mean-square error: In comparison to the ATM algorithm (144.9 ms) and the PCG-based variant (59.4 ms), the proposed method has the lowest error value (44.2 ms). Based on these results, utilising the detected heart sounds considerably improves radar-based heartbeat monitoring, while the achieved performance is also competitive to phonocardiography.
Millimeter-wave sensing using automotive radar imposes high requirements on the applied signal processing in order to obtain the necessary resolution for current imaging radar. High-resolution ...direction of arrival estimation is needed to achieve the desired spatial resolution, limited by the total antenna array aperture. This work gives an overview of the recent progress and work in the field of deep learning based direction of arrival estimation in the automotive radar context, i.e. using only a single measurement snapshot. Additionally, several deep learning models are compared and investigated with respect to their suitability for automotive angle estimation. The models are trained with model- and data-based approaches for data generation, including simulated scenarios as well as real measurement data from more than 400 automotive radar sensors. Finally, their performance is compared to several baseline angle estimation algorithms like the maximum-likelihood estimator. All results are discussed with respect to the estimation error, the resolution of closely spaced targets and the total estimation accuracy. The overall results demonstrate the viability and advantages of the proposed data generation methods, as well as super-resolution capabilities of several architectures.
Severity of COVID-19 shows an extraordinary correlation with increasing age. We generated a mouse model for severe COVID-19 and show that the age-dependent disease severity is caused by the ...disruption of a timely and well-coordinated innate and adaptive immune response due to impaired interferon (IFN) immunity. Aggravated disease in aged mice was characterized by a diminished IFN-γ response and excessive virus replication. Accordingly, adult IFN-γ receptor-deficient mice phenocopied the age-related disease severity, and supplementation of IFN-γ reversed the increased disease susceptibility of aged mice. Further, we show that therapeutic treatment with IFN-λ in adults and a combinatorial treatment with IFN-γ and IFN-λ in aged Ifnar1-/- mice was highly efficient in protecting against severe disease. Our findings provide an explanation for the age-dependent disease severity and clarify the nonredundant antiviral functions of type I, II, and III IFNs during SARS-CoV-2 infection in an age-dependent manner. Our data suggest that highly vulnerable individuals could benefit from immunotherapy combining IFN-γ and IFN-λ.
Bats serve as a reservoir for various, often zoonotic viruses, including significant human pathogens such as Ebola and influenza viruses. However, for unknown reasons, viral infections rarely cause ...clinical symptoms in bats. A tight control of viral replication by the host innate immune defense might contribute to this phenomenon. Transcriptomic studies revealed the presence of the interferon-induced antiviral myxovirus resistance (Mx) proteins in bats, but detailed functional aspects have not been assessed. To provide evidence that bat Mx proteins might act as key factors to control viral replication we cloned
cDNAs from three bat families, Pteropodidae, Phyllostomidae, and Vespertilionidae. Phylogenetically these bat
genes cluster closely with their human ortholog MxA. Using transfected cell cultures, minireplicon systems, virus-like particles, and virus infections, we determined the antiviral potential of the bat Mx1 proteins. Bat Mx1 significantly reduced the polymerase activity of viruses circulating in bats, including Ebola and influenza A-like viruses. The related Thogoto virus, however, which is not known to infect bats, was not inhibited by bat Mx1. Further, we provide evidence for positive selection in bat
genes that might explain species-specific antiviral activities of these proteins. Together, our data suggest a role for Mx1 in controlling these viruses in their bat hosts.
Bats are a natural reservoir for various viruses that rarely cause clinical symptoms in bats but are dangerous zoonotic pathogens, like Ebola or rabies virus. It has been hypothesized that the interferon system might play a key role in controlling viral replication in bats. We speculate that the interferon-induced Mx proteins might be key antiviral factors of bats and have coevolved with bat-borne viruses. This study evaluated for the first time a large set of bat Mx1 proteins spanning three major bat families for their antiviral potential, including activity against Ebola virus and bat influenza A-like virus, and we describe here their phylogenetic relationship, revealing patterns of positive selection that suggest a coevolution with viral pathogens. By understanding the molecular mechanisms of the innate resistance of bats against viral diseases, we might gain important insights into how to prevent and fight human zoonotic infections caused by bat-borne viruses.
A unique example of supramolecular polymerisation in water based on monomers with nanomolar affinities, which yield rod‐like materials with extraordinarily high thermodynamic stability, yet of finite ...length, is reported. A small library of charge‐neutral dendritic peptide amphiphiles was prepared, with a branched nonaphenylalanine‐based core that was conjugated to hydrophilic dendrons of variable steric demand. Below a critical size of the dendron, the monomers assemble into nanorod‐like polymers, whereas for larger dendritic side chains frustrated growth into near isotropic particles is observed. The supramolecular morphologies observed by electron microscopy, X‐ray scattering and diffusion NMR spectroscopy studies are in agreement with the mechanistic insights obtained from fitting polymerisation profiles: non‐cooperative isodesmic growth leads to degrees of polymerisation that match the experimentally determined nanorod contour lengths of close to 70 nm. The reported designs for aqueous self‐assembly into well‐defined anisotropic particles has promising potential for biomedical applications and the development of functional supramolecular biomaterials, with emerging evidence that anisotropic shapes in carrier design outperform conventional isotropic materials for targeted imaging and therapy.
Crowd restrictions: A unique supramolecular polymerisation in water based on non‐ionic dendritic peptide monomers with nanomolar affinities, which yield rod‐like materials with very high thermodynamic stability, yet of finite length, is reported (see figure). Below a critical size of the dendritic side chains, the monomers assemble into nanorod‐like polymers, whereas the use of a larger dendron leads to frustrated growth into small, near‐isotropic particles.
While recombinant adenoviruses (rAds) are widely used in both laboratory and medical gene transfer, library-based applications using this vector platform are not readily available. Recently, we ...developed a new method, the CRISPR-Cas9 mediated in vivo terminal resolution aiding high-efficiency rescue of rAds from recombinant DNA. Here we report on a genetic workflow that allows construction of bacterial artificial chromosome-based rAd libraries reconstituted using highly efficient terminal resolution. We utilized frequent, pre-existing genomic sequences to allow the insertion of a selection marker, complementing two selected target sites into novel endonuclease recognition sites. In the second step, this selection marker is replaced with a transgene or mutation of interest via Gibson assembly. Our approach does not cause unwanted genomic off-target mutations while providing substantial flexibility for the site and nature of the genetic modification. This new genetic workflow, which we termed half site-directed fragment replacement (HFR) allows the introduction of more than 106 unique modifications into rAd encoding BACs using laboratory scale methodology. To demonstrate the power of HFR, we rescued barcoded viral vector libraries yielding a diversity of approximately 2.5 × 104 unique rAds per cm2 of transfected cell culture.
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Ruzsics and colleagues present a novel method for cloning large DNA bacmids based on naturally occurring features. This allows seamless mutagenesis of target sequences, showcased by modification and tagging of an adenoviral genome. Efficiencies are sufficient to generate high diversity viral libraries allowing for in vitro evolution-based screening.
This first pilot trial on external quality assessment (EQA) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) whole-genome sequencing, initiated by the European Society of Clinical ...Microbiology and Infectious Diseases (ESCMID) Study Group for Genomic and Molecular Diagnostics (ESGMD) and the Swiss Society for Microbiology (SSM), aims to build a framework between laboratories in order to improve pathogen surveillance sequencing. Ten samples with various viral loads were sent out to 15 clinical laboratories that had free choice of sequencing methods and bioinformatic analyses. The key aspects on which the individual centers were compared were the identification of (i) single nucleotide polymorphisms (SNPs) and indels, (ii) Pango lineages, and (iii) clusters between samples. The participating laboratories used a wide array of methods and analysis pipelines. Most were able to generate whole genomes for all samples. Genomes were sequenced to various depths (up to a 100-fold difference across centers). There was a very good consensus regarding the majority of reporting criteria, but there were a few discrepancies in lineage and cluster assignments. Additionally, there were inconsistencies in variant calling. The main reasons for discrepancies were missing data, bioinformatic choices, and interpretation of data. The pilot EQA was overall a success. It was able to show the high quality of participating laboratories and provide valuable feedback in cases where problems occurred, thereby improving the sequencing setup of laboratories. A larger follow-up EQA should, however, improve on defining the variables and format of the report. Additionally, contamination and/or minority variants should be a further aspect of assessment.
Influenza A viruses (IAVs) of subtype H9N2 have reached an endemic stage in poultry farms in the Middle East and Asia. As a result, human infections with avian H9N2 viruses have been increasingly ...reported. In 2017, an H9N2 virus was isolated for the first time from Egyptian fruit bats (Rousettus aegyptiacus). Phylogenetic analyses revealed that bat H9N2 is descended from a common ancestor dating back centuries ago. However, the H9 and N2 sequences appear to be genetically similar to current avian IAVs, suggesting recent reassortment events. These observations raise the question of the zoonotic potential of the mammal-adapted bat H9N2. Here, we investigate the infection and transmission potential of bat H9N2 in vitro and in vivo, the ability to overcome the antiviral activity of the human MxA protein, and the presence of N2-specific cross-reactive antibodies in human sera. We show that bat H9N2 has high replication and transmission potential in ferrets, efficiently infects human lung explant cultures, and is able to evade antiviral inhibition by MxA in transgenic B6 mice. Together with its low antigenic similarity to the N2 of seasonal human strains, bat H9N2 fulfils key criteria for pre-pandemic IAVs.
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