Henipaviruses—A constant threat to livestock and humans

In this review, we highlight the risk to livestock and humans from infections with henipaviruses, which belong to the virus family Paramyxoviridae. We provide a comprehensive overview of documented outbreaks of Nipah and Hendra virus infections affecting livestock and humans and assess the burden on the economy and health systems. In an increasingly globalized and interconnected world, attention must be paid to emerging viruses and infectious diseases, as transmission routes can be rapid and worldwide.Peer Reviewe

The Evolution of Complex Muscle Cell In Vitro Models to Study Pathomechanisms and Drug Development of Neuromuscular Disease

Many neuromuscular disease entities possess a significant disease burden and therapeutic options remain limited. Innovative human preclinical models may help to uncover relevant disease mechanisms and enhance the translation of therapeutic findings to strengthen neuromuscular disease precision medicine. By concentrating on idiopathic inflammatory muscle disorders, we summarize the recent evolution of the novel in vitro models to study disease mechanisms and therapeutic strategies. A particular focus is laid on the integration and simulation of multicellular interactions of muscle tissue in disease phenotypes in vitro. Finally, the requirements of a neuromuscular disease drug development workflow are discussed with a particular emphasis on cell sources, co-culture systems (including organoids), functionality, and throughput.Peer Reviewe

A Proteomic Survey of Host and Virus Reveals Differential Dynamics

We studied the dynamics of the proteome of influenza virus A/PR/8/34 (H1N1) infected Madin-Darby canine kidney cells up to 12 hours post infection by mass spectrometry based quantitative proteomics using the approach of stable isotope labeling by amino acids in cell culture (SILAC). We identified 1311 cell proteins and, apart from the proton channel M2, all major virus proteins. Based on their abundance two groups of virus proteins could be distinguished being in line with the function of the proteins in genesis and formation of new virions. Further, the data indicate a correlation between the amount of proteins synthesized and their previously determined copy number inside the viral particle. We employed bioinformatic approaches such as functional clustering, gene ontology, and pathway (KEGG) enrichment tests to uncover co- regulated cellular protein sets, assigned the individual subsets to their biological function, and determined their interrelation within the progression of viral infection. For the first time we are able to describe dynamic changes of the cellular and, of note, the viral proteome in a time dependent manner simultaneously. Through cluster analysis, time dependent patterns of protein abundances revealed highly dynamic up- and/or down-regulation processes. Taken together our study provides strong evidence that virus infection has a major impact on the cell status at the protein level

Pharmacologically induced endolysosomal cholesterol imbalance through clinically licensed drugs itraconazole and fluoxetine impairs Ebola virus infection in vitro

Ebola virus disease (EVD) is a severe and frequently lethal disease caused by Ebola virus (EBOV). The latest occasional EVD outbreak (2013–2016) in Western African, which was accompanied by a high fatality rate, showed the great potential of epidemic and pandemic spread. Antiviral therapies against EBOV are very limited, strain-dependent (only antibody therapies are available) and mostly restricted to symptomatic treatment, illustrating the urgent need for novel antiviral strategies. Thus, we evaluated the effect of the clinically widely used antifungal itraconazole and the antidepressant fluoxetine for a repurposing against EBOV infection. While itraconazole, similar to U18666A, directly binds to and inhibits the endosomal membrane protein Niemann-Pick C1 (NPC1), fluoxetine, which belongs to the structurally unrelated group of weakly basic, amphiphile so-called “functional inhibitors of acid sphingomyelinase” (FIASMA) indirectly acts on the lysosome-residing acid sphingomyelinase via enzyme detachment leading to subsequent lysosomal degradation. Both, the drug-induced endolysosomal cholesterol accumulation and the altered endolysosomal pH, might interfere with the fusion of viral and endolysosomal membrane, preventing infection with EBOV. We further provide evidence that cholesterol imbalance is a conserved cross-species mechanism to hamper EBOV infection. Thus, exploring the endolysosomal host–pathogen interface as a suitable antiviral treatment may offer a general strategy to combat EBOV infection.Peer Reviewe

3D Ex vivo tissue platforms to investigate the early phases of influenza a virus- and SARS-CoV-2-induced respiratory diseases

Pandemic outbreaks of viruses such as influenza virus or SARS-CoV-2 are associated with high morbidity and mortality and thus pose a massive threat to global health and economics. Physiologically relevant models are needed to study the viral life cycle, describe the pathophysiological consequences of viral infection, and explore possible drug targets and treatment options. While simple cell culture-based models do not reflect the tissue environment and systemic responses, animal models are linked with huge direct and indirect costs and ethical questions. Ex vivo platforms based on tissue explants have been introduced as suitable platforms to bridge the gap between cell culture and animal models. We established a murine lung tissue explant platform for two respiratory viruses, influenza A virus (IAV) and SARS-CoV-2. We observed efficient viral replication, associated with the release of inflammatory cytokines and the induction of an antiviral interferon response, comparable to ex vivo infection in human lung explants. Endolysosomal entry could be confirmed as a potential host target for pharmacological intervention, and the potential repurposing potentials of fluoxetine and interferons for host-directed therapy previously seen in vitro could be recapitulated in the ex vivo model.Peer Reviewe

A temporal, spatial and quantitative study on the influenza A virus transcription, translation and virus-host interaction

Die Vermehrung des Influenza A Virus umfasst, neben anderen wichtigen Schritten, die Transkrption der viralen mRNA und die ribosomale Translation der viralen Proteine. Mit großem Aufwand wurde bereits an der Entwicklung von Methoden zur Untersuchung des zeitlichen Verlaufs der Synthese viraler mRNA während des Vermehrungszyklusses in der Wirtszelle geforscht. In der vorliegenden Arbeit wurden sequenzspezifische FIT-PNA-Sonden, welche einen einzelnen, als künstliche fluoreszente Nukleobase dienenden Interkalator tragen, auf die quantitative RT-PCR sowie die Lebendzellmikroskopie angewandt. Die FIT-PNA-Sonden bieten dabei eine hohe Sensitivität und eine enorme Zielspezifität unter nichtstringenten Hybridisierungsbedingungen. Im Speziellen wurden FIT-PNA Sonden mit Sequenzspezifität zur mRNA der Neuraminidase und des Matrixproteins 1 entworfen und untersucht. Die somit erhaltenen Ergebnisse besitzen eine hohe biologische Relevanz und weisen diese Sonden als vielversprechende Methodik in der Virologie und der Zellbiologie aus. Ihre Anwendung konnte bereits auf das Vesikular Stomatitis Virus ausgeweitet werden. Die Kombination aus biologischer Expertise mit modernen Proteomstudien und detaillierten statistischen Analysen ermöglichte einen systemumfassenden Blick auf die durch eine Infektion bedingten Auswirkungen auf die Wirtszelle. Die Markierung von Aminosäuren mit stabilen Isotopen in Zellkultur wurde hierfür benutzt. Es wurden Proben zu verschiedene Zeitpunkten im Infektionszyklus in die Untersuchungen einbezogen, um zeitaufgelöste Detailstudien der zellulären Proteinbiosynthese und Degradation durchzuführen.Replication of the influenza A virus involves, amongst other critical steps, the transcription of viral mRNA and ribosomal translation of viral proteins. Significant efforts have been devoted to the development of methods that allow the investigation of viral mRNA progression during the replication cycle inside the host cell. In the present thesis sequence specific FIT-PNA probes which contain a single intercalator serving as artificial fluorescent nucleobase were introduced for quantitative RT-PCR and live cell imaging. FIT-PNAs provide for both high sensitivity and high target specificity at nonstringent hybridisation conditions (where both matched and mismatched probetarget complexes coexist). In particular, FIT-PNAs specific to the neuraminidase and matrix protein 1 were successfully designed and examined. The obtained results are of high biological importance and suggest the FIT-PNA technique as promising tool in the field of virology and cell biology as this approach was readily applied to Vesicular Stomatitis Virus as well. By combining biological expertise with modern high throughput quantitative proteomics and detailed statistical analysis a system wide view of the effects and dynamics of the early H1N1 infection on the cell proteome was generated. Stable isotope labelling of amino acids in cell culture (SILAC) was employed to globally track changes in gene expression at the protein level. Furthermore, samples at various time points post infection enabling a more detailed timeresolved analysis of host cell protein biosynthesis and degradation during the infection cycle were included. As a result the specific expression characteristics of single genes and functional gene subsets in response to viral infection were bioinformatically analysed

IFITM3 Clusters on Virus Containing Endosomes and Lysosomes Early in the Influenza A Infection of Human Airway Epithelial Cells

Interferon-induced transmembrane proteins (IFITMs) have been shown to strongly affect influenza A virus (IAV) infectivity in tissue culture. Moreover, polymorphisms in IFITM3 have been associated with the severity of the disease in humans. IFITM3 appears to act early in the infection, but its mechanism of action and potential interactions with incoming IAV structures are not yet defined. Here, we visualized endogenous IFITM3 interactions with IAV in the human lung epithelial cell line A549 and in primary human airway epithelial cells employing stimulated emission depletion super-resolution microscopy. By applying an iterative approach for the cluster definition and computational cluster analysis, we found that IFITM3 reorganizes into clusters as IAV infection progresses. IFITM3 cluster formation started at 2-3 h post infection and increased over time to finally coat IAV-containing endosomal vesicles. This IAV-induced phenotype was due to the endosomal recruitment of IFITM3 rather than to an overall increase in the IFITM3 abundance. While the IAV-induced IFITM3 clustering and localization to endosomal vesicles was comparable in primary human airway epithelial cells and the human lung epithelial cell line A549, the endogenous IFITM3 signal was higher in primary cells. Moreover, we observed IFITM3 signals adjacent to IAV-containing recycling endosomes

Structural analysis of the roles of influenza A virus membrane-associated proteins in assembly and morphology

The assembly of influenza A virus at the plasma membrane of infected cells leads to release of enveloped virions that are typically round in tissue culture-adapted strains but filamentous in strains isolated from patients. The viral proteins hemagglutinin (HA), neuraminidase (NA), matrix protein 1 (M1), and M2 ion channel all contribute to virus assembly. When expressed individually or in combination in cells, they can all, under certain conditions, mediate release of membrane-enveloped particles, but their relative roles in virus assembly, release, and morphology remain unclear. To investigate these roles, we produced membrane-enveloped particles by plasmid-derived expression of combinations of HA, NA, and M proteins (M1 and M2) or by infection with influenza A virus. We monitored particle release, particle morphology, and plasma membrane morphology by using biochemical methods, electron microscopy, electron tomography, and cryo-electron tomography. Our data suggest that HA, NA, or HANA (HA plus NA) expression leads to particle release through nonspecific induction of membrane curvature. In contrast, coexpression with the M proteins clusters the glycoproteins into filamentous membrane protrusions, which can be released as particles by formation of a constricted neck at the base. HA and NA are preferentially distributed to differently curved membranes within these particles. Both the budding intermediates and the released particles are morphologically similar to those produced during infection with influenza A virus. Together, our data provide new insights into influenza virus assembly and show that the M segment together with either of the glycoproteins is the minimal requirement to assemble and release membrane-enveloped particles that are truly virus-like