25 research outputs found
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
Fluorescence Imaging of Influenza Virus H1N1 mRNA in Living Infected Cells using Single Chromophore FIT-PNA
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
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
Fluorescence Imaging of Influenza Virus H1N1 mRNA in Living Infected Cells using Single Chromophore FIT-PNA
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
PNA FIT-Probes for the Dual Color Imaging of Two Viral mRNA Targets in Influenza H1N1 Infected Live Cells
Fluorogenic hybridization probes that allow RNA imaging provide
information as to how the synthesis and transport of particular RNA
molecules is orchestrated in living cells. In this study, we explored
the peptide nucleic acid (PNA)-based FIT-probes in the simultaneous
imaging of two different viral mRNA molecules expressed during the
replication cycle of the H1N1 influenza A virus. PNA FIT-probes are
non-nucleotidic, nonstructured probes and contain a single asymmetric
cyanine dye which serves as a fluorescent base surrogate. The fluorochrome
acts as a local intercalator probe and reports hybridization of target
DNA/RNA by enhancement of fluorescence. Though multiplexed hybridization
probes are expected to facilitate the analysis of RNA expression,
there are no previous reports on the dual color imaging of two different
viral mRNA targets. In this work, we developed a set
of two differently colored PNA FIT-probes that allow the spectrally
resolved imaging of mRNA coding for neuraminidase (NA) and matrix
protein 1 (M1); proteins which execute distinct functions during the
replication of the influenza A virus. The probes are characterized
by a wide range of applicable hybridization temperatures. The same
probe sequence enabled live-cell RNA imaging (at 37 °C) as well
as real-time PCR measurements (at 60 °C annealing temperature).
This facilitated a comprehensive analysis of RNA expression by quantitative
(qPCR) and qualitative (imaging) means. Confocal laser scanning microscopy
showed that the viral-RNA specific PNA FIT-probes neither stained
noninfected cells nor cells infected by a control virus. The joint
use of differently colored PNA FIT-probes in this feasibility study
revealed significant differences in the expression pattern of influenza
H1N1 mRNAs coding for NA or M1. These experiments provide evidence
for the usefulness of PNA FIT-probes in investigations on the temporal
and spatial progression of mRNA synthesis in living cells for two
mRNA species
Alteration of Protein Levels during Influenza Virus H1N1 Infection in Host Cells: A Proteomic Survey of Host and Virus Reveals Differential Dynamics
<div><p>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.</p></div