Establishing a trans-packaging system for TBEV replicons to study events of complementation and recombination in the genus Flavivirus

Das FMSE Virusgenom besteht aus einem einzelsträngigen RNA Molekül mit positiver Polarität, welches für drei Strukturproteine und sieben Replikationsproteine codiert. Flavivirusgenome, die keine infektiösen Viruspartikel produzieren, werden durch Deletion eines oder mehrerer Strukturproteine generiert und als Replikons bezeichnet. Das Ziel der präsentierten Arbeit war die Verpackung von FSME Virus Replikons in Viruspartikel, mit welchen verschiedene Zelltypen infiziert werden können um verschiedene Aspekte der Flavivirusbiologie wie RNA Rekombination und Packaging untersuchen zu können. Nach Expression der Strukturproteine CprME im Alphavirusvektor VEEV zusammen mit FSME Virus Replikons in BHK-21 Zellen, konnten Viruspartikel, die Replikon RNA enthielten und ein Zelle nur einmal infizieren konnten, produziert werden. Unterschiedliche Zelltypen wurden mit verschiedenen verpackten Replikons zweifach infiziert um mögliche rekombinante Viren zu erfassen. Hierbei zeigte sich, dass die Infektiosität der verpackten Viruspartikel stark zwischen den untersuchten Zelltypen BHK-21, MEF, HEK 293T, IRE-18 variierte und dass Komplementation in trans nur in HEK 293T Zellen nachgewiesen werden konnte. Da die Anzahl der infizierten Zellen zu gering war um eine effiziente Komplementation zwischen den Replikons in der Wirtszelle nachzuweisen, konnten auch keine Zellpassagen durchgeführt werden um rekombinante Viren nachzuweisen. Daher sollte die Produktionseffizienz bei Verpackung der Replikons optimiert werden indem einerseits die Zelllinie HEK 293T zur Verpackung herangezogen wurde und andererseits die Strukturproteine CprME durch das DNA Expressionsplasmid phSV40 in trans zur Verfügung gestellt wurden. In beiden Fällen wurden erfolgreich FSME Virus Replikons in Viruspartikel verpackt, allerdings konnte eine Effizienzoptimierung der Methode nicht erreicht werden und daher war uns die Anwendung zur Untersuchung von Rekombination nicht möglich.The genome of the flavivirus TBEV consists of a single, positive-strand RNA molecule which encodes three structural and seven non-structural proteins. Flavivirus genomes defective in the production of infectious virus particles are constructed by deletion of one or more structural proteins and are characterized as replicons. We utilized a system to package TBEV replicons into virus particles to allow the infection of different cell types to study different aspects of the flavivirus biology including RNA recombination and packaging. Upon co-expression of TBEV replicons and the structural proteins CprME employing the alphavirus vector VEEV in BHK-21 cells, packaged replicons were recovered which were only capable of infecting a cell once and were termed single round infectious particles (SIPs). The produced SIPs were used in co-infection experiments to test their compatibility for studies on recombination. The infectivity of the produced SIPs strongly varied between the cell types BHK-21, MEF, HEK 293T, IRE-18 and trans-complementation only worked well in HEK 293T cells. However, the number of infected cells was generally too low to allow efficient complementation and therefore no passages could be performed to select recombinant viruses. Consequently, we tried to optimize the production of SIPs first by utilizing HEK 293T cells for packaging of TBEV replicons and secondly by expressing CprME via the plasmid DNA vector phSV40. In both cases trans-packaging of TBEV replicons into SIPs was successful but did not achieve to augment the amounts of produced SIPs to allow investigation of recombination

Viral Respiratory Infections in the Neonatal Intensive Care Unit—A Review

Although infrequent, respiratory viral infections (RVIs) during birth hospitalization have a significant impact on short- and long-term morbidity in term and preterm neonates. RVI have been associated with increased length of hospital stay, severe disease course, unnecessary antimicrobial exposure and nosocomial outbreaks in the neonatal intensive care unit (NICU). Virus transmission has been described to occur via health care professionals, parents and other visitors. Most at risk are infants born prematurely, due to their immature immune system and the fact that they stay in the NICU for a considerable length of time. A prevalence of RVIs in the NICU in symptomatic infants of 6–30% has been described, although RVIs are most probably underdiagnosed, since testing for viral pathogens is not performed routinely in symptomatic patients in many NICUs. Additional challenges are the wide range of clinical presentation of RVIs, their similarity to bacterial infections and the unreliable detection methods prior to the era of molecular biology based technologies. In this review, current knowledge of early-life RVI in the NICU is discussed. Reviewed viral pathogens include human rhinovirus, respiratory syncytial virus and influenza virus, and discussed literature is restricted to reports based on modern molecular biology techniques. The review highlights therapeutic approaches and possible preventive strategies. Furthermore, short- and long-term consequences of RVIs in infants hospitalized in the NICU are discussed

Integrative transcriptome analysis deciphers mechanisms of nickel contact dermatitis

Background Nickel-induced allergic contact dermatitis (nACD) remains a major occupational skin disorder, significantly impacting the quality of life of suffering patients. Complex cellular compositional changes and associated immunological pathways are partly resolved in humans; thus, the impact of nACD on human skin needs to be further elucidated. Methods To decipher involved immunological players and pathways, human skin biopsies were taken at 0, 2, 48, and 96 hours after nickel patch test in six nickel-allergic patients. Gene expression profiles were analyzed via microarray. Results Leukocyte deconvolution of nACD-affected skin identified major leukocyte compositional changes at 48 and 96 hours, including natural killer (NK) cells, macrophage polarization, and T-cell immunity. Gene set enrichment analysis mirrored cellular-linked functional pathways enriched over time. NK cell infiltration and cytotoxic pathways were uniquely found in nACD-affected skin compared to sodium lauryl sulfate-induced irritant skin reactions. Conclusion These results highlight key immunological leukocyte subsets as well as associated pathways in nACD, providing insights into pathophysiology with the potential to unravel novel therapeutic targets.Peer reviewe

Maternal immune activation transgenerationally modulates maternal care and offspring depression-like behavior

AbstractGestational infection is increasingly being recognized for its involvement as causative mechanism in severe developmental brain abnormalities and its contribution to the pathogenesis of psychopathologies later in life. First observations in the widely accepted maternal immune activation (MIA) model based upon the systemic administration of the viral mimetic Polyinosinic:polycytidylic acid (poly(I:C)) have recently suggested a transmission of behavioral and transcriptional traits across generations. Although maternal care behavior (MCB) is known as essential mediator of the transgenerational effects of environmental challenges on offspring brain function and behavior, the possible propagation of alterations of MCB resulting from MIA to following generations has not yet been examined. Here we show that poly(I:C) stimulation at embryonic day 12.5 (E12.5) leads to aberrant MCB and that this effect is transmitted to the female F1 offspring. The transgenerational effects on MCB are paralleled by enhanced depression-like behavior in the second generation F2 offspring with contributions of both maternal and paternal heritages. Examination of offspring hippocampal expression of genes known as targets of MCB and relevant for ensuing non-genetic transmission of altered brain function and behavior revealed transgenerationally conserved and modified expressional patterns in the F1 and F2 generation.Collectively these data firstly demonstrate the transgenerational transmission of the impact of gestational immune activation on the reproductive care behavior of the mother. Behavioral and molecular characteristics of first and second generation offspring suggest transgenerationally imprinted consequences of gestational infection on psychopathological traits related to mood disorders which remain to be examined in future cross-fostering experiments

Bet v 1 triggers antiviral-type immune signalling in birch-pollen-allergic individuals

Background In allergic patients, clinical symptoms caused by pollen remind of symptoms triggered by viral respiratory infections, which are also the main cause of asthmatic exacerbations. In patients sensitized to birch pollen, Bet v 1 is the major symptom-causing allergen. Immune mechanisms driving Bet v 1-related responses of human blood cells have not been fully characterized. Objective To characterize the immune response to Bet v 1 in peripheral blood in patients allergic to birch pollen. Methods The peripheral blood mononuclear cells of birch-allergic (n = 24) and non-allergic (n = 47) adolescents were stimulated ex-vivo followed by transcriptomic profiling. Systems-biology approaches were employed to decipher disease-relevant gene networks and deconvolution of associated cell populations. Results Solely in birch-allergic patients, co-expression analysis revealed activation of networks of innate immunity and antiviral signalling as the immediate response to Bet v 1 stimulation. Toll-like receptors and signal transducer transcription were the main drivers of gene expression patterns. Macrophages and dendritic cells were the main cell subsets responding to Bet v 1. Conclusions and clinical relevance In birch-pollen-allergic patients, the activated innate immune networks seem to be, in part, the same as those activated during viral infections. This tendency of the immune system to read pollens as viruses may provide new insight to allergy prevention and treatment.Peer reviewe

The duration of intrauterine development influences discrimination of speech prosody in infants.

AbstractAuditory speech discrimination is essential for normal language development. Children born preterm are at greater risk of language developmental delays. Using functional near‐infrared spectroscopy at term‐equivalent age, the present study investigated early discrimination of speech prosody in 62 neonates born between week 23 and 41 of gestational age (GA). We found a significant positive correlation between GA at birth and neural discrimination of forward versus backward speech at term‐equivalent age. Cluster analysis identified a critical threshold at around week 32 of GA, pointing out the existence of subgroups. Infants born before week 32 of GA exhibited a significantly different pattern of hemodynamic response to speech stimuli compared to infants born at or after week 32 of GA. Thus, children born before the GA of 32 weeks are especially vulnerable to early speech discrimination deficits. To support their early language development, we therefore suggest a close follow‐up and additional speech and language therapy especially in the group of children born before week 32 of GA

Calving Induced Speedup of Petermann Glacier

This study assesses the response on ice dynamics of Petermann Glacier, a major outlet glacier in northern Greenland, to the 2012 and a possible future calving event. So far Petermann Glacier has been believed to be dynamically stable as another large calving event in 2010 had no significant impact on flow velocity or grounding line retreat. By analyzing a time series of remotely sensed surface velocities, we find an average acceleration of 10% between winter 2011/2012 and winter 2016/2017. This increase in surface velocity is not linear but can be separated into two parts, starting in 2012 and 2016 respectively. By conducting modeling experiments, we show that the first speedup can be directly connected to the 2012 calving event, while the second speedup is not captured. However, on recent remote sensing imagery newly developing fractures are clearly visible ∼12 km upstream from the terminus, propagating from the eastern fjord wall to the center of the ice tongue, indicating a possible future calving event. By including these fracture zones as a new terminus position in the modeling domain, we are able to reproduce the second speedup, suggesting that surface velocities remain on the 2016/2017 level after the anticipated calving event. This indicates that, from a dynamical point of view, the terminus region has already detached from the main ice tongue

Neonatal sepsis is associated with behavioral abnormalities in very low birthweight infants at preschool age

ObjectiveThis study aimed to investigate neonatal sepsis as potential risk factor for adverse behavioral outcome in very low birth weight infants (VLBWI) at preschool age. Regardless of improvements in the obstetric and neonatal intensive care, preterm infants are still at high risk for behavioral problems later in life. The spectrum, origin and potential risk factors of these behavioral problems have not been well-defined.MethodsIn this retrospective observational study, the influence of culture-proven neonatal sepsis on the behavioral outcome of VLBWI born at a gestational age <32 weeks was analyzed at 5 years of age in a multivariable regression model. Behavior was assessed with the Child Behavior Checklist (CBCL). Neonatal morbidities, socioeconomic status and neurodevelopmental outcome served as covariates in the analysis.Results312 VLBWI entered the final analysis, of whom 11% had experienced neonatal sepsis. Neonatal sepsis appeared to be a relevant risk factor for both internalizing, i.e., emotional reactivity and anxiety/depression, as well as externalizing behavioral problems, i.e., oppositional and aggressive behavior in this cohort of VLBWI. Low socioeconomic status and male gender were additional statistically significant risk factors for both internalizing and externalizing behavioral problems. No difference in neurocognitive development was observed between the groups.ConclusionThe study supports the fact that VLBWI are vulnerable to multiple behavioral disorders independent of their cognitive development. In contrast to former assumptions, the results of the study emphasize that not only post-natal environment but also neonatal morbidities, especially neonatal sepsis, have an impact on behavioral outcome of VLBWI at preschool age