OLA-DRB1 microsatellite variants are associated with ovine growth and reproduction traits

The DRB1 intron 2 (GT)n (GA)m microsatellite was genotyped in experimental flocks of seven Merinoland rams and 249 ewes as well as their offspring (381 lambs) from consecutive lambings. A total of 16 DRB1 alleles were detected, ranging between 353 and 857 bp. In comparison with carriers of other alleles, the ewes carrying the predominant 411 bp allele had higher values of all the recorded fertility traits. For ewes carrying the 394 and 857 bp alleles, the birth weight of lambs was about 400 g higher as compared to the residual group of ewes. The observed associations could be due to differences in disease resistance, cell recognition or tissue differentiation between carriers of various MHC haplotypes which can in turn affect individual fertility and growth performance

Hepatitis C virus exploits cyclophilin A to evade PKR

Counteracting innate immunity is essential for successful viral replication. Host cyclophilins (Cyps) have been implicated in viral evasion of host antiviral responses, although the mechanisms are still unclear. Here, we show that hepatitis C virus (HCV) co-opts the host protein CypA to aid evasion of antiviral responses dependent on the effector protein kinase R (PKR). Pharmacological inhibition of CypA rescues PKR from antagonism by HCV NS5A, leading to activation of an interferon regulatory factor-1 (IRF1)-driven cell intrinsic antiviral program that inhibits viral replication. These findings further the understanding of the complexity of Cyp-virus interactions, provide mechanistic insight into the remarkably broad antiviral spectrum of Cyp inhibitors, and uncover novel aspects of PKR activity and regulation. Collectively, our study identifies a novel antiviral mechanism that harnesses cellular antiviral immunity to suppress viral replication

The lysine methyltransferase SMYD3 interacts with hepatitis C virus NS5A and is a negative regulator of viral particle production

Hepatitis C virus (HCV) is a considerable global health and economic burden. The HCV nonstructural protein (NS) 5A is essential for the viral life cycle. The ability of NS5A to interact with different host and viral proteins allow it to manipulate cellular pathways and regulate viral processes, including RNA replication and virus particle assembly. As part of a proteomic screen, we identified several NS5A-binding proteins, including the lysine methyltransferase SET and MYND domain containing protein 3 (SMYD3). We confirmed the interaction in the context of viral replication by co-immunoprecipitation and co-localization studies. Mutational analyses revealed that the MYND-domain of SMYD3 and domain III of NS5A are required for the interaction. Overexpression of SMYD3 resulted in decreased intracellular and extracellular virus titers, whilst viral RNA replication remained unchanged, suggesting that SMYD3 negatively affects HCV particle production in a NS5A-dependent manner. (C) 2014 The Authors. Published by Elsevier Inc

Simulation der Letalität nach verschiedenen Ex-ante- und Ex-post-Triage-Verfahren bei Menschen mit Behinderungen und Vorerkrankungen

Der stetige Anstieg an zu behandelnden Patienten während der COVID-19-Pandemie hat das Gesundheitssystem vor eine Vielzahl an Herausforderungen gestellt. Die Intensivstation ist einer der in diesem Zusammenhang besonders stark betroffenen Bereiche. Nur durch umfangreiche Infektionsschutzmaßnahmen sowie einen enormen logistischen Aufwand konnten in Deutschland selbst in Hochphasen der Pandemie die Behandlung aller Intensivpatienten ermöglicht und eine Triage auch in Regionen mit hohem Patientendruck bei gleichzeitig geringen Kapazitäten verhindert werden. Im Hinblick auf die Pandemievorsorge hat der Deutsche Bundestag ein Gesetz zur Triage verabschiedet, das eine Ex-post-Triage explizit untersagt. Bei einer Ex-post-Triage werden auch Patienten, die bereits auf der Intensivstation behandelt werden, in die Triage-Entscheidung einbezogen und Behandlungskapazitäten nach individueller Erfolgsaussicht verteilt. In der Literatur finden sich rechtliche, ethische und soziale Überlegungen zur Triage bei Pandemien, eine quantitative Bewertung im Hinblick auf verschiedene Patientengruppen auf der Intensivstation gibt es hingegen nicht. Der Fokus der Arbeit liegt auf dieser Forschungslücke, und es wird eine quantitative simulationsbasierte Evaluation von Ex-ante- und Ex-post-Triage-Politiken unter Berücksichtigung von Überlebenswahrscheinlichkeiten, Beeinträchtigungen und Vorerkrankungen durchgeführt. Die Ergebnisse zeigen, dass eine Anwendung von Ex-post-Triage, basierend auf Überlebenswahrscheinlichkeiten in allen Patientengruppen, zu einer Reduktion der Mortalität auf der Intensivstation führt. In dem Szenario, das der realen Situation wohl am nächsten kommt, wird eine Reduktion der Mortalität auf der Intensivstation um ca. 15 % schon bei einer einmaligen Anwendung der Ex-post-Triage erreicht. Dieser mortalitätsreduzierende Effekt ist umso größer, je mehr Patienten auf eine intensivmedizinische Behandlung warten

Identification of {HNRNPK} as Regulator of Hepatitis {C} Virus Particle Production

Hepatitis C virus (HCV) is a major cause of chronic liver disease affecting around 130 million people worldwide. While great progress has been made to define the principle steps of the viral life cycle, detailed knowledge how HCV interacts with its host cells is still limited. To overcome this limitation we conducted a comprehensive whole-virus RNA interference-based screen and identified 40 host dependency and 16 host restriction factors involved in HCV entry/replication or assembly/release. Of these factors, heterogeneous nuclear ribonucleoprotein K (HNRNPK) was found to suppress HCV particle production without affecting viral RNA replication. This suppression of virus production was specific to HCV, independent from assembly competence and genotype, and not found with the related Dengue virus. By using a knock-down rescue approach we identified the domains within HNRNPK required for suppression of HCV particle production. Importantly, HNRNPK was found to interact specifically with HCV RNA and this interaction was impaired by mutations that also reduced the ability to suppress HCV particle production. Finally, we found that in HCV-infected cells, subcellular distribution of HNRNPK was altered; the protein was recruited to sites in close proximity of lipid droplets and colocalized with core protein as well as HCV plus-strand RNA, which was not the case with HNRNPK variants unable to suppress HCV virion formation. These results suggest that HNRNPK might determine efficiency of HCV particle production by limiting the availability of viral RNA for incorporation into virions. This study adds a new function to HNRNPK that acts as central hub in the replication cycle of multiple other viruses

Essential Role of Cyclophilin A for Hepatitis C Virus Replication and Virus Production and Possible Link to Polyprotein Cleavage Kinetics

Viruses are obligate intracellular parasites and therefore their replication completely depends on host cell factors. In case of the hepatitis C virus (HCV), a positive-strand RNA virus that in the majority of infections establishes persistence, cyclophilins are considered to play an important role in RNA replication. Subsequent to the observation that cyclosporines, known to sequester cyclophilins by direct binding, profoundly block HCV replication in cultured human hepatoma cells, conflicting results were obtained as to the particular cyclophilin (Cyp) required for viral RNA replication and the underlying possible mode of action. By using a set of cell lines with stable knock-down of CypA or CypB, we demonstrate in the present work that replication of subgenomic HCV replicons of different genotypes is reduced by CypA depletion up to 1,000-fold whereas knock-down of CypB had no effect. Inhibition of replication was rescued by over-expression of wild type CypA, but not by a mutant lacking isomerase activity. Replication of JFH1-derived full length genomes was even more sensitive to CypA depletion as compared to subgenomic replicons and virus production was completely blocked. These results argue that CypA may target an additional viral factor outside of the minimal replicase contributing to RNA amplification and assembly, presumably nonstructural protein 2. By selecting for resistance against the cyclosporine analogue DEBIO-025 that targets CypA in a dose-dependent manner, we identified two mutations (V2440A and V2440L) close to the cleavage site between nonstructural protein 5A and the RNA-dependent RNA polymerase in nonstructural protein 5B that slow down cleavage kinetics at this site and reduce CypA dependence of viral replication. Further amino acid substitutions at the same cleavage site accelerating processing increase CypA dependence. Our results thus identify an unexpected correlation between HCV polyprotein processing and CypA dependence of HCV replication

A distinctive gene expression fingerprint in mentally retarded male patients reflects disease-causing defects in the histone demethylase KDM5C

Background: Mental retardation is a genetically heterogeneous disorder, as more than 90 genes for this disorder has been found on the X chromosome alone. In addition the majority of patients are non-syndromic in that they do not present with clinically recognisable features. This makes it difficult to determine the molecular cause of this disorder on the basis of the phenotype alone. Mutations in KDM5C (previously named SMCX or JARID1C), a gene that encodes a transcriptional regulator with histone demethylase activity specific for dimethylated and trimethylated H3K4, are a comparatively frequent cause of non-syndromic X-linked mental retardation (NS-XLMR). Specific transcriptional targets of KDM5C, however, are still unknown and the effects of KDM5C deficiency on gene expression have not yet been investigated. Results: By whole-mount in situ hybridisation we showed that the mouse homologue of KDM5C is expressed in multiple tissues during mouse development. We present the results of gene expression profiling performed on lymphoblastoid cell lines as well as blood from patients with mutations in KDM5C. Using whole genome expression arrays and quantitative reverse transcriptase polymerase chain reaction (QRT-PCR) experiments, we identified several genes, including CMKOR1, KDM5B and KIAA0469 that were consistently deregulated in both tissues. Conclusions: Our findings shed light on the pathological mechanisms underlying mental retardation and have implications for future diagnostics of this heterogeneous disorder

Characterization of the mode of action of a potent dengue virus capsid inhibitor

Dengue viruses (DV) represent a significant global health burden, with up to 400 million infections every year and around 500,000 infected individuals developing life-threatening disease. In spite of attempts to develop vaccine candidates and antiviral drugs, there is a lack of approved therapeutics for the treatment of DV infection. We have previously reported the identification of ST-148, a small-molecule inhibitor exhibiting broad and potent antiviral activity against DV in vitro and in vivo (C. M. Byrd et al., Antimicrob. Agents Chemother. 57:15–25, 2013, doi:10 .1128/AAC.01429-12). In the present study, we investigated the mode of action of this promising compound by using a combination of biochemical, virological, and imaging-based techniques. We confirmed that ST-148 targets the capsid protein and obtained evidence of bimodal antiviral activity affecting both assembly/release and entry of infectious DV particles. Importantly, by using a robust bioluminescence resonance energy transfer-based assay, we observed an ST-148-dependent increase of capsid self-interaction. These results were corroborated by molecular modeling studies that also revealed a plausible model for compound binding to capsid protein and inhibition by a distinct resistance mutation. These results suggest that ST-148-enhanced capsid protein self-interaction perturbs assembly and disassembly of DV nucleocapsids, probably by inducing structural rigidity. Thus, as previously reported for other enveloped viruses, stabilization of capsid protein structure is an attractive therapeutic concept that also is applicable to flaviviruses

Real-time imaging of hepatitis C virus infection using a fluorescent cell-based reporter system

Author Manuscript 2010 August 1Hepatitis C virus (HCV), which infects 2–3% of the world population, is a causative agent of chronic hepatitis and the leading indication for liver transplantation1. The ability to propagate HCV in cell culture (HCVcc) is a relatively recent breakthrough and a key tool in the quest for specific antiviral therapeutics. Monitoring HCV infection in culture generally involves bulk population assays, use of genetically modified viruses and/or terminal processing of potentially precious samples. Here we develop a cell-based fluorescent reporter system that allows sensitive distinction of individual HCV-infected cells in live or fixed samples. We demonstrate use of this technology for several previously intractable applications, including live-cell imaging of viral propagation and host response, as well as visualizing infection of primary hepatocyte cultures. Integration of this reporter with modern image-based analysis methods could open new doors for HCV research.New York (State). Dept. of Health (Empire State Stem Cell Fund Contract C023046)United States. Public Health Service (Grant R01 DK56966)National Institutes of Health (U.S.) (Roadmap for Medical Research Grant 1 R01 DK085713-01)Howard Hughes Medical Institute (Investigator