A live cell reporter of human adenovirus chromatin

Die Untersuchung von Viren hat substanziell zur biologischen Revolution im letzten Jahrhundert beigetragen. Die Analyse von Virus-Wirt Interaktionen hat viele zelluläre Prinzipien offenbart wie zum Beispiel die Gesetzmässigkeiten von Genen und deren Spleissung. Ein oft benutztes Modelsystem sind Adenoviren. Dies, weil sie nur beschränkt krankmachend, einfach zu vermehren und gut charakterisiert sind. Trotzdem sind die zugrundeliegenden zellulären Mechanismen, welche eine Infektion ermöglichen, immer noch nicht gut verstanden. Insbesondere Prozesse die zum Abbau viraler Kapside während des Imports führen und wie Viren die Zelle verlassen, war bisher schwierig zu untersuchen. In dieser Arbeit haben wir das Chromatin eines humanen Adenovirus fluoreszent markiert, indem wir das Protein V gegen das fluoreszierende Fusionsprotein GFP-pV ausgetauscht haben, ohne dabei andere virale Gene zu zerstören. Dieses Virus, genannt Ad2-GFP-pV, durchlief den vollen Replikationszyklus und erlaubte es uns daher, viralen Transport und Freisetzung von Viren am Ende des Zyklus zu untersuchen. Wir konnten zeigen, dass während des Eintritts des Kapsids in die Zellen das GFP-pV in zwei Schritten dissoziiert. Innerhalb von 30 Minuten wurden 65% vom GFP-pV ins Zytosol abgegeben und nach 90 Minuten das restliche Protein. Der zweite Schritt war dabei abhängig von einer Interaktion der Viren mit dem Zellkern. Im Weiteren ist es uns gelungen, zum ersten Mal die Dynamik der Virenfreisetzung am Ende des viralen Replikationszykluses zu bestimmen. Wir konnten zeigen, dass dieser Prozess anisotrop ist und in zwei Schritten geschieht. Einem Zerfall der Kernmembran folgt die Lyse der Plasmamembran. Zusammenfassend konnten wir zeigen, dass dieses neue Virus ermöglicht, den vollständigen Replikationszyklus von Adenoviren zu untersuchen. Es hat das Potential, Echtzeituntersuchungen von Infektionen und deren Ausbreitung in Organismen zu machen oder als diagnostisches Virus in der Gentherapie eingesetzt zu werden. SUMMARY Virology has contributed substantially to the biological revolution in the last century. Studying virus-host-interactions has revealed many of the fundamental molecular mechanisms such as the nature of genes and splicing. Adenoviruses have received intense attention in this regard as they are of low pathogenicity, easy to grow and rather well characterized. Still, biological mechanisms enabling adenovirus infections are not completely understood. In particular, processes that lead to disassembly of viral particles during entry as well as release of newly synthesized particles upon cell lysis at the end of the viral replication cycle have been difficult to address. Here we generated a novel human adenovirus with fluorescently tagged chromatin by replacing protein V by GFP-pV while keeping all the other viral genes intact. This virus, named Ad2-GFP-pV, completed the full replication cycle and therefore allowed us to study transport of incoming viral cores as well as viral egress from infected cells. We showed that the viral core component GFP-pV is released from Ad2-GFP- pV during entry and disassembly in two distinct steps. A fast one within 30 min where about 65% of GFP-pV is released and a slower up to 90 min pi leading to full dissociation of GFP-pV from viral particles. The full dissociation is dependent on an interaction of the incoming virus with the nucleus. Using this virus we were also able for the first time to visualize dynamics of viral egress upon cell lysis. We observed that this process is nonisotropic and occurs in two morphologically distinct steps, nuclear disintegration preceding lysis of the plasma membrane. Taken together we showed that this new virus is suitable to study the whole viral replication cycle and thus might prove useful for live analyses of viral infection and spreading in organisms, as well as for applications in gene therapy

A multicenter evaluation of a point of care CRP Test

Point-of-care (POC) C-reactive protein (CRP) testing in the primary healthcare setting is a cost-effective approach for reducing antibiotic prescriptions, but has yet to be widely adopted. Analytical performance of the cobas CRP Test on the cobas b 101 system was evaluated at three POC sites and one reference laboratory. Within-run (repeatability), within-laboratory (intermediate precision), and between-laboratory precision (reproducibility) were assessed. Method comparison (reference test: CRPNX reagent [cobas c 501 module]) and matrix/lot-to-lot comparison experiments were conducted using prospectively collected blood samples from 217 adults (apparently healthy or with clinically relevant conditions). Usability and reliability were assessed by questionnaire and error reporting. Coefficients of variation (CV) for repeatability and intermediate precision ranged from 1.7%-4.0% and 1.9%-4.5%, respectively, for human serum pools containing CRP 4.7-350.7 mg/L; repeatability in clinical samples ranged from 1.6%-5.9% (3.3-360.3 mg/L). CVs for reproducibility ranged from 2.5%-4.0% (4.7-344.3 mg/L). CRP concentrations were comparable for capillary whole blood, serum, Li-heparin whole blood/plasma, K2 and K3 EDTA whole blood/plasma (Pearson's r ≥ 0.996), and among three CRP Test lots (r ≥ 0.993). Clinically relevant CRP concentrations measured with the CRP Test showed good agreement with those measured by CRPNX reagent (serum, weighted Deming regression y = 0.97× + 0.11; Pearson's r ≥ 0.996). The overall mean usability score was 4.18/5 and the error rate across 9378 tests was 1.00%. The cobas CRP Test on the cobas b 101 system demonstrates robust analytic performance when used by healthcare professionals in the POC setting

A multicenter evaluation of a point of care CRP Test

Background: Point-of-care (POC) C-reactive protein (CRP) testing in the primary healthcare setting is a cost-effective approach for reducing antibiotic prescriptions, but has yet to be widely adopted. Methods: Analytical performance of the cobas CRP Test on the cobas b 101 system was evaluated at three POC sites and one reference laboratory. Within-run (repeatability), within-laboratory (intermediate precision), and between-laboratory precision (reproducibility) were assessed. Method comparison (reference test: CRPNX reagent [cobas c 501 module]) and matrix/lot-to-lot comparison experiments were conducted using prospectively collected blood samples from 217 adults (apparently healthy or with clinically relevant conditions). Usability and reliability were assessed by questionnaire and error reporting. Results: Coefficients of variation (CV) for repeatability and intermediate precision ranged from 1.7%–4.0% and 1.9%–4.5%, respectively, for human serum pools containing CRP 4.7–350.7 mg/L; repeatability in clinical samples ranged from 1.6%–5.9% (3.3–360.3 mg/L). CVs for reproducibility ranged from 2.5%–4.0% (4.7–344.3 mg/L). CRP concentrations were comparable for capillary whole blood, serum, Li-heparin whole blood/plasma, K2 and K3 EDTA whole blood/plasma (Pearson's r ≥ 0.996), and among three CRP Test lots (r ≥ 0.993). Clinically relevant CRP concentrations measured with the CRP Test showed good agreement with those measured by CRPNX reagent (serum, weighted Deming regression y = 0.97× + 0.11; Pearson's r ≥ 0.996). The overall mean usability score was 4.18/5 and the error rate across 9378 tests was 1.00%. Conclusions: The cobas CRP Test on the cobas b 101 system demonstrates robust analytic performance when used by healthcare professionals in the POC setting

A multicenter evaluation of a point of care CRP Test

\u3cp\u3eBackground: Point-of-care (POC) C-reactive protein (CRP) testing in the primary healthcare setting is a cost-effective approach for reducing antibiotic prescriptions, but has yet to be widely adopted. Methods: Analytical performance of the cobas CRP Test on the cobas b 101 system was evaluated at three POC sites and one reference laboratory. Within-run (repeatability), within-laboratory (intermediate precision), and between-laboratory precision (reproducibility) were assessed. Method comparison (reference test: CRPNX reagent [cobas c 501 module]) and matrix/lot-to-lot comparison experiments were conducted using prospectively collected blood samples from 217 adults (apparently healthy or with clinically relevant conditions). Usability and reliability were assessed by questionnaire and error reporting. Results: Coefficients of variation (CV) for repeatability and intermediate precision ranged from 1.7%–4.0% and 1.9%–4.5%, respectively, for human serum pools containing CRP 4.7–350.7 mg/L; repeatability in clinical samples ranged from 1.6%–5.9% (3.3–360.3 mg/L). CVs for reproducibility ranged from 2.5%–4.0% (4.7–344.3 mg/L). CRP concentrations were comparable for capillary whole blood, serum, Li-heparin whole blood/plasma, K2 and K3 EDTA whole blood/plasma (Pearson's r ≥ 0.996), and among three CRP Test lots (r ≥ 0.993). Clinically relevant CRP concentrations measured with the CRP Test showed good agreement with those measured by CRPNX reagent (serum, weighted Deming regression y = 0.97× + 0.11; Pearson's r ≥ 0.996). The overall mean usability score was 4.18/5 and the error rate across 9378 tests was 1.00%. Conclusions: The cobas CRP Test on the cobas b 101 system demonstrates robust analytic performance when used by healthcare professionals in the POC setting.\u3c/p\u3