Mutagenesis of the Herpesvirus saimiri genome cloned as a bacterial artificial chromosome and further characterization of an orf51 insertional mutant

Herpesvirus saimiri (HVS) ist ein lymphotropes Virus aus der Familie der Gamma-Herpesviren und gehört zur Untergruppe der Rhadinoviren. In seinem natürlichen Wirt, dem Totenkopfäffchen (Saimiri sciureus), ist es apathogen, verursacht aber in anderen Neuwelt-Primaten rasch wachsende, polyklonale T-Zell-Lymphome. Im Gegensatz zu den Subtypen A und B ist HVS der Subgruppe C – wie auch das hier verwendete Isolat C488 – in der Lage, primäre humane T-Zellen zu einem stabilen, antigen-unabhängigen Wachstum in Zellkultur zu transformieren. In der vorliegenden Arbeit berichte ich über die Transposon-Mutagenese einer transformations-kompetenten Variante des Virusgenoms von HVS C488, vorliegend als Bacterial Artificial Chromosome in E. coli. Dazu wurde mit einem auf einem Tn5-Transposon basierenden System eine Zufalls-Mutagenese durchgeführt. Die hiermit generierte große Anzahl an Mutanten mit jeweils einer Insertion an einer zufälligen Position im Genom wurden geordnet asserviert und mittels eines PCR-Screening-Verfahrens die gewünschte Mutante identifiziert. Exemplarisch soll hier die Inaktivierung des Gens von orf51 (Open Reading Frame 51) durch Insertion beschrieben werden. Es kodiert für ein virales Glykoprotein, das sich zwischen den einzelnen Virus-Untergruppen deutlich unterscheidet und dadurch als möglicher Grund für die phänotypischen Unterschiede in Frage kommt. Beim Humanen Herpesvirus Typ 8 (HHV-8 oder Kaposi’s Sarcoma associated Herpesvirus, KSHV) steht an homologer Stelle das Gen K8.1, welches für ein immunologisch entscheidendes Glykoprotein in der Virushülle kodiert. Auch andere Gamma-Herpesviren besitzen dort entsprechende Gene, wie gp350/220 beim Epstein-Barr-Virus (EBV) oder gp150 beim Murinen Herpesvirus 68 (MHV-68). Wir können zeigen, dass orf51 für ein Glykoprotein mit etwa 55 kDa Größe kodiert, die Expression durch eine Unterbrechung dieses Gens durch Insertion eines Transposon-Elements inaktiviert, nach Rekonstitution mittels homologer Rekombination jedoch wieder aktiviert werden kann. Die Inaktivierung von orf51 bewirkt einen Rückgang der Effizienz der Zell-zu-Zell Ausbreitung der Infektion in Zellkultur um 1 bis 2 Log-Stufen. Deshalb halten wir orf51 für einen entscheidenden aber nicht essentiellen Faktor für die Ausbreitung des Virus von Zelle zu Zelle.Herpesvirus saimiri (HVS), the rhadinovirus prototype, is apathogenic in the persistently infected natural host, the squirrel monkey, but causes acute T-cell lymphoma in other new world primate species. In contrast to subgroups A and B, only strains of HVS subgroup C such as C488 are capable of transforming primary human T cells to stable antigen-independent growth in culture. Here, we report the manipulation of the genome of HVS, available as a bacterial artificial chromosome (BAC) by transposon mutagenesis, demonstrated by the insertional inactivation of the orf51 gene that encodes a virus glycoprotein. The ORF51 glycoprotein is one of only a few genes that are highly divergent and may account for phenotypic differences between viruses of different subgroups. At the homologous genomic location, an immunodominant envelope glycoprotein is encoded by the k8.1 gene of the Kaposi’s sarcoma associated human herpesvirus (KSHV, HHV-8). Other related gamma-herpesviruses also have glycoproteins at the homologous position of their genomes, like gp350/220 in EBV or the gp150 in MHV-68. We show that orf51 encodes a glycoprotein of approximately 55 kDa molecular weight, that interruption of the orf51 by insertion of a Tn5 transposable element is associated with loss of ORF51 glycoprotein expression, and that ORF51 protein expression is reinstituted after repair by homologous recombination. Inactivation of orf51 was associated with decrease of virus titer by 1 to 2 logs, with decreased cell-to-cell spread and plaque size. Thus, orf51 is important for cell-to-cell spread and efficient production of infectious particles but not essential

The Latency-Associated Nuclear Antigen Homolog of Herpesvirus Saimiri Inhibits Lytic Virus Replication

Herpesvirus saimiri (HVS), a T-lymphotropic tumor virus of neotropical primates, and the Kaposi's sarcoma-associated human herpesvirus 8 (KSHV) belong to the gamma-(2)-herpesvirus (Rhadinovirus) subfamily and share numerous features of genome structure and organization. The KSHV latency-associated nuclear antigen (LANA) protein appears to be relevant for viral persistence, latency, and transformation. It binds to DNA, colocalizes with viral episomal DNA, and presumably mediates efficient persistence of viral genomes. LANA further represses the transcriptional and proapoptotic activities of the p53 tumor suppressor protein. Here we report on the ORF73 gene of HVS strain C488, which is the positional and structural homolog of KSHV LANA. The ORF73 gene in OMK cells can encode a 62-kDa protein that localizes to the nucleus in a pattern similar to that of LANA. We show that the ORF73 gene product can regulate viral gene expression by acting as a transcriptional modulator of latent and lytic viral promoters. To define the HVS ORF73 function in the background of a replication-competent virus, we constructed a viral mutant that expresses ORF73 under the transcriptional control of a mifepristone (RU-486)-inducible promoter. The HVS ORF73 gene product efficiently suppresses lytic viral replication in permissive cells, indicating that it defines a critical control point between viral persistence and lytic replication

Improved visualization of peripherally inserted central catheters on chest radiographs of neonates using fractional multiscale image processing

Abstract Background Peripherally inserted central catheters (PICCs) provide secure intravenous access for the delivery of life-sustaining medications and nutrition. They are commonly used in pediatrics. Confirmation of correct central catheter tip position is crucial. Verification is usually done by a radiograph. The aim of this study is to evaluate the ability of Fractional Multiscale image Processing (FMP) to detect PICC tips on the digital chest radiographs of neonates. Methods A total of 94 radiographs of 47 patients were included in the study. 29 patients were male, 18 were female. The mean age of all examined children was 9.2 days (range 0–99 days). In total, six readers (two radiologists, two residents in radiology, one last year medical student, one neonatologist) evaluated 94 unprocessed and catheter-enhanced radiographs using a 5-point Likert scale (1 = poor catheter tip visualization, 5 = excellent catheter tip visualization). Additionally, the two radiologists evaluated the diagnostic confidence for chest pathologies using a 5-point Likert scale (1 = poor diagnostic confidence, 5 = excellent diagnostic confidence). Radiographs were evaluated on a dedicated workstation. Results In all cases, the catheter-enhanced radiograph rated higher than (n = 471), or equal (n = 93) to, the unprocessed radiograph when visualizing catheter tips. 87% of the catheter-enhanced radiographs obtained a rating of 4 or higher, while only 42% of unprocessed radiographs received 4 or more points. Regarding diagnostic confidence for chest pathologies one radiologist rated two catheter-enhanced radiographs higher than the unprocessed radiographs, while all other 186 evaluations rated the catheter-enhanced radiographs equal to (n = 78) or lower than (n = 108) the unprocessed radiographs. Only 60% of the catheter-enhanced radiographs yielded a diagnostic confidence of 4 or higher, while 90% of the unprocessed images received 4 or more points. Conclusion Catheter-enhanced digital chest radiographs demonstrate improved visualization of low contrast PICC tips in neonates compared to unprocessed radiographs. Furthermore, they enable detection of accompanying chest pathologies. However, definitive diagnosis of chest pathologies should be made on unprocessed radiographs

“Multisystem Inflammatory Syndrome in Children” (MIS-C) after COVID-19 Infection in the Metropolitan Area of Nuremberg-Erlangen, Germany—Expectations and Results of a Two-Year Period

Background: Multisystemic Inflammatory Syndrome in children (MIS-C) is a rare autoimmune disorder occurring after a latency period following acute SARS-CoV-2 infection. The therapeutic regime of MIS-C is adapted to the therapy of the Kawasaki disease, as clinical symptoms are similar. Since the Kawasaki disease can potentially result in severe symptoms, which may even affect long-term health, it is essential to gain further knowledge about MIS-C. Thus, we aimed to investigate the incidence, symptoms, therapeutical procedure and outcome of MIS-C patients in the metropolitan area of Nuremberg-Erlangen during the SARS-CoV2 pandemic. Material and Methods: Retrospective analysis of clinical charts of MIS-C patients was carried out at three children’s hospitals covering the medical care of the metropolitan area of Nuremberg-Erlangen in Germany. Demographic characteristics and symptoms at first visit, their clinical course, therapeutic regime and outcome were recorded within the time period January 2021–December 2022. Results: Analysis of 10 patients (5 male, 5 female) with MIS-C resulting in an incidence of 2.14/100.000 children. The median time between COVID-19 infection and admission to hospital was 5 weeks. The median age was 7 years. Symptoms comprised fever (100%), rash (70%), bilateral non-purulent conjunctivitis (70%) and urticaria (20%). At the time of presentation, diagnosis-defining inflammation parameters were increased and the range for C-reactive protein was 4.13 mg/dL to 28 mg/dL, with a median of 24.7 mg/dL. Procalcitonin was initially determined in six patients (1.92 ng/mL to 21.5 ng/mL) with a median value of 5.5 pg/mL. Two patients displayed leukocytosis and two displayed leukopenia. None of the patients presented coronary pathologies. Nine of the ten patients received intravenous immunoglobulin (IVIG) therapy. In addition, patients received intravenous steroids (80%) and acetylsalicylic acid (80%). Conclusion: SARS-CoV virus may rarely exert multiorgan manifestations due to hyperinflammatory immunological processes. Within two years of the COVID-19 pandemic, we identified ten patients with COVID-induced MIS-C in the metropolitan area Nuremberg-Erlangen. In the description of the patient collective, we can confirm that MIS-C is distinguished from the Kawasaki disease by the lack of coronary manifestations. Interestingly, although having monitored all pediatric facilities in the investigated area, we find lower incidences of MIS-C compared to findings in the literature. In conclusion, an overestimation of incidences in the upcoming MIS-C during the pandemic needs to be considered