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The emergence of SARS-CoV-2 in 2019 led to a severe pandemic situation. Treatment options are limited and the efficacy of vaccines decreases due to mutations in SARS-CoV-2 strains. Therefore, new treatment options are urgently needed, and computational compound screenings are used to predict drugs quickly. One of these screenings revealed farnesyltransferase inhibitors (FTIs) as potential candidates. We demonstrated that the FTIs lonafarnib and tipifarnib have an impact on SARS-CoV-2 Wildtype and the Delta variant. Both FTIs dose-dependently reduced morphological changes and the formation of cytopathic effects in SARS-CoV-2 infected Calu-3 cells.

Purpose Omicron is rapidly spreading as a new SARS-CoV-2 variant of concern (VOC). The question whether this new variant has an impact on SARS-CoV-2 rapid antigen test (RAT) performance is of utmost importance. To obtain an initial estimate regarding differences of RATs in detecting omicron and delta, seven commonly used SARS-CoV-2 RATs from different manufacturers were analysed using cell culture supernatants and clinical specimens. Methods For this purpose, cell culture-expanded omicron and delta preparations were serially diluted in Dulbecco’s modified Eagle’s Medium (DMEM) and the Limit of Detection (LoD) for both VOCs was determined. Additionally, clinical specimens stored in viral transport media or saline (n = 51) were investigated to complement in vitro results with cell culture supernatants. Ct values and RNA concentrations were determined via quantitative reverse transcription polymerase chain reaction (RT-qPCR). Results The in vitro determination of the LoD showed no obvious differences in detection of omicron and delta for the RATs examined. The LoD in this study was at a dilution level of 1:1,000 (corresponding to 3.0—5.6 × 106 RNA copies/mL) for tests I–V and at a dilution level of 1:100 (corresponding to 3.7—4.9 × 107 RNA copies/mL) for tests VI and VII. Based on clinical specimens, no obvious differences were observed between RAT positivity rates when comparing omicron to delta in this study setting. Overall positivity rates varied between manufacturers with 30–81% for omicron and 42–71% for delta. Test VII was only conducted in vitro with cell culture supernatants for feasibility reasons. In the range of Ct Conclusion In this study, RATs from various manufacturers were investigated, which displayed no obvious differences in terms of analytical LoD in vitro and RAT positivity rates based on clinical samples comparing the VOCs omicron and delta. However, differences between tests produced by various manufacturers were detected. In terms of clinical samples, a focus of this study was on specimens with high virus concentrations. Further systematic, clinical and laboratory studies utilizing large datasets are urgently needed to confirm reliable performance in terms of sensitivity and specificity for all individual RATs and SARS-CoV-2 variants.

Rapid antigen tests (RATs) are an integral part of SARS-CoV-2 containment strategies. As emerging variants of concern (VOCs) displace the initially circulating strains, it is crucial that RATs do not fail to detect these new variants. In this study, four RATs for nasal swab testing were investigated using cultured strains of B.1.1 (non-VOC), B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma), and B.1.617.2 (Delta). Based on dilution series in cell culture medium and pooled saliva, the limit of detection of these RATs was determined in a laboratory setting. Further investigations on cross-reactivity were conducted using recombinant N-protein from seasonal human coronaviruses (hCoVs). RATs evaluated showed an overall comparable performance with cultured strains of the non-VOC B.1.1 and the VOCs Alpha, Beta, Gamma, and Delta. No cross-reactivity was detected with recombinant N-protein of the hCoV strains HKU1, OC43, NL63, and 229E. A continuous evaluation of SARS-CoV-2 RAT performance is required, especially with regard to evolving mutations. Moreover, cross-reactivity and interference with pathogens and other substances on the test performance of RATs should be consistently investigated to ensure suitability in the context of SARS-CoV-2 containment.

A versatile portfolio of diagnostic tests is essential for the containment of the severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) pandemic. Besides nucleic acid-based test systems and point-of-care (POCT) antigen (Ag) tests, quantitative, laboratory-based nucleocapsid Ag tests for SARS-CoV-2 have recently been launched. Here, we evaluated four commercial Ag tests on automated platforms and one POCT to detect SARS-CoV-2. We evaluated PCR-positive (n = 107) and PCR-negative (n = 303) respiratory swabs from asymptomatic and symptomatic patients at the end of the second pandemic wave in Germany (February–March 2021) as well as clinical isolates EU1 (B.1.117), variant of concern (VOC) Alpha (B.1.1.7) or Beta (B.1.351), which had been expanded in a biosafety level 3 laboratory. The specificities of automated SARS-CoV-2 Ag tests ranged between 97.0 and 99.7% (Lumipulse G SARS-CoV-2 Ag (Fujirebio): 97.03%, Elecsys SARS-CoV-2 Ag (Roche Diagnostics): 97.69%; LIAISON® SARS-CoV-2 Ag (Diasorin) and SARS-CoV-2 Ag ELISA (Euroimmun): 99.67%). In this study cohort of hospitalized patients, the clinical sensitivities of tests were low, ranging from 17.76 to 52.34%, and analytical sensitivities ranged from 420,000 to 25,000,000 Geq/ml. In comparison, the detection limit of the Roche Rapid Ag Test (RAT) was 9,300,000 Geq/ml, detecting 23.58% of respiratory samples. Receiver-operating-characteristics (ROCs) and Youden’s index analyses were performed to further characterize the assays’ overall performance and determine optimal assay cutoffs for sensitivity and specificity. VOCs carrying up to four amino acid mutations in nucleocapsid were detected by all five assays with characteristics comparable to non-VOCs. In summary, automated, quantitative SARS-CoV-2 Ag tests show variable performance and are not necessarily superior to a standard POCT. The efficacy of any alternative testing strategies to complement nucleic acid-based assays must be carefully evaluated by independent laboratories prior to widespread implementation.

Hepatitis E is the major enterically transmitted viral hepatitis after hepatitis A. There are four known genotypes (GT) of the hepatitis E virus (HEV) affecting human beings. HEV GT1 causes outbreaks of acute hepatitis in developing countries of Africa and Asia, whereas HEV GT3 has been recognized as an emergent pathogen in industrialized countries characterized by zoonotic transmission and the possible development of chronic hepatitis in immunocompromised individuals. The aim of this work was to evaluate if the major antigen of HEV, the ORF2 capsid protein, can be used in combination with immunobiotic bacterium like particles (IBLP) for oral vaccination in a mouse model. We have cloned and expressed the RGS-His5-tagged HEV GT 3 capsid protein (ORF2) in E. coli and purified it by NiNTA under native and denaturing conditions. IBLP were prepared from two immunobiotic Lactobacillus rhamnosus strains by heat and acid treatments. Two different doses of protein were combined with IBLP and orally administered to Balb/c mice. After three oral immunizations separated for 14-day intervals, blood, intestinal fluid, Peyer´s patches and spleen samples were drawn. IgA and IgG specific antibodies were determined by ELISA. Mononuclear cell populations were analyzed by flow cytometry, and the cytokine profiles of isolated cells stimulated with ORF2 were determined by ELISA on the supernatants.Orally administered recombinant ORF2 protein combined with IBLP from two L. rhamnosus strains (CRL1505 and IBL027) induced both antigen specific humoral and cellular immune responses in mice. Interestingly, the adjuvant particles showed different behaviors, being BLP027 more effective inducing specific secretory IgA in the gut lumen. IFN-ɣ, TNF-ɑ and IL-4 were produced by Peyer?s plaques lymphocytes stimulated with the capsid antigen ex vivo suggesting a mixed Th1/Th2-type adaptive immune response in immunized mice.Oral vaccines are attractive because they are not invasive, do not need to be administered by specialized personal and, most important, elicit both systemic and local immune responses at the port of entry of the pathogens. Here we present a promising experimental oral vaccine for HEV genotype 3, which could be further developed for human and/or veterinary use. Fil: Arce, Lorena Paola. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina. Universidad Nacional de Tucumán. Facultad de Medicina. Departamento Biomédico. Cátedra de Orientación Genética; Argentina Fil: Raya Tonetti, María Fernanda. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina. Universidad Nacional de Tucumán. Facultad de Medicina. Departamento Biomédico. Cátedra de Orientación Genética; Argentina Fil: Raimondo, María Paula. Universidad Nacional de Tucumán. Facultad de Medicina. Departamento Biomédico. Cátedra de Orientación Genética; Argentina Fil: Müller, Melisa Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina. Universidad Nacional de Tucumán. Facultad de Medicina. Departamento Biomédico. Cátedra de Orientación Genética; Argentina Fil: Salva, Maria Susana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina Fil: Alvarez, Gladis Susana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina Fil: Baiker, Armin. Bavarian Health and Food Safety Authority; Alemania Fil: Villena, Julio Cesar. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina Fil: Vizoso Pinto, María Guadalupe. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina. Universidad Nacional de Tucumán. Facultad de Medicina. Departamento Biomédico. Cátedra de Orientación Genética; Argentina

For the analytical surveillance of genetic engineering operations suitable methods for characterization of used (recombinant) cell lines and used transfer vector plasmids are needed. The purpose of the described method is the identification of retroviral DNA-isoforms based on lenti-or gammaretrovirus (HIV-1 or MLV). The primers, used in an optimized touch-down PCR protocol, specifically amplify transfer-vector encoded vector genomes and proviruses integrated into stably transduced vertebrate cell lines, distinguishing them from the background of endogenous retroviral sequences. The resulting PCR products were isolated by preparative agarose electrophoresis and subsequently used for sequence identification by Sanger sequencing. In addition, the amplification and sequencing of PCR products corresponding to the HIV-1- and MLV-based vector-genomes was successful from residual transfer vector plasmids in cell culture supernatants and basic polyethylene glycol-precipitated vector preparations. The methods were validated using 19 samples (cell lines, plasmids and viral vector preparations). No false-positive PCR results were observed, while both retroviral DNA-isoforms were detected with the respective assay. The successful implementation in routine analysis is presented using two routine samples.

Several commercial and novel synthetic β-carbolines (βCs) were evaluated for their antiviral activity against herpes simplex virus type 1 (HSV-1) using an adapted MTS assay. Of 21 drugs tested, although 11 exerted antiviral activity at non-cytotoxic concentrations, only 3 of them [9-methyl-norharmane (9-Me-nHo), 9-methyl-harmane (9-Me-Ho) and 6-methoxy-harmane (6-MeO-Ho)] completely avoided virus-driven cytopathic effects. Half-maximal effective concentrations (EC 50 values) (4.9 ± 0.4, 5.9 ± 0.8 and 19.5 ± 0.3 µM, respectively) and selectivity indexes (88.8, 40.2 and 7.0, respectively) of the latter three βCs against HSV-1 were determined by MTS, flow cytometry and plaque reduction assays. The mode of action of these drugs was also evaluated. According to time-of-addition assays, the selected compounds were not virucidal and did not interfere with attachment or penetration of HSV-1, but interfered with later events of virus infection. Western blot studies showed that early and late protein expression was significantly delayed or even suppressed. Herpes simplex virus type 2 (HSV-2) was also inhibited by the selected substances in a similar manner. Interestingly, 6-MeO-Ho, 9-Me-Ho and 9-Me-nHo restricted HSV-1 ICP0 localisation to the nucleus during later stages of infection, possibly affecting its functionality in the cytoplasm where ICP0 normally inhibits antiviral signalling and promotes viral replication. In silico prediction of ADME (Absorption, Distribution, Metabolism and Excretion) properties showed that all compounds fulfilled Lipinski's rule and their calculated absorptions were >95%. Fil: Gonzalez, Maria Micaela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentina. Ludwig Maximilians Universitat; Alemania Fil: Cabrerizo, Franco Martín. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentina Fil: Baiker, Armin. Bavarian Health and Food Safety Authority; Alemania Fil: Erra Balsells, Rosa. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Centro de Investigaciones en Hidratos de Carbono. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Centro de Investigaciones en Hidratos de Carbono; Argentina Fil: Osterman, Andreas. Ludwig Maximilians Universitat; Alemania. German Center for Infection Research; Alemania Fil: Nitschko, Hans. Ludwig Maximilians Universitat; Alemania. German Center for Infection Research; Alemania Fil: Vizoso Pinto, María Guadalupe. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina. Universidad Nacional de Tucumán. Facultad de Medicina. Departamento Biomédico; Argentina. Ludwig Maximilians Universitat; Alemania

Murine leukemia virus (MLV) and murine stem cell virus (MSCV) and derived retroviral vectors are widely used to study retrovirus biology and as tools for gene delivery. The method described here represents a quantitative real time PCR (qPCR) with hydrolysis probe that can be applied within classical qPCR as well as in digital droplet PCR (ddPCR). The method targets a 60 bp long fragment located within the U5 region of the MLV/MSCV genome sequence. For the here described method a LOD95% of 25 copies per PCR reaction (DNA) and 80 copies per PCR reaction (RNA) was determined, and PCR efficiencies of 92.5 % and 98.5 %, respectively, were observed. This method enables the fast and simple titration of viral genomic RNA present in retroviral vector stocks for accurate and consistent transduction experiments. Furthermore, it enables the detection of proviral and transfer plasmid derived DNA sequences and can be modified to differentiate between retroviral RNA and DNA.

Die Synthetische Biologie (SynBio) ist ein interdisziplinares Forschungs- und Anwendungsgebiet, welches ein breites Spektrum an Naturwissenschaften zusammenzufuhrt und dabei ingenieurwissenschaftliche Prinzipien verfolgt. Ein wichtiges Merkmal der SynBio ist, dass sie biologische Systeme wesentlich verandert und zum Teil auch mit chemisch synthetisierten Komponenten so kombiniert, wie dies bei naturlich vorkommenden Organismen nicht der Fall ist. Die Entwicklung der SynBio korreliert mit den Entwicklungen der Gentechnik. Da aber die beiden Gebiete nicht strikt voneinander abzugrenzen sind, kann die SynBio als eine Weiterentwicklung der „klassischen“ Gentechnik verstanden werden.

Das Bayerische Landesamt fur Gesundheit und Lebensmittelsicherheit (LGL) ist als zentrale Gentechnik-Fachbehorde des Freistaats Bayern zustandig fur die analytische Uberwachung gentechnischer Arbeiten in gentechnischen Anlagen. In diesem Zusammenhang entnimmt und untersucht das LGL Proben aus gentechnischen Anlagen. Die Schwerpunkte der Untersuchungen sind dabei unter anderem die Uberprufung von Sicherheit und Hygiene am Arbeitsplatz, die Uberprufung der Einschliesungsmasnahmen und die Uberprufung der Konformitat der gentechnischen Arbeiten mit den Zulassungsbescheiden. Die Herstellung und Anwendung viraler Vektoren zahlt dabei zu den haufig durchgefuhrten gentechnische Arbeiten. Die fur den Gentransfer eingesetzten viralen Vektoren werden dabei in der Regel in die Risikogruppen 1 oder 2 (gemas EU-Richtlinie 2000/54/EG) eingestuft. Um eine analytische Uberwachung dieser Arbeiten zu ermoglichen, entwickelt das LGL Strategien zum Nachweis haufig genutzter viraler Vektorsysteme zur Identifizierung der darin enthaltenen Transgene. Diese Strategien beinhalten derzeit Methoden zum Nachweis von Nukleinsauren der viralen Vektoren mittels real-time PCR, Methoden zum Nachweis viraler Vektorpartikel mittels Antigen-ELISA und Methoden zur Transgenidentifizierung mittels Endpunkt-PCR und Sequenzierung. Der Grosteil der angewendeten molekularbiologischen Methoden wurde dabei im Rahmen von Forschungsprojekten am LGL entwickelt. Die am LGL eingesetzten Antigen-ELISAs werden von kommerziellen Anbietern bezogen. In diesem Artikel werden die derzeit am LGL angewendeten Strategien zum Nachweis und der Identifizierung HIV-1-basierter lentiviraler Vektoren, AAV2-basierter Vektoren und adenoviraler Vektoren der ersten Generation vorgestellt.

The purpose of the described method is the detection of and differentiation between RNA and DNA of human immunodeficiency virus (HIV)-derived lentiviral vectors (LV) in cell culture supernatants and swab samples. For the analytical surveillance of genetic engineering, operations methods for the detection of the HIV-1-based LV generations are required. Furthermore, for research issues, it is important to prove the absence of LV particles for downgrading experimental settings in terms of the biosafety level. Here, a quantitative polymerase chain reaction method targeting the long terminal repeat U5 subunit and the start sequence of the packaging signal ψ is described. Numerous controls are included in order to monitor the technical procedure.

The purpose of the described method is the detection of and differentiation between RNA and DNA of HIV-derived lentiviral vectors (LV) in cell culture supernatants and swab samples. For the analyti...

The production and application of viral vectors are frequently performed genetic engineering operations. HIV-1-based lentiviral vectors, AAV2-based, and adenoviral vectors are amongst the most abundant viral vectors utilized for gene delivery. They are generally classified into risk group 1 or 2 (according to EU directive 2000/54/EC on the protection of workers from risks related to exposure to biological agents at work).

INTRODUCTION In contrast to varicella zoster virus (VZV) primary infection, VZV vaccination does not seem to provide lifelong immunity against varicella. Because more people get vaccinated every year, the development of sensitive serological test systems for the detection of protective anti-VZV IgG will become important in the future. METHODS We have previously developed a novel VZV line assay based on 5 different recombinant VZV antigens. In this study, we compared this novel assay with a commercially available glycoprotein enzyme immunoassay (RIDASCREEN VZV IgG) in detecting anti-VZV IgG of children with previous varicella infection and VZV vaccination. RESULTS One hundred twenty-five children were included in this study, 72 with a history of varicella infection and 53 with VZV vaccination. Both assays detected anti-VZV IgG antibodies in both study groups with similar sensitivities. The VZV line assay revealed striking differences in the anti-VZV IgG composition against the VZV open reading frames, 4, 14 and 49, between both study groups, indicating that wild-type varicella infection causes a more diverse immune response against VZV than does vaccination. The exploitation of these results enabled the discrimination of both study groups with a sensitivity of 0.93 and a specificity of 0.83, indicating that the serologic differentiation of children with previous varicella infection and VZV vaccination might be possible. CONCLUSION The VZV line assay enables the detection of anti-VZV IgG with sensitivities comparable to glycoprotein enzyme immunoassays and might be suitable for the serologic discrimination between children with a history of varicella infection and VZV vaccination.

Hepatitis E Virus (HEV) is an emerging virus causing epidemic acute hepatitis in developing countries as well as sporadic cases in industrialized countries. The life cycle of HEV is still poorly understood and the lack of efficient cell culture systems and animal models are the principal limitations for a detailed study of the viral replication cycle. Here we exhaustively examine all possible intraviral protein-protein interactions (PPIs) of HEV by systematic Yeast two-hybrid (Y2H) and LuMPIS screens, providing a basis for studying the function of these proteins in the viral replication cycle. Key PPIs correlate with the already published HEV 3D structure. Furthermore, we report 20 novel PPIs including the homodimerization of the RNA dependent RNA polymerase (RdRp), the self-interaction of the papain like protease, and ORF3 interactions with the papain-like protease and putative replicase components: RdRp, methylase and helicase. Furthermore, we determined the dissociation constant (Kd) of ORF3 interactions with the viral helicase, papain-like protease and methylase, which suggest a regulatory function for ORF3 in orchestrating the formation of the replicase complex. These interactions may represent new targets for antiviral drugs. Fil: Osterman, Andreas. Ludwig Maximilians Universitat. Max Von Pettenkofer Institute; Alemania Fil: Stellberger, Thorsten. Bavarian Health And Food Safety Authority (lgl); Alemania Fil: Gebhardt, Anna. Ludwig Maximilians Universitat. Max Von Pettenkofer Institute; Alemania Fil: Kurz, M.arisa. Ludwig Maximilians Universitat. Max Von Pettenkofer Institute; Alemania Fil: Friedel, Caroline C.. Ludwig Maximilians Universitat; Alemania Fil: Uetz, Peter. Center For The Study Of Biological Complexity, Vcu, Usa; Estados Unidos Fil: Nitschko, Hans. Ludwig Maximilians Universitat. Max Von Pettenkofer Institute; Alemania Fil: Baiker, Armin. Bavarian Health And Food Safety Authority (lgl); Alemania Fil: Vizoso Pinto, María Guadalupe. Ludwig Maximilians Universitat. Max Von Pettenkofer Institute; Alemania. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina

The immediate-early 63-kDa (IE63) protein of varicella-zoster virus (VZV) is a phosphoprotein encoded by open reading frame (ORF) ORF63/ORF70. To identify functional domains, 22 ORF63 mutations were evaluated for effects on IE63 binding to the major VZV transactivator, IE62, and on IE63 phosphorylation and nuclear localization in transient transfections, and after insertion into the viral genome with VZV cosmids. The IE62 binding site was mapped to IE63 amino acids 55 to 67, with R59/L60 being critical residues. Alanine substitutions within the IE63 center region showed that S165, S173, and S185 were phosphorylated by cellular kinases. Four mutations that changed two putative nuclear localization signal (NLS) sequences altered IE63 distribution to a cytoplasmic/nuclear pattern. Only three of 22 mutations in ORF63 were compatible with recovery of infectious VZV from our cosmids, but infectivity was restored by inserting intact ORF63 into each mutated cosmid. The viable IE63 mutants had a single alanine substitution, altering T171, S181, or S185. These mutants, rOKA/ORF63rev[T171], rOKA/ORF63rev[S181], and rOKA/ORF63rev[S185], produced less infectious virus and had a decreased plaque phenotype in vitro. ORF47 kinase protein and glycoprotein E (gE) synthesis was reduced, indicating that IE63 contributed to optimal expression of early and late gene products. The three IE63 mutants replicated in skin xenografts in the SCIDhu mouse model, but virulence was markedly attenuated. In contrast, infectivity in T-cell xenografts was not altered. Comparative analysis suggested that IE63 resembled the herpes simplex virus type 1 U S 1.5 protein, which is expressed colinearly with ICP22 (U S 1). In summary, most mutations of ORF63 made with our VZV cosmid system were lethal for infectivity. The few IE63 changes that were tolerated resulted in VZV mutants with an impaired capacity to replicate in vitro. However, the IE63 mutants were attenuated in skin but not T cells in vivo, indicating that the contribution of the IE63 tegument/regulatory protein to VZV pathogenesis depends upon the differentiated human cell type which is targeted for infection within the intact tissue microenvironment.

Declining learning and memory function is associated with the attenuation of adult hippocampal neurogenesis. As in humans, chronic stress or depression in animals is accompanied by hippocampal dysfunction, and neurogenesis is correspondingly down regulated, in part, by the activity of the hypothalamic-pituitary-adrenal axis as well as glutamatergic and serotonergic networks. Antidepressants can reverse this effect over time but one of the most clinically effective moderators of stress or depression and robust stimulators of neurogenesis is simple voluntary physical exercise such as running. Curiously, running also elevates circulating stress hormone levels yet neurogenesis is doubled in running animals. In evaluating the signalling that running provides to the central nervous system in mice, we have found that peripheral vascular endothelial growth factor (VEGF) is necessary for the effects of running on adult hippocampal neurogenesis. Peripheral blockade of VEGF abolished running-induced neurogenesis but had no detectable effect on baseline neurogenesis in non-running animals. These data suggest that VEGF is an important element of a 'somatic regulator' of adult neurogenesis and that these somatic signalling networks can function independently of the central regulatory networks that are typically considered in the context of hippocampal neurogenesis.

Background Virus-host interactions result in altered gene expression profiles in host cell nuclei and enable virus particle production, thus obligatorily involving changes in their epigenomes. Neither such epigenome changes nor their response to antiviral treatment have been extensively studied to date, although viral infections are known to contribute to the long-term development of severe secondary diseases, for example, hepatocellular carcinoma. This may be causally linked to virus-induced persistent plastic chromatin deformations. Results We studied whether impaired hepatitis B virus (HBV) replication can lead to the restitution of epigenome signatures hypothesizing that hepatocytes alternatively could adopt a ‘memory’ state of the infection; that is, the chromatin could persist in a HBV-induced configuration potentially inheritable between dividing hepatocytes. We therefore determined epigenomic signatures and gene expression changes altered by HBV and the effects of suppressed HBV replication in nontransformed hepatocytes of newborn mice. Further we investigated differential histone acetyltransferase and histone deacetylase activities in HBV-negative and HBVpositive hepatocytes, as well as the effects of HBV suppression on gene expression and the chromatin landscape. We show that the expression of several genes and the chromatin landscape become altered upon HBV infection, including global hypoacetylation of H2A.Z and H3K9. Reporter assays monitoring the activities of histone acetyltransferases or histone deacetylases, respectively, suggest that hypoacetylation most probably depends on elevated sirtuin deacetylase activity, but not on class I/II histone deacetylases. Using Micrococcus nuclease to study the chromatin accessibility in met murine-D3 and hepatitis B virus met murine hepatocytes, we demonstrate that the observed differences in H2A.Z/H3K9 acetylation lead to global chromatin structure changes. At all selected sites examined by chromatin immunoprecipitation and quantitative real-time PCR, these effects can be partly restituted via the nucleoside analog reverse transcriptase inhibitor 3TC or using anti-HBV microRNA-like molecules. Conclusions Increased sirtuin activity might lead to global histone hypoacetylation signatures, which could contribute to the HBV-induced pathomechanism in nontransformed hepatocytes. Using several techniques to suppress HBV replication, we observed restituted gene expression and chromatin signature patterns reminiscent of noninfected hepatocytes. Importantly, ectopic expression of antiviral short-hairpin RNA, but not microRNA-like molecules, provoked intolerable off-target effects on the gene expression level. Electronic supplementary material The online version of this article (doi:10.1186/1868-7083-6-26) contains supplementary material, which is available to authorized users.

Varicella-zoster virus (VZV) open reading frame 63 (ORF63), located between nucleotides 110581 and 111417 in the internal repeat region, encodes a nuclear phosphoprotein which is homologous to herpes simplex virus type 1 (HSV-1) ICP22 and is duplicated in the terminal repeat region as ORF70 (nucleotides 118480 to 119316). We evaluated the role of ORFs 63 and 70 in VZV replication, using recombinant VZV cosmids and PCR-based mutagenesis to make single and dual deletions of these ORFs. VZV was recovered within 8 to 10 days when cosmids with single deletions were transfected into melanoma cells along with the three intact VZV cosmids. In contrast, VZV was not detected in transfections carried out with a dual deletion cosmid. Infectious virus was recovered when ORF63 was cloned into a nonnative Avr II site in this cosmid, confirming that failure to generate virus was due to the dual ORF63/70 deletion and that replication required at least one gene copy. This requirement may be related to our observation that ORF63 interacts directly with ORF62, the major immediate-early transactivating protein of VZV. ORF64 is located within the inverted repeat region between nucleotides 111565 and 112107; it has some homology to the HSV-1 Us10 gene and is duplicated as ORF69 (nucleotides 117790 to 118332). ORF64 and ORF69 were deleted individually or simultaneously using the VZV cosmid system. Single deletions of ORF64 or ORF69 yielded viral plaques with the same kinetics and morphology as viruses generated with the parental cosmids. The dual deletion of ORF64 and ORF69 was associated with an abnormal plaque phenotype characterized by very large, multinucleated syncytia. Finally, all of the deletion mutants that yielded recombinants retained infectivity for human T cells in vitro and replicated efficiently in human skin in the SCIDhu mouse model of VZV pathogenesis.

In dividing cells, the two aims a gene therapeutic approach should accomplish are efficient nuclear delivery and retention of therapeutic DNA. For stable transgene expression, therapeutic DNA can either be maintained by somatic integration or episomal persistence of which the latter approach would diminish the risk of insertional mutagenesis. As most monosystems fail to fulfill both tasks with equal efficiency, hybrid-vector systems represent promising alternatives. Our hybrid-vector system synergizes high-capacity adenoviral vectors (HCAdV) for efficient delivery and the scaffold/matrix attachment region (S/MAR)-based pEPito plasmid replicon for episomal persistence. After proving that this plasmid replicon can be excised from adenovirus in vitro, colony forming assays were performed. We found an increased number of colonies of up to sevenfold in cells that received the functional plasmid replicon proving that the hybrid-vector system is functional. Transgene expression could be maintained for 6 weeks and the extrachromosomal plasmid replicon was rescued. To show efficacy in vivo, the adenoviral hybrid-vector system was injected into C57Bl/6 mice. We found that the plasmid replicon can be released from adenoviral DNA in murine liver resulting in long-term transgene expression. In conclusion, we demonstrate the efficacy of our novel HCAdV-pEPito hybrid-vector system in vitro and in vivo.Molecular Therapy-Nucleic Acids (2013) 2, e83; doi:10.1038/mtna.2013.11; published online 2 April 2013.

Varicella-zoster virus (VZV) is the etiological agent of chickenpox and shingles. Due to the virus's restricted host and cell type tropism and the lack of tools for VZV proteomics, it is one of the least-characterized human herpesviruses. We generated 251 monoclonal antibodies (MAbs) against 59 of the 71 (83%) currently known unique VZV proteins to characterize VZV protein expression in vitro and in situ . Using this new set of MAbs, 44 viral proteins were detected by Western blotting (WB) and indirect immunofluorescence (IF); 13 were detected by WB only, and 2 were detected by IF only. A large proportion of viral proteins was analyzed for the first time in the context of virus infection. Our study revealed the subcellular localization of 46 proteins, 14 of which were analyzed in detail by confocal microscopy. Seven viral proteins were analyzed in time course experiments and showed a cascade-like temporal gene expression pattern similar to those of other herpesviruses. Furthermore, selected MAbs tested positive on human skin lesions by using immunohistochemistry, demonstrating the wide applicability of the MAb collection. Finally, a significant portion of the VZV-specific antibodies reacted with orthologs of simian varicella virus (SVV), thus enabling the systematic analysis of varicella in a nonhuman primate model system. In summary, this study provides insight into the potential function of numerous VZV proteins and novel tools to systematically study VZV and SVV pathogenesis.

Hepatitis E Virus (HEV) causes epidemic infections in regions of poor hygiene in the developing world. Over the last years, however, increasing numbers of autochthonous infections in industrialized countries have been described, leading to new interest in this pathogen. Currently available serological test formats to detect IgG and IgM antibodies are mainly based on bacterially expressed ORF2 and ORF3 antigens and often give ambiguous results. The objective of this study was the development of a different assay format for HEV diagnosis—a HEV immunofluorescence test (HEV-IFT) based on mammalian cells transiently expressing recombinant HEV ORF2 protein with a simple production and staining protocol and the investigation of its performance and methodical feasibility under diagnostic laboratory conditions. 31 sera of patients at different phases of HEV infection and 40 control sera from a non-endemic region were analyzed for anti-HEV IgG, IgM, and IgA antibodies. The HEV-IFT detected successfully anti-HEV IgG and IgA, but not anti-HEV IgM antibodies. In the study group the HEV-IFT was able to confirm HEV infections and to support diagnosis when ambiguous results were obtained by commercial assays. Signal localization and staining patterns helped to gather additional information about reactive antibodies present in patient sera. In conclusion the developed IFT for the detection of anti-HEV IgG and IgA antibodies can be used for diagnosis and for the serological confirmation of HEV infections. Fil: Osterman, Andreas. Ludwig Maximilians Universitat. Max Von Pettenkofer Institute; Alemania Fil: Vizoso Pinto, María Guadalupe. Ludwig Maximilians Universitat. Max Von Pettenkofer Institute; Alemania. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Tucumán; Argentina Fil: Jung, Jette. Ludwig Maximilians Universitat. Max Von Pettenkofer Institute; Alemania Fil: Jaeger, Gundula. Ludwig Maximilians Universitat. Max Von Pettenkofer Institute; Alemania Fil: Eberle, Josef. Ludwig Maximilians Universitat. Max Von Pettenkofer Institute; Alemania Fil: Nitschko, Hans. Ludwig Maximilians Universitat. Max Von Pettenkofer Institute; Alemania Fil: Baiker, Armin. Ludwig Maximilians Universitat. Max Von Pettenkofer Institute; Alemania. Bavarian Health and Food Safety Authority; Alemania

The episomal status of S/MAR (scaffold/matrix attached region)-based vectors can be confirmed by several methods including Southern blots, fluorescence in situ hybridization (FISH) analysis, or plasmid rescue experiments. In rescue experiments, genomic DNA (gDNA) or DNA from Hirt extracts is isolated from cell clones or mixed populations in which S/MAR plasmids are stably established. Bacteria are transformed with this DNA and if episomal plasmid DNA (pDNA) is present, resistant bacterial colonies will form.

Nonviral episomal vectors represent attractive alternatives to currently used virus-based expression systems. In the late 1990s, it was shown that a plasmid containing an expression cassette linked to a scaffold/matrix attached region (S/MAR) replicates as a low copy number episome in all cell lines tested, as well as primary cells, and can be used for the genetic modification of higher animals. Once established in the cell, the S/MAR vector replicates early during S-phase and, in the absence of selection, is stably retained in the cells for an unlimited period of time. This vector can therefore be regarded as a minimal model system for studying the epigenetic regulation of replication and functional nuclear architecture. In theory, this construct represents an almost “ideal” expression system for gene therapy. In practice, S/MAR-based vectors stably modify mammalian cells with efficiencies far below those of virus-based constructs. Consequently, they have not yet found application in gene therapy trials. Furthermore, S/MAR vector systems are not trivial to handle and several critical technical issues have to be considered when modifying these vectors for various applications.

Glioblastoma multiforme is a highly aggressive tumor with a median survival of 14 months despite all standard therapies. Focusing on alternative treatment strategies, we evaluated the oncolytic potential of varicella zoster virus (VZV) in malignant glioma cell cultures.Replication of wildtype and mutant VZV was comparatively analyzed in glioma cell lines (U87, U251 and U373) and in primary malignant glioma cells (n=10) in vitro by infectious foci assay, immunofluorescence microscopy and western blot analysis. Additionally, the tumor-targeting potential of VZV-infected human mesenchymal stem cells was evaluated.VZV replicated efficiently in all the glioma cells studied here followed by rapid oncolysis in vitro. The attenuated vaccine VZV mutant rOKA/ORF63rev[T171] exhibited most efficient replication. Human mesenchymal stem cells were found suitable for targeting VZV to sites of tumor growth.VZV exhibits an intrinsic oncolytic potential in malignant glioma cell cultures and might be a novel candidate for virotherapy in glioblastoma multiforme.

Analyzing the putative interaction partners of an individual protein is one approach to elucidate its function. In the LuMPIS protocol, bait and prey proteins are expressed with N-terminal maltose binding protein (MBP)- and eGFP-luciferase (eGFP-luc) tags, respectively. Positive protein-protein interactions (PPIs) can be detected after pull-down of the MBP-tagged prey protein using amylose beads followed by the bioluminescence detection of the bound eGFP-luc-tagged bait protein. The LuMPIS technology offers the following advantages: the PPIs are detected in the mammalian cell context, the use of two long protein tags (i.e., MBP and eGFP-luc) increases the expression levels of genes whose gene expression levels are known to be frequently impaired, the use of amylose beads for pull-down is much more economic as compared to sepharose beads in combination with monoclonal antibodies and finally, the use of an eGFP-luc-tag enables the qualitative control of transfection efficiencies by fluorescence microscopy prior to starting the assay.

Varicella zoster virus (VZV) ORF25 is a 156 amino acid protein belonging to the approximately 40 core proteins that are conserved throughout the Herpesviridae. By analogy to its functional orthologue UL33 in Herpes simplex virus 1 (HSV-1), ORF25 is thought to be a component of the terminase complex. To investigate how cleavage and encapsidation of viral DNA links to the nuclear egress of mature capsids in VZV, we tested 10 VZV proteins that are predicted to be involved in either of the two processes for protein interactions against each other using three independent protein-protein interaction (PPI) detection systems: the yeast-two-hybrid (Y2H) system, a luminescence based MBP pull-down interaction screening assay (LuMPIS), and a bioluminescence resonance energy transfer (BRET) assay. A set of 20 interactions was consistently detected by at least 2 methods and resulted in a dense interaction network between proteins associated in encapsidation and nuclear egress. The results indicate that the terminase complex in VZV consists of ORF25, ORF30, and ORF45/42 and support a model in which both processes are closely linked to each other. Consistent with its role as a central hub for protein interactions, ORF25 is shown to be essential for VZV replication. Fil: Vizoso Pinto, María Guadalupe. Ludwig Maximilians Universitat. Max Von Pettenkofer Institute. Cátedra Virology; Alemania. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina Fil: Pothineni, Venkata R.. Ludwig Maximilians Universitat. Max Von Pettenkofer Institute. Cátedra Virology; Alemania Fil: Haase, Rudolf. Ludwig Maximilians Universitat. Max Von Pettenkofer Institute. Cátedra Virology; Alemania Fil: Woidy, Mathias. Ludwig Maximilians Universitat; Alemania Fil: Lotz Havla, Amelie. Ludwig Maximilians Universitat; Alemania Fil: Gersting, Soren W.. Ludwig Maximilians Universitat; Alemania Fil: Muntau, Ania C.. Ludwig Maximilians Universitat; Alemania Fil: Haas, Jurgen. Ludwig Maximilians Universitat. Max Von Pettenkofer Institute. Cátedra Virology; Alemania Fil: Sommer, Marvin. University of Stanford; Estados Unidos Fil: Arvin, Ann M.. University of Stanford; Estados Unidos Fil: Baiker, Armin. Bavarian Health and Food Safety Authority; Alemania

Analyzing the putative interaction partners of an individual protein is one approach to elucidate its function. In the LuMPIS protocol, bait and prey proteins are expressed with N-terminal maltose binding protein (MBP)- and eGFP-luciferase (eGFP-luc) tags, respectively. Positive protein-protein interactions (PPIs) can be detected after pull-down of the MBP-tagged prey protein using amylose beads followed by the bioluminescence detection of the bound eGFP-luc-tagged bait protein. The LuMPIS technology offers the following advantages: the PPIs are detected in the mammalian cell context, the use of two long protein tags (i.e., MBP and eGFP-luc) increases the expression levels of genes whose gene expression levels are known to be frequently impaired, the use of amylose beads for pull-down is much more economic as compared to sepharose beads in combination with monoclonal antibodies and finally, the use of an eGFP-luc-tag enables the qualitative control of transfection efficiencies by fluorescence microscopy prior to starting the assay.

The herpesviruses, like most other DNA viruses, replicate in the host cell nucleus. Subnuclear domains known as promyelocytic leukemia protein nuclear bodies (PML-NBs), or ND10 bodies, have been implicated in restricting early herpesviral gene expression. These viruses have evolved countermeasures to disperse PML-NBs, as shown in cells infected in vitro, but information about the fate of PML-NBs and their functions in herpesvirus infected cells in vivo is limited. Varicella-zoster virus (VZV) is an alphaherpesvirus with tropism for skin, lymphocytes and sensory ganglia, where it establishes latency. Here, we identify large PML-NBs that sequester newly assembled nucleocapsids (NC) in neurons and satellite cells of human dorsal root ganglia (DRG) and skin cells infected with VZV in vivo. Quantitative immuno-electron microscopy revealed that these distinctive nuclear bodies consisted of PML fibers forming spherical cages that enclosed mature and immature VZV NCs. Of six PML isoforms, only PML IV promoted the sequestration of NCs. PML IV significantly inhibited viral infection and interacted with the ORF23 capsid surface protein, which was identified as a target for PML-mediated NC sequestration. The unique PML IV C-terminal domain was required for both capsid entrapment and antiviral activity. Similar large PML-NBs, termed clastosomes, sequester aberrant polyglutamine (polyQ) proteins, such as Huntingtin (Htt), in several neurodegenerative disorders. We found that PML IV cages co-sequester HttQ72 and ORF23 protein in VZV infected cells. Our data show that PML cages contribute to the intrinsic antiviral defense by sensing and entrapping VZV nucleocapsids, thereby preventing their nuclear egress and inhibiting formation of infectious virus particles. The efficient sequestration of virion capsids in PML cages appears to be the outcome of a basic cytoprotective function of this distinctive category of PML-NBs in sensing and safely containing nuclear aggregates of aberrant proteins., Author Summary Many DNA viruses, including varicella-zoster virus (VZV), a herpesvirus that causes varicella (chickenpox) and zoster (shingles), replicate in the host cell nucleus. Here, we have identified an intrinsic antiviral mechanism that specifically targets newly assembled VZV capsids and contains these essential viral structures in a nuclear “safe house”. Using immuno-electron microscopy, PML (promyelocytic leukemia) protein fibers that formed filamentous spherical cages were shown to trap virion capsids very efficiently, preventing their transport out of the nucleus and inhibiting the formation of infectious virus particles. PML cages containing virion capsids were found in VZV-infected neurons and satellite cells in human sensory ganglia and in skin cells, which are major targets during VZV pathogenesis. Similar PML nuclear bodies that sequester abnormal proteins have been reported in neurodegenerative disorders, like Huntington's disease. We found that cages formed by PML isoform IV sequestered both the virion capsids of VZV, which is a neurotropic herpesvirus, and the mutant Huntington's disease protein. This work provides the first evidence that PML, which is abundant in mammalian cell nuclei, can function both to contain potentially damaging cellular protein aggregates and as an intrinsic host defense against a herpesvirus during nuclear virion assembly.

Varicella zoster virus (VZV) is a human herpesvirus encoding at least 69 distinct viral proteins which causes chickenpox after primary infection and shingles during reactivation and which is particularly important in pregnancy and immunocompromised patients. Current serodiagnostic tests are either based on whole cell lysates or glycoprotein preparations. In order to investigate the humoral immune response to VZV infection or vaccination in more detail, and to improve the currently available diagnostic assays, we developed a nucleic acid programmable protein array (NAPPA) containing all 69 VZV proteins and performed a detailed analysis of 68 sera from individuals with either no, a previous or an acute VZV infection. In addition to the known reactive glycoprotein antigens (ORF 5, ORF 14, ORF 31, ORF 37, ORF 68), we discovered IgG antibodies against a variety of other membrane (ORF 2, ORF 24), capsid (ORF 20, ORF 23, ORF 43) and tegument (ORF 53, ORF 9, ORF 11) proteins, as well as other proteins involved in virus replication and assembly (ORF 25, ORF 26, ORF 28) and the transactivator proteins ORF 12, ORF 62 and ORF 63. All of these antigens were only reactive in a subset of VZV-positive individuals. A subset of the newly identified VZV antigens was validated by western blot analysis. Using these seroreactive new VZV antigens, more sensitive assays and tests distinguishing between different clinical entities may be developed.

Background Yeast two-hybrid (Y2H) screens have been among the most powerful methods to detect and analyze protein-protein interactions. However, they suffer from a significant degree of false negatives, i.e. true interactions that are not detected, and to a certain degree from false positives, i.e. interactions that appear to take place only in the context of the Y2H assay. While the fraction of false positives remains difficult to estimate, the fraction of false negatives in typical Y2H screens is on the order of 70-90%. Here we present novel Y2H vectors that significantly decrease the number of false negatives and help to mitigate the false positive problem. Results We have constructed two new vectors (pGBKCg and pGADCg) that allow us to make both C-terminal fusion proteins of DNA-binding and activation domains. Both vectors can be combined with existing vectors for N-terminal fusions and thus allow four different bait-prey combinations: NN, CC, NC, and CN. We have tested all ~4,900 pairwise combinations of the 70 Varicella-Zoster-Virus (VZV) proteins for interactions, using all possible combinations. About ~20,000 individual Y2H tests resulted in 182 NN, 89 NC, 149 CN, and 144 CC interactions. Overlap between screens ranged from 17% (NC-CN) to 43% (CN-CC). Performing four screens (i.e. permutations) instead of one resulted in about twice as many interactions and thus much fewer false negatives. In addition, interactions that are found in multiple combinations confirm each other and thus provide a quality score. This study is the first systematic analysis of such N- and C-terminal Y2H vectors. Conclusions Permutations of C- and N-terminal Y2H vectors dramatically increase the coverage of interactome studies and thus significantly reduce the number of false negatives. We suggest that future interaction screens should use such vector combinations on a routine basis, not the least because they provide a built-in quality score for Y2H interactions that can provide a measure of reproducibility without additional assays.

The increasing knowledge of the molecular and genetic background of many different human diseases has led to the vision that genetic engineering might be used one day for their phenotypic correction. The main goal of gene therapy is to treat loss-of-function genetic disorders by delivering correcting therapeutic DNA sequences into the nucleus of a cell, allowing its long-term expression at physiologically relevant levels. Manifold different vector systems for the therapeutic gene delivery have been described over the recent years. They all have their individual advantages but also their individual limitations and must be judged on a careful risk/benefit analysis. Integrating vector systems can deliver genetic material to a target cell with high efficiency enabling long-term expression of an encoded transgene. The main disadvantage of integrating vector systems, however, is their potential risk of causing insertional mutagenesis. Episomal vector systems have the potential to avoid these undesired side effects, since they behave as separate extrachromosomal elements in the nucleus of a target cell. Within this article we present a comprehensive survey of currently available episomal vector systems for the genetic modification of mammalian cells. We will discuss their advantages and disadvantages and their applications in the context of basic research, biotechnology and gene therapy.

We have developed an episomal replicating expression vector in which the SV40 gene coding for the large T-antigen was replaced by chromosomal scaffold/matrix attached regions. Southern analysis as well as vector rescue experiments in CHO cells and in Escherichia coli demonstrate that the vector replicates episomally in CHO cells. It occurs in a very low copy number in the cells and is stably maintained over more than 100 generations without selection pressure.

In case using viral vectors as therapeutics in context of gene therapy small changes in vector dose can have fatal outcome for the patient. It has been shown in earlier clinical studies that there is a non-linear dose response for most of the vectors, so the therpeutical window is narrow. Precise vector titration is of extraordinary high importance. The widely distributed standard real time PCR (qPCR) may not provide this in a satisfactory way. In this study we were aiming to address this challenge by implementing a more reliable and accurate strategy for vector quantification. We implemented a new digital PCR (ddPCR) strategy based on droplet formation as tool for vector quantification. By forming around 20.000 individual PCR reactions using sample partitioning in a water oil emulsion the system is able to detect after end point PCR if every single droplet is negative or positive for the target DNA. Importantly, providing a standard is not necessary since quantification is absolute whereas normal qPCR provides standard dependent relative quantification. By choosing primers in the region of adenoviral ITR and fiber, we were able to quantify in one single reaction high capacity adenoviral vector (HCAdV) and the helper virus, which is needed during production of the viral gene deleted HCAdV. We applied this new strategy to quantify HCAdV produced with a shortened protocol using column purification instead of traditional caesium chloride centrifugation. The virus was purified from the same characterized stock using different columns, which were also compared in this study. Once the virus was purified we infected various cell lines with different volumes of the final preparation, after 3h total DNA was isolated and different PCR strategies were performed. First our data showed that this protocol leads to sufficient HCAdV for preclinical in vitro studies. Besides the transducing units calculated from ddPCR vs. qPCR data (≈10^6 transducing units per ml), this was also confirmed by EGFP expression analysis and the number of physical particles measured via UV-vis. Also the helper virus concentration was within an acceptable range (

The comprehensive yeast two-hybrid analysis of intraviral protein interactions in two members of the herpesvirus family, Kaposi sarcoma–associated herpesvirus (KSHV) and varicella-zoster virus (VZV), revealed 123 and 173 interactions, respectively. Viral protein interaction networks resemble single, highly coupled modules, whereas cellular networks are organized in separate functional submodules. Predicted and experimentally verified interactions between KSHV and human proteins were used to connect the viral interactome into a prototypical human interactome and to simulate infection. The analysis of the combined system showed that the viral network adopts cellular network features and that protein networks of herpesviruses and possibly other intracellular pathogens have distinguishing topologies.

The inhibition of gene expression by RNA interference harbors a high potential for application in the therapy of human diseases. However, while exogenous application of siRNAs efficiently inhibits gene expression, these effects are only transient in mammalian cells. We designed a short hairpin RNA-expression cassette to target the bcr-abl oncogene that was then introduced into the nonviral vector system pEPI-1, which replicates episomally in the absence of selection in the bcr-abl-positive cell line K562. Forty-two days after transfection the bcr-abl- but not the cytokine-dependent growth rate was found to be drastically reduced in K562 cells. Western analysis revealed a more than 90% reduction in the expression of the fusion protein bcr-abl while the expression of the bcr protein remained unaffected. In addition, we show that the level of bcr-abl mRNA was specifically reduced in these cells for more than 90%. These results demonstrate that the vector system pEPI-1 allows specific and efficient long term gene suppression by using a short hairpin RNA transcription unit.