Replication Properties Research Articles

Immunogenic and replicative properties of classical swine fever virus replicon particles modified to induce IFN-α/β and carry foreign genes

Virus replicon particles (VRP) are genetically engineered infectious virions incapable of generating progeny virus due to partial or complete deletion of at least one structural gene. VRP fulfil the criteria of a safe vaccine and gene delivery system. With VRP derived from classical swine fever virus (CSF-VRP), a single intradermal vaccination protects from disease. Spreading of the challenge virus in the host is however not completely abolished. Parameters that are critical for immunogenicity of CSF-VRP are not well characterized. Considering the importance of type I interferon (IFN-α/β) to immune defence development, we generated IFN-α/β–inducing VRP to determine how this would influence vaccine efficacy. We also evaluated the effect of co-expressing granulocyte macrophage colony-stimulating factor (GM-CSF) in the vaccine context. The VRP were capable of long-term replication in cell culture despite the presence of IFN-α/β. In vivo, RNA replication was essential for the induction of an immune response. IFN-α/β–inducing and GM-CSF–expressing CSF-VRP were similar to unmodified VRP in terms of antibody and peripheral T-cell responses, and in reducing the blood levels of challenge virus RNA. Importantly, the IFN-α/β–inducing VRP did show increased efficacy over the unmodified VRP in terms of B-cell and T-cell responses, when tested with secondary immune responses by in vitro restimulation assay.

CD133+ Acute Myeloid Leukemia (AML) Cells Exhibit Higher Clonogenic Capacity and Higher Levels of pAKT and Bcl-2 Proteins Than Their Negative Counterpart

AbstractAbstract 1721 Introduction:CD133 is a transmembrane protein expressed by hematopoietic stem cells and other cell types, whose physiological functions remain unknown. To determine its potential prognostic value in AML, we analyzed its expression on blasts from 76 patients at diagnosis. Moreover, since leukemic stem cells play a critical role in the resistance to chemotherapeutic drugs and may be responsible for relapse, we compared the growth potential and activated cellular pathways of CD133+ and CD133- cells from 20 AML patients. Methods:First, bone marrow samples were collected at diagnosis from 76 AML patients (median age: 65, range: 1–84). CD133 expression was evaluated on blasts cells and CD34+ cells using 6-color flow cytometry (FC) and the monoclonal antibody AC133. The mean fluorescence intensity ratio (MFIR) was calculated by dividing the MFI of CD133 by the MFI of its isotypic control. Second, purified CD34+, CD34+CD133+ and CD34+CD133- cells were isolated from 20 AML bone marrows using the magnetic separation system Minimacs (Miltenyi Biotec). These fractions were submitted to an in vitro colony forming assay during 14 days. Finally, the level of expression of survival proteins involved in the PI3K/AKT, Mitogen-Activated Protein Kinase (MAPK) and Bcl-2 pathways, as well as CXCR4 and Focal Adhesion Kinase (FAK) migration proteins, was analyzed by FC in these fractions. Results:The level of CD133 expression on blasts cells was significantly different between AML patients with favorable (n=12), intermediate (n=38) and unfavorable karyotypes (n=23) (p=0.007). In particular, it was significantly higher in patients with unfavorable karyotype than in patients with intermediate karyotype (p=0.032) or favorable karyotype (p=0.005). Moreover, when considering NPM1 and FLT3 mutation status in patients with intermediate karyotype, the level of CD133 expression was significantly higher in patients with an intermediate molecular profile than in patients with a favorable molecular profile (p=0.004). Most interestingly, we showed that the level of CD133 expression has an impact on overall survival in multivariate analysis, independently of leukocytosis, karyotype and achievement of complete remission after induction therapy (p=0.04). Second, in 20 patients, we found that AML progenitor cells (AML CFU) were present in all sorted fractions, but the CD34+CD133+ fraction gave rise to larger and more numerous colonies (74.9±39.6) than the CD34+CD133- fraction (11,25±6). Moreover, the CD34+CD133+ fraction was the only one able to form secondary colonies (52.1±31.8). Finally, we found that the expression of PI3K (mean percentage of positive cells: 78±20.1), pAKT (80±19.8), pERK (80.1±19), CXCR4 (71.6±22.7), pFAK (81.2±19.8), Bcl-2 (72.9±26.1), Bcl-xL (45.7±24.8), and pBad (63.9±36.1) was significantly upregulated in CD34+CD133+ cells compared to CD34+CD133- cells, whereas Bax was downregulated. Discussion:CD133 is known to be expressed in hematopoietic progenitors, but its role in the resistance to chemotherapy is not well studied. In our work, the level of expression of CD133 on AML blasts appeared as an independent immunophenotypic prognostic factor, encouraging its further evaluation in a larger number of cases. We also showed that CD133+ cells have greater replicative properties than CD133- cells, and exhibit activation of various signaling pathways. Since CD133+ cells might escape from apoptosis through activation of PI3K/AKT and ERK, these proteins could represent potential therapeutic targets. In conclusion, our results revealed that CD133+ cells exhibit more aggressive behavior than CD133- ones, and that CD133+ AML progenitors might contribute to resistance to chemotherapy through preferential activation of PI3K/AKT, MAPK and Bcl-2 survival response. Disclosures:No relevant conflicts of interest to declare.

Cloning of the Epstein-Barr Virus-Related Rhesus Lymphocryptovirus as a Bacterial Artificial Chromosome: a Loss-of-Function Mutation of the rhBARF1 Immune Evasion Gene

Rhesus macaques are naturally infected with a gammaherpesvirus which is in the same lymphocryptovirus (LCV) genus as and closely related to Epstein-Barr virus (EBV). The rhesus macaque LCV (rhLCV) contains a repertoire of genes identical to that of EBV, and experimental rhLCV infection of naive rhesus macaques accurately models acute and persistent EBV infection of humans. We cloned the LCL8664 rhLCV strain as a bacterial artificial chromosome to create recombinant rhLCV for investigation in this animal model system. A recombinant rhLCV (clone 16 rhLCV) carrying a mutation in the putative immune evasion gene rhBARF1 was created along with a rescued wild-type (rWT) rhLCV in which the rhBARF1 open reading frame (ORF) was repaired. The rWT rhLCV molecular clone demonstrated viral replication and B-cell immortalization properties comparable to those of the naturally derived LCL8664 rhLCV. Qualitatively, clone 16 rhLCV carrying a mutated rhBARF1 was competent for viral replication and B-cell immortalization, but quantitative assays showed that clone 16 rhLCV immortalized B cells less efficiently than LCL8664 and rWT rhLCV. Functional studies showed that rhBARF1 could block CSF-1 cytokine signaling as well as EBV BARF1, whereas the truncated rhBARF1 from clone 16 rhLCV was a loss-of-function mutant. These recombinant rhLCV can be used in the rhesus macaque animal model system to better understand how a putative viral immune evasion gene contributes to the pathogenesis of acute and persistent EBV infection. The development of a genetic system for making recombinant rhLCV constitutes a major advance in the study of EBV pathogenesis in the rhesus macaque animal model.

Implications of Efavirenz for Neuropsychiatry: A Review

ABSTRACTAntiretroviral therapy has revolutionized the treatment of the human immunodeficiency virus because it has improved the clinical outcomes of patients. It is essential that these drugs cross the blood–brain barrier, since the virus is present in the central nervous system (CNS). Efavirenz passes through this barrier satisfactorily and can reduce the deleterious central effects of the human immunodeficiency virus. However, patients treated with efavirenz have been observed to experience psychiatric symptoms such as mania, depression, suicidal thoughts, psychosis, and hallucinations. The aim of this review is to describe the pharmacokinetic and pharmacodynamic properties of efavirenz and its major neuropsychiatric symptoms and the neurochemical pathways associated with these changes in the CNS. The databases Medline and Lilacs were used to search for review articles and preclinical and clinical research articles published from January 1996 to 2010. The search terms used were efavirenz, central nervous system, neuropsychiatry, neurotransmitters, adverse effects, and neurochemistry. Subject categories considered included effects on viral replication, pharmacokinetic and pharmacodynamic properties of efavirenz, and neuropsychiatric adverse effects including time course, duration, and probable mechanisms involved. The mechanisms involved in these changes include interference with cytochrome P450 enzymes, cytokines, tryptophan-2–3-dioxygenase, and brain creatine kinase.

Comparison of the replication properties of murine and human calicivirus RNA-dependent RNA polymerases

The human caliciviruses (CV), norovirus (NoV) and sapovirus (SaV), are major causes of outbreak gastroenteritis worldwide. To date, the investigation of human NoV and SaV replication cycles has been impeded as neither is culturable. Consequently, the recently discovered murine NoV (MNV) has been adopted as a surrogate replication model for the human CVs. In this study, we sought to compare the biochemical properties of the MNV RNA-dependent RNA polymerase (RdRp) with related human NoV and SaV-RdRps to address the suitability of MNV as a model for the human CVs. Three human NoV-RdRps (GII.b, GII.4 and GII.7), an MNV-RdRp and two human SaV-RdRps (GI and GII) were overexpressed in Escherichia coli, purified and their enzymatic activity and fidelity compared. Despite ~70% amino acid variation between the RdRp from the two different CV genera, the majority of the physiological characteristics of the RdRps were similar. All RdRps exhibited co-operative dimerisation and had optimal activity at 25°C, a pH range between 7 and 8, required 2-5mM MnCl(2) and were inhibited with increasing NaCl concentrations. We observed RdRp activity at temperatures as low as 5°C and as high as 65°C. Using an in vitro fidelity assay, similar mutation rates were observed for the separate RdRps (1×10(-4)-1×10(-5)). This is the first report to compare the physiological, biochemical and mutational properties of the MNV-RdRp to those of the human CV-RdRps and it suggests that MNV may be directly applicable to the study of human NoV.

Replication and pathogenesis of the pandemic (H1N1) 2009 influenza virus in mammalian models

This study aimed to characterize the replication and pathogenic properties of a Korean pandemic (H1N1) 2009 influenza virus isolate in ferrets and mice. Ferrets infected with A/Korea/01/2009 (H1N1) virus showed mild clinical signs. The virus replicated well in lungs and slightly in brains with no replication in any other organs. Severe bronchopneumonia and thickening of alveolar walls were detected in the lungs. Viral antigens were detected in the bronchiolar epithelial cells, in peribronchial glands with severe peribronchitis and in cells present in the alveoli. A/Korea/01/2009 (H1N1) virus-infected mice showed weight loss and pathological lung lesions including perivascular cuffing, interstitial pneumonia and alveolitis. The virus replicated highly in the lungs and slightly in the nasal tissues. Viral antigens were detected in bronchiolar epithelial cells, pneumocytes and interstitial macrophages. However, seasonal H1N1 influenza virus did not replicate in the lungs of ferrets, and viral antigens were not detected. Thus, this Korean pandemic (H1N1) 2009 isolate infected the lungs of ferrets and mice successfully and caused more pathological lesions than did the seasonal influenza virus.

DNA Polymerase Recognition of Unnatural Base Pair: 1-Methoxy-3-methylbenzene and 6-Methyl-1(2H)-isoquinolinone

The faithful replication of DNA by DNA polymerases is necessary for the expansion of both the genetic alphabet and the genetic code. 1 The stability and sequence specificity of a natural DNA duplex are based on the complementary and orthogonal Watson-Crick hydrogen-bonding patterns of dA:dT and dC:dG base pairs. Although inter-nucleobase hydrogen bonding and shape complementarity are important for the stability and faithful copying of DNA, there is no reason to assume that the pairing of two unnatural bases could not be driven by other intermolecular interactions, for example, those based on hydrophobic interactions and van der Waals forces. Some groups have used this concept to explore the development of unnatural nucleotides bearing nucleobase analogs that pair via hydrophobic interactions and packing forces. 2-5 Recently, Romesberg’s group found that the heteropair formed between 1-methoxy-3-methylbenzene 1 and 6-methyl-1(2H)-isoquinolinethione 2 is a very promising, predominantly hydrophobic base pair. This base pair is synthesized by the insertion of one unnatural triphosphate opposite the other unnatural nucleotide in the template, and it is then extended by the insertion of the next correct dNTP (natural triphosphates), with relatively high efficiency and fidelity, by diverse DNA polymerases (Figure 1). 5 However, the slowest step in the replication of DNA containing this heteropair is the incorporation of 1 triphosphate opposite 2 in the template. Also, dG inserts competitively opposite 2 in the template, which may limit the use of this heteropair in biological and biotechnological applications. We are therefore interested in the modifications of 1 or 2 to optimize the formation of unnatural heteropairs. Herein, we report the DNA replication results for two nucleotides, 6-methyl-1(2H)-isoquinolinone 3, in which an oxygen atom is substituted for the sulfur atom in 2 to compare the effect of the oxygen atom on replication properties, and 7-methyl-1(2H)isoquinolinone 4, in which, as well as replacing the sulfur atom in 2 by an oxygen atom, the methyl substituent is moved from position 6 to position 7. The substitution at position 7 is expected to bury the methyl group within the duplex, as shown

Evaluation of replication properties on moulded surface by ultrasonic injection moulding system

An ultrasonic injection moulding (UIM) system, which applies ultrasonic waves to injection moulding, as a precision injection moulding technology was developed. Replication properties of the microstructure of the moulded surface were evaluated by the UIM system. In particular, the effects of oscillation conditions on the replication ratio of the moulded surface were investigated. As a result, the replication ratio of the moulded surface was significantly improved in UIM compared with that in conventional moulding. The replication ratio increased when the ultrasonic wave was applied immediately after the resin was filled in a cavity. Results showed that, by applying ultrasonic waves, oscillatory flow was generated inside the cavity and consequently the surface replication was increased. The surface replication during packing and holding stages was improved by the oscillatory flow provided by the ultrasonic vibration.

Single-Molecule Analysis Reveals Changes in the DNA Replication Program for the POU5F1 Locus upon Human Embryonic Stem Cell Differentiation

Human embryonic stem cells (hESCs), due to their pluripotent nature, represent a particularly relevant model system to study the relationship between the replication program and differentiation state. Here, we define the basic properties of the replication program in hESCs and compare them to the programs of hESC-derived multipotent cells (neural rosette cells) and primary differentiated cells (microvascular endothelial cells [MECs]). We characterized three genomic loci: two pluripotency regulatory genes, POU5F1 (OCT4) and NANOG, and the IGH locus, a locus that is transcriptionally active specifically in B-lineage cells. We applied a high-resolution approach to capture images of individual replicated DNA molecules. We demonstrate that for the loci studied, several basic properties of replication, including the average speed of replication forks and the average density of initiation sites, were conserved among the cells analyzed. We also demonstrate, for the first time, the presence of initiation zones in hESCs. However, significant differences were evident in other aspects of replication for the DNA segment containing the POU5F1 gene. Specifically, the locations of centers of initiation zones and the direction of replication fork progression through the POU5F1 gene were conserved in two independent hESC lines but were different in hESC-derived multipotent cells and MECs. Thus, our data identify features of the replication program characteristic of hESCs and define specific changes in replication during hESC differentiation.

Epigenetically-Inherited Centromere and Neocentromere DNA Replicates Earliest in S-Phase

Eukaryotic centromeres are maintained at specific chromosomal sites over many generations. In the budding yeast Saccharomyces cerevisiae, centromeres are genetic elements defined by a DNA sequence that is both necessary and sufficient for function; whereas, in most other eukaryotes, centromeres are maintained by poorly characterized epigenetic mechanisms in which DNA has a less definitive role. Here we use the pathogenic yeast Candida albicans as a model organism to study the DNA replication properties of centromeric DNA. By determining the genome-wide replication timing program of the C. albicans genome, we discovered that each centromere is associated with a replication origin that is the first to fire on its respective chromosome. Importantly, epigenetic formation of new ectopic centromeres (neocentromeres) was accompanied by shifts in replication timing, such that a neocentromere became the first to replicate and became associated with origin recognition complex (ORC) components. Furthermore, changing the level of the centromere-specific histone H3 isoform led to a concomitant change in levels of ORC association with centromere regions, further supporting the idea that centromere proteins determine origin activity. Finally, analysis of centromere-associated DNA revealed a replication-dependent sequence pattern characteristic of constitutively active replication origins. This strand-biased pattern is conserved, together with centromere position, among related strains and species, in a manner independent of primary DNA sequence. Thus, inheritance of centromere position is correlated with a constitutively active origin of replication that fires at a distinct early time. We suggest a model in which the distinct timing of DNA replication serves as an epigenetic mechanism for the inheritance of centromere position.

Generation of VSV pseudotypes using recombinant ΔG-VSV for studies on virus entry, identification of entry inhibitors, and immune responses to vaccines

Vesicular stomatitis virus (VSV) is a prototypic enveloped animal virus that has been used extensively to study virus entry, replication and assembly due to its broad host range and robust replication properties in a wide variety of mammalian and insect cells. Studies on VSV assembly led to the creation of a recombinant VSV in which the glycoprotein (G) gene was deleted. This recombinant (rVSV-ΔG) has been used to produce VSV pseudotypes containing the envelope glycoproteins of heterologous viruses, including viruses that require high-level biocontainment; however, because the infectivity of rVSV-ΔG pseudotypes is restricted to a single round of replication the analysis can be performed using biosafety level 2 (BSL-2) containment. As such, rVSV-ΔG pseudotypes have facilitated the analysis of virus entry for numerous viral pathogens without the need for specialized containment facilities. The pseudotypes also provide a robust platform to screen libraries for entry inhibitors and to evaluate the neutralizing antibody responses following vaccination. This manuscript describes methods to produce and titer rVSV-ΔG pseudotypes. Procedures to generate rVSV-ΔG stocks and to quantify virus infectivity are also described. These protocols should allow any laboratory knowledgeable in general virological and cell culture techniques to produce successfully replication-restricted rVSV-ΔG pseudotypes for subsequent analysis.

Construction of Self-Transmissible Green Fluorescent Protein-Based Biosensor Plasmids and Their Use for Identification ofN-Acyl Homoserine-Producing Bacteria in Lake Sediments

Many bacteria utilize quorum sensing (QS) systems to communicate with each other by means of the production, release, and response to signal molecules. N-Acyl homoserine lactone (AHL)-based QS systems are particularly widespread among the Proteobacteria, in which they regulate various functions. It has become evident that AHLs can also serve as signals for interspecies communication. However, knowledge on the impact of AHLs for the ecology of bacteria in their natural habitat is scarce, due mainly to the lack of tools that allow the study of QS in bacterial communities in situ. Here, we describe the construction of self-mobilizable green fluorescent protein (GFP)-based AHL sensors that utilize the conjugation and replication properties of the broad-host-range plasmid RP4. We show that these novel AHL sensor plasmids can be easily transferred to different bacterial species by biparental mating and that they give rise to green fluorescent cells in case the recipient is an AHL producer. We also demonstrate that these sensor plasmids are capable of self-spreading within mixed biofilms and are a suitable tool for the identification of AHL-producing bacteria in lake sediment.

Targeting of p53-Transcriptional Dysfunction by Conditionally Replicating Adenovirus Is Not Limited by p53-Homologues

A hallmark of human tumors is the loss of p53 or its transcriptional functions. In this study, we describe the generation of the conditionally replicating adenovirus Adp53sensor for the treatment of p53-dysfunctional tumors. p53-selective attenuation of viral replication was achieved by using p53-dependent expression of the transcriptional repressor Gal4-KRAB that was directed against the adenoviral E1A locus. Adp53sensor shows efficient replication in p53-dysfunctional, but not in p53-active cells. In p53-dysfunctional cells, p53-analogous transcriptional activity by other p53 family members was not sufficient to compromise replication of Adp53sensor. In comparison with a genetically similar, but p53-insensitive virus, Adp53sensor replication was inhibited after systemic infection of p53-wt-mice, but not in p53-ko-mice thus confirming the correct function of the chosen approach. Adp53sensor showed efficient lytic and replicative properties in all investigated cells with p53-dysfunction and successfully inhibited the growth of subcutaneous xenotransplants in vivo. We further demonstrated that intravenous injection of Adp53sensor lead to significantly reduced liver damage compared to the control virus. Together, our data show that Adp53sensor is an oncolytic, p53-selective adenovirus for efficient treatment of p53-dysfunctional tumors with a favorable toxicity profile. Moreover, Adp53sensor provides a strategy that should be applicable to other transcriptionally regulated DNA viruses.

Replicator selection and the extended phenotype.

Adaptations are often spoken of as ‘for the good of’ some entity, but what is that entity? Groups and species are now rightly unfashionable, so what are we left with? The prevailing answer is Darwin's: ‘the individual’. Individuals clearly do not maximise their own survival so the concept of fitness had to be invented. If fitness is correctly defined in Hamilton's way as ‘inclusive fitness’ it ceases to matter whether we speak of individuals maximising their inclusive fitness or of genes maximising their survival. The two formulations are mutually inter-translatable. Yet some serious mistranslations are quoted from the literature, which have led their authors into actual biological error. The present paper blames the prevailing concentration on the individual for these errors, and advocates a reversion to the replicator as the proper focus of evolutionary attention. A gene is an obvious replicator, but there are others, and the general properties of replicators are discussed. Defenders of the individual as the unit of selection often point to the unity and integration of the genome as expressed phenotypically. This paper ends by attacking even this assumption, not by a reductionist fragmentation of the phenotype, but, on the contrary, by extending it to include more than one individual. Replicators survive by virtue of their effects on the world, and these effects are not restricted to one individual body but constitute a wider ‘extended phenotype’.

Abstract 1383: The effect of a novel angiogenesis inhibitor (TM) on the efficacy of HSV-1 oncolytic virus

Abstract Oncolytic viruses (OVs) are modified viruses specifically designed to lyse cancerous cells while leaving surrounding tissue intact. Solid tumors such as glioma represent an excellent candidate for this novel cancer therapy. We are investigating barriers towards oncolytic viral therapy in vivo. The tumor microenvironment plays an inhibitory role in viral propagation and spread. In particular, the angiogenic and inflammatory response that follows OV inoculation have been demonstrated to significantly limit OV efficacy. The goal of our current project is to determine if the novel copper-chelating angiostatic/anti-inflammatory drug, tetrathiomolybdate (TM), represents a potential co-therapy that can be used in conjunction with OV to enhance overall efficacy in a glioma model. Based on previous findings from our laboratory, the anti-cancerous effects of oncolytic viruses can be significantly increased by pre-treatment with an angiostatic agent. Apart from supporting angiogenesis, copper found in serum has also been shown to inhibit wild-type HSV infection and replication. Due it its copper chelating properties, TM has been FDA approved for Wilson's disease and is currently under clinical investigation for efficacy as an angiostatic and antineoplastic agent. Here we tested if copper can inhibit OV mediated glioma cell killing. 1): We found a significant and dose dependant inhibitory effect of copper on the cytotoxic ability of OV against multiple glioma and head and neck SCC cells. 2): The inhibitory effects of copper were apparent even at physiologically relevant concentrations of copper (1 mg/L in serum). 3): Further we found that Cu mediated inhibition of OV killing of glioma cell could be rescued by TM mediated copper chelation. 4): Finally we have tested that TM does not interfere with viral replication or tumor cell killing properties in vitro. Given the potential angiostatic and anti-inflammatory properties of TM, these studies support further in vivo testing of TM in conjunction with oncolytic viral therapy. 5): We are currently testing therapeutic efficacy of combining TM and OV for anti-glioma efficacy in vivo. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1383.

Comparison of egg and high yielding MDCK cell-derived live attenuated influenza virus for commercial production of trivalent influenza vaccine: In vitro cell susceptibility and influenza virus replication kinetics in permissive and semi-permissive cells

Currently MedImmune manufactures cold-adapted ( ca) live, attenuated influenza vaccine (LAIV) from specific-pathogen free (SPF) chicken eggs. Difficulties in production scale-up and potential exposure of chicken flocks to avian influenza viruses especially in the event of a pandemic influenza outbreak have prompted evaluation and development of alternative non-egg based influenza vaccine manufacturing technologies. As part of MedImmune's effort to develop the live attenuated influenza vaccine (LAIV) using cell culture production technologies we have investigated the use of high yielding, cloned MDCK cells as a substrate for vaccine production by assessing host range and virus replication of influenza virus produced from both SPF egg and MDCK cell production technologies. In addition to cloned MDCK cells the indicator cell lines used to evaluate the impact of producing LAIV in cells on host range and replication included two human cell lines: human lung carcinoma (A549) cells and human muco-epidermoid bronchiolar carcinoma (NCI H292) cells. The influenza viruses used to infect the indicators cell lines represented both the egg and cell culture manufacturing processes and included virus strains that composed the 2006–2007 influenza seasonal trivalent vaccine (A/New Caledonia/20/99 (H1N1), A/Wisconsin/67/05 (H3N2) and B/Malaysia/2506/04). Results from this study demonstrate remarkable similarity between influenza viruses representing the current commercial egg produced and developmental MDCK cell produced vaccine production platforms. MedImmune's high yielding cloned MDCK cells used for the cell culture based vaccine production were highly permissive to both egg and cell produced ca attenuated influenza viruses. Both the A549 and NCI H292 cells regardless of production system were less permissive to influenza A and B viruses than the MDCK cells. Irrespective of the indicator cell line used the replication properties were similar between egg and the cell produced influenza viruses. Based on these study results we conclude that the MDCK cell produced and egg produced vaccine strains are highly comparable.

Effect of the Neuraminidase Mutation H274Y Conferring Resistance to Oseltamivir on the Replicative Capacity and Virulence of Old and Recent Human Influenza A(H1N1) Viruses

The viral fitness of neuraminidase inhibitor (NAI)-resistant influenza viruses is believed to be impaired. Unexpectedly, an oseltamivir-resistant A(H1N1) variant containing the H274Y neuraminidase (NA) mutation recently disseminated worldwide, suggesting that the replication and virulence properties of this mutant virus were not compromised. In vitro replicative capacities were determined for old (A/WSN/33, A/Mississipi/3/01, A/New Caledonia/20/99, and A/Solomon Islands/03/06) and recent (A/Brisbane/59/2007-like) influenza A(H1N1) viruses either harboring or not harboring the H274Y NA mutation. Ferrets were infected with the A/Brisbane/59/2007-like wild-type (WT) isolate and its H274Y NA variant. Old A(H1N1) WT viruses grew at higher titers than did the A/Brisbane/59/2007-like viruses in vitro. The H274Y mutation was associated with reduced viral plaque areas in cells infected with A/WSN/33 and A/Mississippi/3/01, whereas the 2 A/Brisbane/59/2007-like isolates showed similar plaque sizes. In ferrets, the pyrexic response induced by the A/Brisbane/59/2007-like H274Y mutant was significantly higher than that induced by the WT isolate. Nasal wash viral titers were significantly greater for the mutant isolate on day 2 after inoculation, whereas the 2 viruses showed similar titers between days 3 and 7 after inoculation. The viral fitness of the recent A/Brisbane/59/2007-like H274Y variant is not impaired, consistent with its global dissemination. These results reinforce the need for new antiviral strategies.

Targeting of P53-transcriptional dysfunction by conditionally replicating adenovirus is not limited by P53-homologues

A hallmark of hepatocellular carcinomas is the loss of p53 or its transcriptional functions. In this study we describe the generation of the conditionally-replicating adenovirus Adp53sensor for the treatment of p53-dysfunctional HCC. p53-selective attenuation of viral replication was achieved by using p53-dependent expression of the transcriptional repressor GAL4-KRAB that was directed against the adenoviral E1A locus. Adp53sensor shows efficient replication in p53-dysfunctional, but not in p53-active cells. In p53-dysfunctional cells, p53-analogous transcriptional activity by other p53-family members was not sufficient to compromise replication of Adp53sensor. Furthermore RNAi experiments suggest that endogenous p73 is able to efficiently interfere with attenuated p53-dependent transcription for fine-tuning of the transcriptional response, but p73 is neither able to interfere with a fully-developed p53-transcriptional response nor capable to significantly induce the p53-depedent promoter in Adp53sensor under conditions of p53-dysfunction. In comparison with a genetically similar, but p53-insensitive virus, Adp53-sensor replication was strongly inhibited in the normal liver of p53-wt-mice, but not in p53-ko-mice thus confirming the correct function of the p53-dependent inhibitory loop. Adp53sensor showed efficient lytic and replicative properties in all cells with p53-dysfunction and successfully inhibited the growth of HCC xenotransplants. Intravenous injection of Adp53sensor lead to significantly reduced liver damage compared to the control virus. Adp53sensor provides a strategy that should be broadly applicable to other transcriptionally regulated DNA-viruses.

Prion dynamics with size dependency–strain phenomena

Models for the polymerization process involved in prion self-replication are well-established and studied [H. Engler, J. Pruss, and G.F. Webb, Analysis of a model for the dynamics of prions II, J. Math. Anal. Appl. 324 (2006), pp. 98–117; M.L. Greer, L. Pujo-Menjouet, and G.F. Webb, A mathematical analysis of the dynamics of prion proliferation, J. Theoret. Biol. 242 (2006), pp. 598–606; J. Pruss, L. Pujo-Menjouet, G.F. Webb, and R. Zacher, Analysis of a model for the dynamics of prions, Discrete Cont. Dyn. Sys. Ser. B 6(1) (2006), pp. 215–225] in the case where the dynamics coefficients do not depend on the size of polymers. However, several experimental studies indicate that the structure and size of the prion aggregates are determinant for their pathological effect. This motivated the analysis in Calvez et al. [Size distribution dependence of prion aggregates infectivity, Math Biosci. 217 (2009), pp. 88–99] where the authors take into account size-dependent replicative properties of prion aggregates. We first improve a result concerning the dynamics of prion aggregates when a pathological state exists (high production of the normal protein). Then we study the strain phenomena and more specifically we wonder what specific replicative properties are determinant in strain propagation. We propose to interpret it also as a dynamical property of size repartitions.

Identification of the Cellular Prohibitin 1/Prohibitin 2 Heterodimer as an Interaction Partner of the C-Terminal Cytoplasmic Domain of the HIV-1 Glycoprotein

Our studies aim to elucidate the functions carried out by the very long, and in its length highly conserved, C-terminal cytoplasmic domain (Env-CT) of the HIV-1 glycoprotein. Mass spectrometric analysis of cellular proteins bound to a tagged version of the HIV Env-CT led to the identification of the prohibitin 1 and 2 proteins (Phb1 and Phb2). These ubiquitously expressed proteins, which exist as stable heterodimers, have been shown to have multiple functions within cells and to localize to multiple cellular and extracellular compartments. The specificity of binding of the Phb1/Phb2 complex to the Env-CT was confirmed in various manners, including coimmunoprecipitation with authentic provirally encoded, full-length Env. Strong binding was dependent on Env residues 790 to 800 and could be severely inhibited by the double mutation L799R/L800Q but not by mutation of these amino acids individually. Analysis of the respective mutant virions revealed that their different abilities to bind Phb1/Phb2 correlated with their replicative properties. Thus, mutated virions with single mutations [HIV-Env-(L799R) and HIV-Env-(L800Q)] replicated similarly to wild-type HIV, but HIV-Env-(L799R/L800Q) virions, which cannot bind Phb1/Phb2, exhibited a cell-dependent replicative phenotype similar to that of HIV-Env-Tr712, lacking the entire Env-CT domain. Thus, replicative spread was achieved, although somewhat delayed, in "permissive" MT-4 cells but failed to occur in "nonpermissive" H9 T cells. These results point to binding of the Phb1/Phb2 complex to the Env-CT as being of importance for replicative spread in nonpermissive cells, possibly by modulating critical Phb-dependent cellular process(es).